Science.gov

Sample records for ccl3-mediated neutrophil recruitment

  1. Interferon-gamma coordinates CCL3-mediated neutrophil recruitment in vivo

    PubMed Central

    Bonville, Cynthia A; Percopo, Caroline M; Dyer, Kimberly D; Gao, Jiliang; Prussin, Calman; Foster, Barbara; Rosenberg, Helene F; Domachowske, Joseph B

    2009-01-01

    Background We have shown previously that acute infection with the respiratory pathogen, pneumonia virus of mice (PVM), results in local production of the proinflammatory chemokine, CCL3, and that neutrophil recruitment in response to PVM infection is reduced dramatically in CCL3 -/- mice. Results In this work, we demonstrate that CCL3-mediated neutrophil recruitment is coordinated by interferon-gamma (IFNγ). Neutrophil recruitment in response to PVM infection was diminished five-fold in IFNγ receptor gene-deleted mice, although neutrophils from IFNγR -/- mice expressed transcripts for the CCL3 receptor, CCR1 and responded functionally to CCL3 ex vivo. Similarly, in the absence of PVM infection, CCL3 overexpression alone could not elicit neutrophil recruitment in the absence of IFNγ. Interestingly, although supplemental IFNγ restored neutrophil recruitment and resulted in a sustained weight loss among CCL3-overexpressing IFNγ -/- mice, CCL3-mediated neutrophil recruitment alone did not result in the pulmonary edema or respiratory failure characteristic of severe viral infection, suggesting that CCL3 and IFN-γ together are sufficient to promote neutrophil recruitment but not pathologic activation. Conclusion Our findings reveal a heretofore unrecognized hierarchical interaction between the IFNγ and CCL3, which demonstrate that IFNγ is crucial for CCL3-mediated neutrophil recruitment in vivo. PMID:19298652

  2. CFTR RECRUITMENT TO PHAGOSOMES IN NEUTROPHILS

    PubMed Central

    Zhou, Yun; Song, Kejing; Painter, Richard G.; Aiken, Martha; Reiser, Jakob; Stanton, Bruce A.; Nauseef, William M.; Wang, Guoshun

    2013-01-01

    Optimal microbicidal activity of human polymorphonuclear leukocytes (PMN) relies on generation of toxic agents such as hypochlorous acid (HOCl) in phagosomes. HOCl formation requires H2O2 produced by the NADPH oxidase, myeloperoxidase derived from azurophilic granules, and chloride ion. Chloride transport from cytoplasm into phagosomes requires chloride channels which include cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chloride channel. However, the phagosomal targeting of CFTR in PMN has not been defined. Using human peripheral blood PMN, we determined that ~95–99% of LAMP-1 positive mature phagosomes were CFTR-positive, as judged by immunostaining and flow cytometric analysis. To establish a model cell system to evaluate CFTR phagosomal recruitment, we stably expressed EGFP alone, EGFP-wt-CFTR and EGFP-ΔF508-CFTR fusion proteins in promyelocytic PLB-985 cells, respectively. After differentiation into neutrophil-like cells, CFTR presentation to phagosomes was examined. EGFP-wt-CFTR was observed to associate with phagosomes and co-localize with LAMP-1. Flow cytometric analysis of the isolated phagosomes indicated that such a phagosomal targeting was determined by the CFTR portion of the fusion protein. In contrast, significantly less EGFP-ΔF508-CFTR was found in phagosomes, indicating a defective targeting of the molecule to the organelle. Importantly, CFTR corrector compound VRT-325 facilitated the recruitment of ΔF508-CFTR to phagosomes. These data demonstrate the possibility of pharmacologic correction of impaired recruitment of mutant CFTR, thereby providing a potential means to augment chloride supply to the phagosomes of PMN in patients with cystic fibrosis to enhance their microbicidal function. PMID:23486169

  3. The role of tissue resident cells in neutrophil recruitment

    PubMed Central

    Kim, Nancy D.; Luster, Andrew D.

    2015-01-01

    Neutrophils are first responders of the immune system, rapidly migrating into affected tissues in response to injury or infection. To effectively call in this first line of defense, strategically placed cells within the vasculature and tissue respond to noxious stimuli by sending out coordinated signals that recruit neutrophils. Regulation of organ-specific neutrophil entry occurs at two levels. First, the vasculature supplying the organ provides cues for neutrophil egress out of the bloodstream in a manner dependent upon its unique cellular composition and architectural features. Second, resident immune cells and stromal cells within the organ send coordinated signals that guide neutrophils to their final destination. Here, we review recent findings that highlight the importance of these tissue-specific responses in the regulation of neutrophil recruitment and the initiation and resolution of inflammation. PMID:26297103

  4. Neutrophils recruited to sites of infection protect from virus challenge by releasing neutrophil extracellular traps.

    PubMed

    Jenne, Craig N; Wong, Connie H Y; Zemp, Franz J; McDonald, Braedon; Rahman, Masmudur M; Forsyth, Peter A; McFadden, Grant; Kubes, Paul

    2013-02-13

    Neutrophils mediate bacterial clearance through various mechanisms, including the release of mesh-like DNA structures or neutrophil extracellular traps (NETs) that capture bacteria. Although neutrophils are also recruited to sites of viral infection, their role in antiviral innate immunity is less clear. We show that systemic administration of virus analogs or poxvirus infection induces neutrophil recruitment to the liver microvasculature and the release of NETs that protect host cells from virus infection. After systemic intravenous poxvirus challenge, mice exhibit thrombocytopenia and the recruitment of both neutrophils and platelets to the liver vasculature. Circulating platelets interact with, roll along, and adhere to the surface of adherent neutrophils, forming large, dynamic aggregates. These interactions facilitate the release of NETs within the liver vasculature that are able to protect host cells from poxvirus infection. These findings highlight the role of NETs and early tissue-wide responses in preventing viral infection.

  5. Perivascular macrophages mediate neutrophil recruitment during bacterial skin infection

    PubMed Central

    Abtin, Arby; Jain, Rohit; Mitchell, Andrew J.; Roediger, Ben; Brzoska, Anthony J.; Tikoo, Shweta; Cheng, Qiang; Ng, Lai Guan; Cavanagh, Lois L.; von Andrian, Ulrich H.; Hickey, Michael J.; Firth, Neville; Weninger, Wolfgang

    2014-01-01

    Transendothelial migration of neutrophils in post-capillary venules is a key event in the inflammatory response against pathogens and tissue damage. The precise regulation of this process is incompletely understood. We report that perivascular macrophages are critical for neutrophil migration into skin infected with the pathogen Staphylococcus aureus. Using multiphoton intravital microscopy we show that neutrophils extravasate from inflamed dermal venules in close proximity to perivascular macrophages, which are a major source of neutrophil chemoattractants. The virulence factor alpha-hemolysin lyses perivascular macrophages leading to decreased neutrophil transmigration. Our data illustrate a previously unrecognized role for perivascular macrophages in neutrophil recruitment to inflamed skin, and indicate that Staphylococcus aureus uses hemolysin-dependent killing of these cells as an immune evasion strategy. PMID:24270515

  6. Perivascular macrophages mediate neutrophil recruitment during bacterial skin infection.

    PubMed

    Abtin, Arby; Jain, Rohit; Mitchell, Andrew J; Roediger, Ben; Brzoska, Anthony J; Tikoo, Shweta; Cheng, Qiang; Ng, Lai Guan; Cavanagh, Lois L; von Andrian, Ulrich H; Hickey, Michael J; Firth, Neville; Weninger, Wolfgang

    2014-01-01

    Transendothelial migration of neutrophils in postcapillary venules is a key event in the inflammatory response against pathogens and tissue damage. The precise regulation of this process is incompletely understood. We report that perivascular macrophages are critical for neutrophil migration into skin infected with the pathogen Staphylococcus aureus. Using multiphoton intravital microscopy we showed that neutrophils extravasate from inflamed dermal venules in close proximity to perivascular macrophages, which are a major source of neutrophil chemoattractants. The virulence factor α-hemolysin produced by S. aureus lyses perivascular macrophages, which leads to decreased neutrophil transmigration. Our data illustrate a previously unrecognized role for perivascular macrophages in neutrophil recruitment to inflamed skin and indicate that S. aureus uses hemolysin-dependent killing of these cells as an immune evasion strategy. PMID:24270515

  7. Mediators of neutrophil recruitment in human abdominal aortic aneurysms

    PubMed Central

    Houard, Xavier; Touat, Ziad; Ollivier, Véronique; Louedec, Liliane; Philippe, Monique; Sebbag, Uriel; Meilhac, Olivier; Rossignol, Patrick; Michel, Jean-Baptiste

    2009-01-01

    Aims Neutrophils/platelet interactions are involved in abdominal aortic aneurysm (AAA). The intraluminal thrombus (ILT) is a human model of platelet/neutrophil interactions. The present study focused on mediators involved in neutrophil recruitment in AAA. Methods and results Conditioned media from luminal, intermediate, and abluminal layers of 29 human ILTs were analysed for neutrophil markers [elastase/α1-antitrypsin and MMP9/NGAL complexes, myeloperoxidase (MPO), and α-defensin peptides], RANTES, platelet factor 4 (PF4), and interleukin-8 (IL-8). Their time-dependent release into serum from clots generated in vitro and their plasma concentrations in AAA patients and controls were determined. Immunohistochemistry for neutrophils, platelets, IL-8, PF4, and RANTES on AAA sections was performed; and molecules involved in ILT neutrophil chemotactic function were analysed in vitro. Neutrophils and platelets colocalized in the luminal layer of the thrombus. Consistently, neutrophil markers and platelet-derived RANTES and PF4 were released predominantly by the luminal thrombus pole, where their concentrations were significantly correlated. The luminal ILT layer was also the main source of IL-8, whose immunostaining colocalized with neutrophils. All were also released time dependently from clots and were increased in plasma of AAA patients. Luminal ILT layers displayed potent neutrophil chemotactic activity in vitro, which was inhibited by RANTES- and IL-8-blocking antibodies as well as by reparixin, an antagonist of the IL-8 receptors CXCR1 and CXCR2. Conclusion Taken together, these results suggest that platelet-derived RANTES and neutrophil-derived IL-8 are involved in attracting neutrophils to the luminal layer of AAA ILT. PMID:19201759

  8. Chemokine CXCL1 mediated neutrophil recruitment: Role of glycosaminoglycan interactions.

    PubMed

    Sawant, Kirti V; Poluri, Krishna Mohan; Dutta, Amit K; Sepuru, Krishna Mohan; Troshkina, Anna; Garofalo, Roberto P; Rajarathnam, Krishna

    2016-01-01

    The chemokine CXCL1/MGSA plays a pivotal role in the host immune response by recruiting and activating neutrophils for microbial killing at the tissue site. CXCL1 exists reversibly as monomers and dimers, and mediates its function by binding glycosaminoglycans (GAG) and CXCR2 receptor. We recently showed that both monomers and dimers are potent CXCR2 agonists, the dimer is the high-affinity GAG ligand, lysine and arginine residues located in two non-overlapping domains mediate GAG interactions, and there is extensive overlap between GAG and receptor-binding domains. To understand how these structural properties influence in vivo function, we characterized peritoneal neutrophil recruitment of a trapped monomer and trapped dimer and a panel of WT lysine/arginine to alanine mutants. Monomers and dimers were active, but WT was more active indicating synergistic interactions promote recruitment. Mutants from both domains showed reduced GAG heparin binding affinities and reduced neutrophil recruitment, providing compelling evidence that both GAG-binding domains mediate in vivo trafficking. Further, mutant of a residue that is involved in both GAG binding and receptor signaling showed the highest reduction in recruitment. We conclude that GAG interactions and receptor activity of CXCL1 monomers and dimers are fine-tuned to regulate neutrophil trafficking for successful resolution of tissue injury. PMID:27625115

  9. Chemokine CXCL1 mediated neutrophil recruitment: Role of glycosaminoglycan interactions

    PubMed Central

    Sawant, Kirti V.; Poluri, Krishna Mohan; Dutta, Amit K.; Sepuru, Krishna Mohan; Troshkina, Anna; Garofalo, Roberto P.; Rajarathnam, Krishna

    2016-01-01

    The chemokine CXCL1/MGSA plays a pivotal role in the host immune response by recruiting and activating neutrophils for microbial killing at the tissue site. CXCL1 exists reversibly as monomers and dimers, and mediates its function by binding glycosaminoglycans (GAG) and CXCR2 receptor. We recently showed that both monomers and dimers are potent CXCR2 agonists, the dimer is the high-affinity GAG ligand, lysine and arginine residues located in two non-overlapping domains mediate GAG interactions, and there is extensive overlap between GAG and receptor-binding domains. To understand how these structural properties influence in vivo function, we characterized peritoneal neutrophil recruitment of a trapped monomer and trapped dimer and a panel of WT lysine/arginine to alanine mutants. Monomers and dimers were active, but WT was more active indicating synergistic interactions promote recruitment. Mutants from both domains showed reduced GAG heparin binding affinities and reduced neutrophil recruitment, providing compelling evidence that both GAG-binding domains mediate in vivo trafficking. Further, mutant of a residue that is involved in both GAG binding and receptor signaling showed the highest reduction in recruitment. We conclude that GAG interactions and receptor activity of CXCL1 monomers and dimers are fine-tuned to regulate neutrophil trafficking for successful resolution of tissue injury. PMID:27625115

  10. CXCL5 Drives Neutrophil Recruitment in TH17-Mediated GN

    PubMed Central

    Disteldorf, Erik M.; Krebs, Christian F.; Paust, Hans-Joachim; Turner, Jan-Eric; Nouailles, Geraldine; Tittel, André; Meyer-Schwesinger, Catherine; Stege, Gesa; Brix, Silke; Velden, Joachim; Wiech, Thorsten; Helmchen, Udo; Steinmetz, Oliver M.; Peters, Anett; Bennstein, Sabrina B.; Kaffke, Anna; Llanto, Chrystel; Lira, Sergio A.; Mittrücker, Hans-Willi; Stahl, Rolf A.K.; Kurts, Christian; Kaufmann, Stefan H.E.

    2015-01-01

    Neutrophil trafficking to sites of inflammation is essential for the defense against bacterial and fungal infections, but also contributes to tissue damage in TH17-mediated autoimmunity. This process is regulated by chemokines, which often show an overlapping expression pattern and function in pathogen- and autoimmune-induced inflammatory reactions. Using a murine model of crescentic GN, we show that the pathogenic TH17/IL-17 immune response induces chemokine (C-X-C motif) ligand 5 (CXCL5) expression in kidney tubular cells, which recruits destructive neutrophils that contribute to renal tissue injury. By contrast, CXCL5 was dispensable for neutrophil recruitment and effective bacterial clearance in a murine model of acute bacterial pyelonephritis. In line with these findings, CXCL5 expression was highly upregulated in the kidneys of patients with ANCA-associated crescentic GN as opposed to patients with acute bacterial pyelonephritis. Our data therefore identify CXCL5 as a potential therapeutic target for the restriction of pathogenic neutrophil infiltration in TH17-mediated autoimmune diseases while leaving intact the neutrophil function in protective immunity against invading pathogens. PMID:24904089

  11. Visualization of Signaling Molecules During Neutrophil Recruitment in Transgenic Mice Expressing FRET Biosensors.

    PubMed

    Mizuno, Rei; Kamioka, Yuji; Sakai, Yoshiharu; Matsuda, Michiyuki

    2016-01-01

    A number of chemical mediators regulate neutrophil recruitment to inflammatory sites either positively or negatively. Although the actions of each chemical mediator on the intracellular signaling networks controlling cell migration have been studied with neutrophils cultured in vitro, how such chemical mediators act cooperatively or counteractively in vivo remains largely unknown. To understand the mechanisms regulating neutrophil recruitment to the inflamed intestine in vivo, we recently generated transgenic mice expressing biosensors based on FRET (Förster resonance energy transfer) and set up two-photon excitation microscopy to observe the gastrointestinal tract in living mice. By measuring FRET in neutrophils, we showed activity changes of protein kinases in the neutrophils recruited to inflamed intestines. In this chapter, we describe the protocol used to visualize the protein kinase activities in neutrophils of the inflamed intestine of transgenic mice expressing the FRET biosensors. PMID:27246030

  12. Visualization of Signaling Molecules During Neutrophil Recruitment in Transgenic Mice Expressing FRET Biosensors.

    PubMed

    Mizuno, Rei; Kamioka, Yuji; Sakai, Yoshiharu; Matsuda, Michiyuki

    2016-01-01

    A number of chemical mediators regulate neutrophil recruitment to inflammatory sites either positively or negatively. Although the actions of each chemical mediator on the intracellular signaling networks controlling cell migration have been studied with neutrophils cultured in vitro, how such chemical mediators act cooperatively or counteractively in vivo remains largely unknown. To understand the mechanisms regulating neutrophil recruitment to the inflamed intestine in vivo, we recently generated transgenic mice expressing biosensors based on FRET (Förster resonance energy transfer) and set up two-photon excitation microscopy to observe the gastrointestinal tract in living mice. By measuring FRET in neutrophils, we showed activity changes of protein kinases in the neutrophils recruited to inflamed intestines. In this chapter, we describe the protocol used to visualize the protein kinase activities in neutrophils of the inflamed intestine of transgenic mice expressing the FRET biosensors.

  13. Neutrophil recruitment to lymph nodes limits local humoral response to Staphylococcus aureus.

    PubMed

    Kamenyeva, Olena; Boularan, Cedric; Kabat, Juraj; Cheung, Gordon Y C; Cicala, Claudia; Yeh, Anthony J; Chan, June L; Periasamy, Saravanan; Otto, Michael; Kehrl, John H

    2015-04-01

    Neutrophils form the first line of host defense against bacterial pathogens. They are rapidly mobilized to sites of infection where they help marshal host defenses and remove bacteria by phagocytosis. While splenic neutrophils promote marginal zone B cell antibody production in response to administered T cell independent antigens, whether neutrophils shape humoral immunity in other lymphoid organs is controversial. Here we investigate the neutrophil influx following the local injection of Staphylococcus aureus adjacent to the inguinal lymph node and determine neutrophil impact on the lymph node humoral response. Using intravital microscopy we show that local immunization or infection recruits neutrophils from the blood to lymph nodes in waves. The second wave occurs temporally with neutrophils mobilized from the bone marrow. Within lymph nodes neutrophils infiltrate the medulla and interfollicular areas, but avoid crossing follicle borders. In vivo neutrophils form transient and long-lived interactions with B cells and plasma cells, and their depletion augments production of antigen-specific IgG and IgM in the lymph node. In vitro activated neutrophils establish synapse- and nanotube-like interactions with B cells and reduce B cell IgM production in a TGF-β1 dependent manner. Our data reveal that neutrophils mobilized from the bone marrow in response to a local bacterial challenge dampen the early humoral response in the lymph node. PMID:25884622

  14. Slit2 prevents neutrophil recruitment and renal ischemia-reperfusion injury.

    PubMed

    Chaturvedi, Swasti; Yuen, Darren A; Bajwa, Amandeep; Huang, Yi-Wei; Sokollik, Christiane; Huang, Liping; Lam, Grace Y; Tole, Soumitra; Liu, Guang-Ying; Pan, Jerry; Chan, Lauren; Sokolskyy, Yaro; Puthia, Manoj; Godaly, Gabriela; John, Rohan; Wang, Changsen; Lee, Warren L; Brumell, John H; Okusa, Mark D; Robinson, Lisa A

    2013-07-01

    Neutrophils recruited to the postischemic kidney contribute to the pathogenesis of ischemia-reperfusion injury (IRI), which is the most common cause of renal failure among hospitalized patients. The Slit family of secreted proteins inhibits chemotaxis of leukocytes by preventing activation of Rho-family GTPases, suggesting that members of this family might modulate the recruitment of neutrophils and the resulting IRI. Here, in static and microfluidic shear assays, Slit2 inhibited multiple steps required for the infiltration of neutrophils into tissue. Specifically, Slit2 blocked the capture and firm adhesion of human neutrophils to inflamed vascular endothelial barriers as well as their subsequent transmigration. To examine whether these observations were relevant to renal IRI, we administered Slit2 to mice before bilateral clamping of the renal pedicles. Assessed at 18 hours after reperfusion, Slit2 significantly inhibited renal tubular necrosis, neutrophil and macrophage infiltration, and rise in plasma creatinine. In vitro, Slit2 did not impair the protective functions of neutrophils, including phagocytosis and superoxide production, and did not inhibit neutrophils from killing the extracellular pathogen Staphylococcus aureus. In vivo, administration of Slit2 did not attenuate neutrophil recruitment or bacterial clearance in mice with ascending Escherichia coli urinary tract infections and did not increase the bacterial load in the livers of mice infected with the intracellular pathogen Listeria monocytogenes. Collectively, these results suggest that Slit2 may hold promise as a strategy to combat renal IRI without compromising the protective innate immune response.

  15. Fatty acids as modulators of neutrophil recruitment, function and survival.

    PubMed

    Rodrigues, Hosana G; Takeo Sato, Fabio; Curi, Rui; Vinolo, Marco A R

    2016-08-15

    Neutrophils are well-known to act in the destruction of invading microorganisms. They have also been implicated in the activation of other immune cells including B- and T-lymphocytes and in the resolution of inflammation and tissue regeneration. Neutrophils are produced in the bone marrow and released into the circulation from where they migrate to tissues to perform their effector functions. Neutrophils are in constant contact with fatty acids that can modulate their function, activation and fate (survival or cell death) through different mechanisms. In this review, the effects of fatty acids pertaining to five classes, namely, long-chain saturated fatty acids (LCSFAs), short-chain fatty acids (SCFAs), and omega-3 (n-3), omega-6 (n-6) and omega-9 (n-9) unsaturated fatty acids, on neutrophils and the relevance of these effects for disease development are discussed.

  16. Inhibitors of neutrophil recruitment identified using transgenic zebrafish to screen a natural product library

    PubMed Central

    Wang, Xingang; Robertson, Anne L.; Li, Jingyu; Chai, Ruth Jinfen; Haishan, Wang; Sadiku, Pranvera; Ogryzko, Nikolay V.; Everett, Martin; Yoganathan, Kanagasundaram; Luo, Hongbo Robert; Renshaw, Stephen A.; Ingham, Philip W.

    2014-01-01

    Cell migration is fundamental to the inflammatory response, but uncontrolled cell migration and excess recruitment of neutrophils and other leukocytes can cause damage to the tissue. Here we describe the use of an in vivo model – the Tg(mpx:GFP)i114 zebrafish line, in which neutrophils are labelled by green fluorescent protein (GFP) – to screen a natural product library for compounds that can affect neutrophil migratory behaviour. Among 1040 fungal extracts screened, two were found to inhibit neutrophil migration completely. Subfractionation of these extracts identified sterigmatocystin and antibiotic PF1052 as the active components. Using the EZ-TAXIScan chemotaxis assay, both compounds were also found to have a dosage-dependent inhibitory effect on murine neutrophil migration. Furthermore, neutrophils treated with PF1052 failed to form pseudopods and appeared round in shape, suggesting a defect in PI3-kinase (PI3K) signalling. We generated a transgenic neutrophil-specific PtdIns(3,4,5)P3 (PIP3) reporter zebrafish line, which revealed that PF1052 does not affect the activation of PI3K at the plasma membrane. In human neutrophils, PF1052 neither induced apoptosis nor blocked AKT phosphorylation. In conclusion, we have identified an antibiotic from a natural product library with potent anti-inflammatory properties, and have established the utility of the mpx:GFP transgenic zebrafish for high-throughput in vivo screens for novel inhibitors of neutrophil migration. PMID:24291762

  17. Inhibitors of neutrophil recruitment identified using transgenic zebrafish to screen a natural product library.

    PubMed

    Wang, Xingang; Robertson, Anne L; Li, Jingyu; Chai, Ruth Jinfen; Haishan, Wang; Sadiku, Pranvera; Ogryzko, Nikolay V; Everett, Martin; Yoganathan, Kanagasundaram; Luo, Hongbo Robert; Renshaw, Stephen A; Ingham, Philip W

    2014-01-01

    Cell migration is fundamental to the inflammatory response, but uncontrolled cell migration and excess recruitment of neutrophils and other leukocytes can cause damage to the tissue. Here we describe the use of an in vivo model - the Tg(mpx:GFP)(i114) zebrafish line, in which neutrophils are labelled by green fluorescent protein (GFP) - to screen a natural product library for compounds that can affect neutrophil migratory behaviour. Among 1040 fungal extracts screened, two were found to inhibit neutrophil migration completely. Subfractionation of these extracts identified sterigmatocystin and antibiotic PF1052 as the active components. Using the EZ-TAXIScan chemotaxis assay, both compounds were also found to have a dosage-dependent inhibitory effect on murine neutrophil migration. Furthermore, neutrophils treated with PF1052 failed to form pseudopods and appeared round in shape, suggesting a defect in PI3-kinase (PI3K) signalling. We generated a transgenic neutrophil-specific PtdIns(3,4,5)P3 (PIP3) reporter zebrafish line, which revealed that PF1052 does not affect the activation of PI3K at the plasma membrane. In human neutrophils, PF1052 neither induced apoptosis nor blocked AKT phosphorylation. In conclusion, we have identified an antibiotic from a natural product library with potent anti-inflammatory properties, and have established the utility of the mpx:GFP transgenic zebrafish for high-throughput in vivo screens for novel inhibitors of neutrophil migration.

  18. A Novel Mechanism of Neutrophil Recruitment in a Coculture Model of the Rheumatoid Synovium

    PubMed Central

    Lally, Frank; Smith, Emily; Filer, Andrew; Stone, Michael A.; Shaw, John S.; Nash, Gerard B.; Buckley, Christopher D.; Rainger, G. Ed

    2011-01-01

    Objective Rheumatoid arthritis (RA) is classically thought of as a Th1, T lymphocyte–driven disease of the adaptive immune system. However, cells of the innate immune system, including neutrophils, are prevalent within the diseased joint, and accumulate in large numbers. This study was undertaken to determine whether cells of the rheumatoid stromal microenvironment could establish an inflammatory environment in which endothelial cells are conditioned in a disease-specific manner to support neutrophil recruitment. Methods Human umbilical vein endothelial cells (ECs) and fibroblasts isolated from the synovium or skin of RA patients were established in coculture on opposite sides of porous transwell filters. After 24 hours of EC conditioning, the membranes were incorporated into a parallel-plate, flow-based adhesion assay and levels of neutrophil adhesion to ECs were measured. Results ECs cocultured with synovial, but not skin, fibroblasts could recruit neutrophils in a manner that was dependent on the number of fibroblasts. Antibody blockade of P-selectin or E-selectin reduced neutrophil adhesion, and an antibody against CD18 (the β2 integrin) abolished adhesion. Blockade of CXCR2, but not CXCR1, also greatly inhibited neutrophil recruitment. Interleukin-6 (IL-6) was detectable in coculture supernatants, and both IL-6 and neutrophil adhesion were reduced in a dose-dependent manner by hydrocortisone added to cocultures. Antibody blockade of IL-6 also effectively abolished neutrophil adhesion. Conclusion Synovial fibroblasts from the rheumatoid joint play an important role in regulating the recruitment of inflammatory leukocytes during active disease. This process may depend on a previously unsuspected route of IL-6–mediated crosstalk between fibroblasts and endothelial cells. PMID:16255036

  19. G Protein-Coupled Receptor 43 Modulates Neutrophil Recruitment during Acute Inflammation

    PubMed Central

    Nicholls, Alyce J.; Oliveira, Ana Carolina; Mason, Linda J.; Binge, Lauren; Mackay, Charles R.; Wong, Connie H. Y.

    2016-01-01

    Fermentation of dietary fibre in the gut yields large amounts of short chain fatty acids (SCFAs). SCFAs can impart biological responses in cells through their engagement of ‘metabolite-sensing’ G protein-coupled receptors (GPCRs). One of the main SCFA receptors, GPR43, is highly expressed by neutrophils, which suggests that the actions of GPR43 and dietary fibre intake may affect neutrophil recruitment during inflammatory responses in vivo. Using intravital imaging of the small intestine, we found greater intravascular neutrophil rolling and adhesion in Gpr43−/−mice in response to LPS at 1 h. After 4 h of LPS challenge, the intravascular rolling velocity of GPR43-deficient neutrophils was reduced significantly and increased numbers of neutrophils were found in the lamina propria of Gpr43−/−mice. Additionally, GPR43-deficient leukocytes demonstrated exacerbated migration into the peritoneal cavity following fMLP challenge. The fMLP-induced neutrophil migration was significantly suppressed in wildtype mice that were treated with acetate, but not in Gpr43−/−mice, strongly suggesting a role for SCFAs in modulating neutrophil migration via GPR43. Indeed, neutrophils of no fibre-fed wildtype mice exhibited elevated migratory behaviour compared to normal chow-fed wildtype mice. Interestingly, this elevated migration could also be reproduced through simple transfer of a no fibre microbiota into germ-free mice, suggesting that the composition and function of microbiota stemming from a no fibre diet mediated the changes in neutrophil migration. Therefore, GPR43 and a microbiota composition that allows for SCFA production function to modulate neutrophil recruitment during inflammatory responses. PMID:27658303

  20. CARD9-Dependent Neutrophil Recruitment Protects against Fungal Invasion of the Central Nervous System

    PubMed Central

    Swamydas, Muthulekha; Rodriguez, Carlos A.; Lim, Jean K.; Mendez, Laura M.; Fink, Danielle L.; Hsu, Amy P.; Zhai, Bing; Karauzum, Hatice; Mikelis, Constantinos M.; Rose, Stacey R.; Ferre, Elise M. N.; Yockey, Lynne; Lemberg, Kimberly; Kuehn, Hye Sun; Rosenzweig, Sergio D.; Lin, Xin; Chittiboina, Prashant; Datta, Sandip K.; Belhorn, Thomas H.; Weimer, Eric T.; Hernandez, Michelle L.; Hohl, Tobias M.; Kuhns, Douglas B.; Lionakis, Michail S.

    2015-01-01

    Candida is the most common human fungal pathogen and causes systemic infections that require neutrophils for effective host defense. Humans deficient in the C-type lectin pathway adaptor protein CARD9 develop spontaneous fungal disease that targets the central nervous system (CNS). However, how CARD9 promotes protective antifungal immunity in the CNS remains unclear. Here, we show that a patient with CARD9 deficiency had impaired neutrophil accumulation and induction of neutrophil-recruiting CXC chemokines in the cerebrospinal fluid despite uncontrolled CNS Candida infection. We phenocopied the human susceptibility in Card9-/- mice, which develop uncontrolled brain candidiasis with diminished neutrophil accumulation. The induction of neutrophil-recruiting CXC chemokines is significantly impaired in infected Card9-/- brains, from both myeloid and resident glial cellular sources, whereas cell-intrinsic neutrophil chemotaxis is Card9-independent. Taken together, our data highlight the critical role of CARD9-dependent neutrophil trafficking into the CNS and provide novel insight into the CNS fungal susceptibility of CARD9-deficient humans. PMID:26679537

  1. Interleukin-1 alpha modulates neutrophil recruitment in chronic inflammation induced by hydrocarbon oil1

    PubMed Central

    Lee, Pui Y.; Kumagai, Yutaro; Xu, Yuan; Li, Yi; Barker, Tolga; Liu, Chao; Sobel, Eric S.; Takeuchi, Osamu; Akira, Shizuo; Satoh, Minoru; Reeves, WestleyH.

    2012-01-01

    Exposure to naturally-occurring hydrocarbon oils is associated with the development of chronic inflammation and a wide spectrum of pathological findings in humans and animal models. The mechanism underlying the unremitting inflammatory response to hydrocarbons remains largely unclear. The medium-length alkane 2,6,10,14 tetramethylpentadecane (TMPD; also known as pristane) is a hydrocarbon that potently elicits chronic peritonitis characterized by persistent infiltration of neutrophils and monocytes. In this study, we reveal the essential role of interleukin (IL)-1α in sustaining the chronic recruitment of neutrophils following TMPD treatment. IL-1α and IL-1 receptor signaling promote the migration of neutrophils to the peritoneal cavity in a CXC chemokine receptor-2 (CXCR2)-dependent manner. This mechanism is at least partially dependent on the production of the neutrophil chemoattractant CXCL5. Moreover, although chronic infiltration of inflammatory monocytes is dependent on a different pathway requiring Toll-like receptor (TLR)-7, type-I interferon receptor, and CC-chemokine receptor-2 (CCR2), the adaptor molecules MyD88, IRAK-4, IRAK1 and IRAK2 are shared in regulating the recruitment of both monocytes and neutrophils. Taken together, our findings uncover an IL-1α-dependent mechanism of neutrophil recruitment in hydrocarbon-induced peritonitis and illustrate the interactions of innate immune pathways in chronic inflammation. PMID:21191074

  2. Protection from septic peritonitis by rapid neutrophil recruitment through omental high endothelial venules

    PubMed Central

    Buscher, Konrad; Wang, Huiyu; Zhang, Xueli; Striewski, Paul; Wirth, Benedikt; Saggu, Gurpanna; Lütke-Enking, Stefan; Mayadas, Tanya N.; Ley, Klaus; Sorokin, Lydia; Song, Jian

    2016-01-01

    Acute peritonitis is a frequent medical condition that can trigger severe sepsis as a life-threatening complication. Neutrophils are first-responders in infection but recruitment mechanisms to the abdominal cavity remain poorly defined. Here, we demonstrate that high endothelial venules (HEVs) of the greater omentum constitute a main entry pathway in TNFα-, Escherichia coli (E. coli)- and caecal ligation and puncture-induced models of inflammation. Neutrophil transmigration across HEVs is faster than across conventional postcapillary venules and requires a unique set of adhesion receptors including peripheral node addressin, E-, L-selectin and Mac-1 but not P-selectin or LFA-1. Omental milky spots readily concentrate intra-abdominal E. coli where macrophages and recruited neutrophils collaborate in phagocytosis and killing. Inhibition of the omental neutrophil response exacerbates septic progression of peritonitis. This data identifies HEVs as a clinically relevant vascular recruitment site for neutrophils in acute peritonitis that is indispensable for host defence against early systemic bacterial spread and sepsis. PMID:26940548

  3. Protection from septic peritonitis by rapid neutrophil recruitment through omental high endothelial venules.

    PubMed

    Buscher, Konrad; Wang, Huiyu; Zhang, Xueli; Striewski, Paul; Wirth, Benedikt; Saggu, Gurpanna; Lütke-Enking, Stefan; Mayadas, Tanya N; Ley, Klaus; Sorokin, Lydia; Song, Jian

    2016-01-01

    Acute peritonitis is a frequent medical condition that can trigger severe sepsis as a life-threatening complication. Neutrophils are first-responders in infection but recruitment mechanisms to the abdominal cavity remain poorly defined. Here, we demonstrate that high endothelial venules (HEVs) of the greater omentum constitute a main entry pathway in TNFα-, Escherichia coli (E. coli)- and caecal ligation and puncture-induced models of inflammation. Neutrophil transmigration across HEVs is faster than across conventional postcapillary venules and requires a unique set of adhesion receptors including peripheral node addressin, E-, L-selectin and Mac-1 but not P-selectin or LFA-1. Omental milky spots readily concentrate intra-abdominal E. coli where macrophages and recruited neutrophils collaborate in phagocytosis and killing. Inhibition of the omental neutrophil response exacerbates septic progression of peritonitis. This data identifies HEVs as a clinically relevant vascular recruitment site for neutrophils in acute peritonitis that is indispensable for host defence against early systemic bacterial spread and sepsis. PMID:26940548

  4. Protection from septic peritonitis by rapid neutrophil recruitment through omental high endothelial venules.

    PubMed

    Buscher, Konrad; Wang, Huiyu; Zhang, Xueli; Striewski, Paul; Wirth, Benedikt; Saggu, Gurpanna; Lütke-Enking, Stefan; Mayadas, Tanya N; Ley, Klaus; Sorokin, Lydia; Song, Jian

    2016-01-01

    Acute peritonitis is a frequent medical condition that can trigger severe sepsis as a life-threatening complication. Neutrophils are first-responders in infection but recruitment mechanisms to the abdominal cavity remain poorly defined. Here, we demonstrate that high endothelial venules (HEVs) of the greater omentum constitute a main entry pathway in TNFα-, Escherichia coli (E. coli)- and caecal ligation and puncture-induced models of inflammation. Neutrophil transmigration across HEVs is faster than across conventional postcapillary venules and requires a unique set of adhesion receptors including peripheral node addressin, E-, L-selectin and Mac-1 but not P-selectin or LFA-1. Omental milky spots readily concentrate intra-abdominal E. coli where macrophages and recruited neutrophils collaborate in phagocytosis and killing. Inhibition of the omental neutrophil response exacerbates septic progression of peritonitis. This data identifies HEVs as a clinically relevant vascular recruitment site for neutrophils in acute peritonitis that is indispensable for host defence against early systemic bacterial spread and sepsis.

  5. Tumor-Derived CXCL1 Promotes Lung Cancer Growth via Recruitment of Tumor-Associated Neutrophils

    PubMed Central

    Zhu, Ha; Xu, Junfang; Zheng, Yuanyuan; Cao, Xuetao

    2016-01-01

    Neutrophils have a traditional role in inflammatory process and act as the first line of defense against infections. Although their contribution to tumorigenesis and progression is still controversial, accumulating evidence recently has demonstrated that tumor-associated neutrophils (TANs) play a key role in multiple aspects of cancer biology. Here, we detected that chemokine CXCL1 was dramatically elevated in serum from 3LL tumor-bearing mice. In vitro, 3LL cells constitutively expressed and secreted higher level of CXCL1. Furthermore, knocking down CXCL1 expression in 3LL cells significantly hindered tumor growth by inhibiting recruitment of neutrophils from peripheral blood into tumor tissues. Additionally, tumor-infiltrated neutrophils expressed higher levels of MPO and Fas/FasL, which may be involved in TAN-mediated inhibition of CD4+ and CD8+ T cells. These results demonstrate that tumor-derived CXCL1 contributes to TANs infiltration in lung cancer which promotes tumor growth. PMID:27446967

  6. Activated human valvular interstitial cells sustain interleukin-17 production to recruit neutrophils in infective endocarditis.

    PubMed

    Yeh, Chiou-Yueh; Shun, Chia-Tung; Kuo, Yu-Min; Jung, Chiau-Jing; Hsieh, Song-Chou; Chiu, Yen-Ling; Chen, Jeng-Wei; Hsu, Ron-Bin; Yang, Chia-Ju; Chia, Jean-San

    2015-06-01

    The mechanisms that underlie valvular inflammation in streptococcus-induced infective endocarditis (IE) remain unclear. We previously demonstrated that streptococcal glucosyltransferases (GTFs) can activate human heart valvular interstitial cells (VIC) to secrete interleukin-6 (IL-6), a cytokine involved in T helper 17 (Th17) cell differentiation. Here, we tested the hypothesis that activated VIC can enhance neutrophil infiltration through sustained IL-17 production, leading to valvular damage. To monitor cytokine and chemokine production, leukocyte recruitment, and the induction or expansion of CD4(+) CD45RA(-) CD25(-) CCR6(+) Th17 cells, primary human VIC were cultured in vitro and activated by GTFs. Serum cytokine levels were measured using an enzyme-linked immunosorbent assay (ELISA), and neutrophils and Th17 cells were detected by immunohistochemistry in infected valves from patients with IE. The expression of IL-21, IL-23, IL-17, and retinoic acid receptor-related orphan receptor C (Rorc) was upregulated in GTF-activated VIC, which may enhance the proliferation of memory Th17 cells in an IL-6-dependent manner. Many chemokines, including chemokine (C-X-C motif) ligand 1 (CXCL1), were upregulated in GTF-activated VIC, which might recruit neutrophils and CD4(+) T cells. Moreover, CXCL1 production in VIC was induced in a dose-dependent manner by IL-17 to enhance neutrophil chemotaxis. CXCL1-expressing VIC and infiltrating neutrophils could be detected in infected valves, and serum concentrations of IL-17, IL-21, and IL-23 were increased in patients with IE compared to healthy donors. Furthermore, elevated serum IL-21 levels have been significantly associated with severe valvular damage, including rupture of chordae tendineae, in IE patients. Our findings suggest that VIC are activated by bacterial modulins to recruit neutrophils and that such activities might be further enhanced by the production of Th17-associated cytokines. Together, these factors can amplify

  7. Comparative Ability of Mesenchymal Stromal Cells from Different Tissues to Limit Neutrophil Recruitment to Inflamed Endothelium

    PubMed Central

    Munir, Hafsa; Luu, Nguyet-Thin; Clarke, Lewis S. C.; Nash, Gerard B.; McGettrick, Helen M.

    2016-01-01

    Mesenchymal stromal cells (MSC) are tissue-resident stromal cells capable of modulating immune responses, including leukocyte recruitment by endothelial cells (EC). However, the comparative potency of MSC from different sources in suppressing recruitment, and the necessity for close contact with endothelium remain uncertain, although these factors have implications for use of MSC in therapy. We thus compared the effects of MSC isolated from bone marrow, Wharton’s jelly, and trabecular bone on neutrophil recruitment to cytokine-stimulated EC, using co-culture models with different degrees of proximity between MSC and EC. All types of MSC suppressed neutrophil adhesion to inflamed endothelium but not neutrophil transmigration, whether directly incorporated into endothelial monolayers or separated from them by thin micropore filters. Further increase in the separation of the two cell types tended to reduce efficacy, although this diminution was least for the bone marrow MSC. Immuno-protective effects of MSC were also diminished with repeated passage; with BMMSC, but not WJMSC, completing losing their suppressive effect by passage 7. Conditioned media from all co-cultures suppressed neutrophil recruitment, and IL-6 was identified as a common bioactive mediator. These results suggest endogenous MSC have a homeostatic role in limiting inflammatory leukocyte infiltration in a range of tissues. Since released soluble mediators might have effects locally or remotely, infusion of MSC into blood or direct injection into target organs might be efficacious, but in either case, cross-talk between EC and MSC appears necessary. PMID:27171357

  8. Dual oxidase regulates neutrophil recruitment in allergic airways.

    PubMed

    Chang, Sandra; Linderholm, Angela; Franzi, Lisa; Kenyon, Nicholas; Grasberger, Helmut; Harper, Richart

    2013-12-01

    Enhanced reactive oxygen species production in allergic airways is well described and correlates with increased airway contractions, inflammatory cell infiltration, goblet cell metaplasia, and mucus hypersecretion. There is also an abundance of interleukin-4/interleukin-13 (IL-4/IL-13)- or interleukin-5-secreting cells that are thought to be central to the pathogenesis of allergic asthma. We postulated that the dual oxidases (DUOX1 and DUOX2), members of the nicotinamide adenine dinucleotide phosphate oxidase family that release hydrogen peroxide (H2O2) in the respiratory tract, are critical proteins in the pathogenesis of allergic airways. DUOX activity is regulated by cytokines, including IL-4 and IL-13, and DUOX-mediated H2O2 influences several important features of allergic asthma: mucin production, IL-8 secretion, and wound healing. The objective of this study was to establish the contribution of DUOXs to the development of allergic asthma in a murine model. To accomplish this goal, we utilized a DUOXA-deficient mouse model (Duoxa(-/-)) that lacked maturation factors for both DUOX1 and DUOX2. Our results are the first to demonstrate evidence of DUOX protein and DUOX functional activity in murine airway epithelium. We also demonstrate that DUOXA maturation factors are required for airway-specific H2O2 production and localization of DUOX to cilia of fully differentiated airway epithelial cells. We compared wild-type and Duoxa(-/-) mice in an ovalbumin exposure model to determine the role of DUOX in allergic asthma. In comparison to DUOX-intact mice, Duoxa(-/-) mice had reduced mucous cell metaplasia and lower levels of TH2 cytokine levels in bronchoalveolar fluid. In addition, increased airway resistance in response to methacholine was observed in Duoxa(+/+) mice, as expected, but was absent in Duoxa(-/-) mice. Surprisingly, Duoxa(-/-) mice had decreased influx of neutrophils in bronchoalveolar fluid and lung tissue sections associated with a lower level of the

  9. Febrile-range hyperthermia augments pulmonary neutrophil recruitment and amplifies pulmonary oxygen toxicity.

    PubMed

    Hasday, Jeffrey D; Garrison, Allen; Singh, Ishwar S; Standiford, Theodore; Ellis, Garrettson S; Rao, Srinivas; He, Ju-Ren; Rice, Penny; Frank, Mariah; Goldblum, Simeon E; Viscardi, Rose M

    2003-06-01

    Febrile-range hyperthermia (FRH) improves survival in experimental infections by accelerating pathogen clearance, but may also increase collateral tissue injury. We hypothesized that FRH would worsen the outcome of inflammation stimulated by a non-replicating agonist and tested this hypothesis in a murine model of pulmonary oxygen toxicity. Using a conscious, temperature-controlled mouse model, we showed that maintaining a core temperature at FRH (39 degrees C to 40 degrees C) rather than at euthermic levels (36.5 degrees C to 37 degrees C) during hyperoxia exposure accelerated lethal pulmonary vascular endothelial injury, reduced the inspired oxygen threshold for lethality, induced expression of granulocyte-colony stimulating factor, and expanded the circulating neutrophil pool. In these same mice, FRH augmented pulmonary expression of the ELR(+) CXC chemokines, KC and LPS-induced CXC chemokine, enhanced recruitment of neutrophils, and changed the histological pattern of lung injury to a neutrophilic interstitial pneumonitis. Immunoblockade of CXC receptor-2 abrogated neutrophil recruitment, reduced pulmonary vascular injury, and delayed death. These combined data demonstrate that FRH may enlist distinct mediators and effector cells to profoundly shift the host response to a defined injurious stimulus, in part by augmenting delivery of neutrophils to sites of inflammation, such as may occur in infections. In certain conditions, such as in the hyperoxic lung, this process may be deleterious.

  10. Platelet serotonin promotes the recruitment of neutrophils to sites of acute inflammation in mice.

    PubMed

    Duerschmied, Daniel; Suidan, Georgette L; Demers, Melanie; Herr, Nadine; Carbo, Carla; Brill, Alexander; Cifuni, Stephen M; Mauler, Maximilian; Cicko, Sanja; Bader, Michael; Idzko, Marco; Bode, Christoph; Wagner, Denisa D

    2013-02-01

    The majority of peripheral serotonin is stored in platelets, which secrete it on activation. Serotonin releases Weibel-Palade bodies (WPBs) and we asked whether absence of platelet serotonin affects neutrophil recruitment in inflammatory responses. Tryptophan hydroxylase (Tph)1–deficient mice, lacking non-neuronal serotonin, showed mild leukocytosis compared with wild-type (WT), primarily driven by an elevated neutrophil count. Despite this, 50% fewer leukocytes rolled on unstimulated mesenteric venous endothelium of Tph1(-/-) mice. The velocity of rolling leukocytes was higher in Tph1(-/-) mice, indicating fewer selectin-mediated interactions with endothelium. Stimulation of endothelium with histamine, a secretagogue of WPBs, or injection of serotonin normalized the rolling in Tph1(-/-) mice. Diminished rolling in Tph1(-/-) mice resulted in reduced firm adhesion of leukocytes after lipopolysaccharide treatment. Blocking platelet serotonin uptake with fluoxetine in WT mice reduced serum serotonin by > 80% and similarly reduced leukocyte rolling and adhesion. Four hours after inflammatory stimulation, neutrophil extravasation into lung, peritoneum, and skin wounds was reduced in Tph1(-/-) mice, whereas in vitro neutrophil chemotaxis was independent of serotonin. Survival of lipopolysaccharide-induced endotoxic shock was improved in Tph1(-/-) mice. In conclusion, platelet serotonin promotes the recruitment of neutrophils in acute inflammation, supporting an important role for platelet serotonin in innate immunity. PMID:23243271

  11. Circumventing Y. pestis Virulence by Early Recruitment of Neutrophils to the Lungs during Pneumonic Plague.

    PubMed

    Vagima, Yaron; Zauberman, Ayelet; Levy, Yinon; Gur, David; Tidhar, Avital; Aftalion, Moshe; Shafferman, Avigdor; Mamroud, Emanuelle

    2015-05-01

    Pneumonic plague is a fatal disease caused by Yersinia pestis that is associated with a delayed immune response in the lungs. Because neutrophils are the first immune cells recruited to sites of infection, we investigated the mechanisms responsible for their delayed homing to the lung. During the first 24 hr after pulmonary infection with a fully virulent Y. pestis strain, no significant changes were observed in the lungs in the levels of neutrophils infiltrate, expression of adhesion molecules, or the expression of the major neutrophil chemoattractants keratinocyte cell-derived chemokine (KC), macrophage inflammatory protein 2 (MIP-2) and granulocyte colony stimulating factor (G-CSF). In contrast, early induction of chemokines, rapid neutrophil infiltration and a reduced bacterial burden were observed in the lungs of mice infected with an avirulent Y. pestis strain. In vitro infection of lung-derived cell-lines with a YopJ mutant revealed the involvement of YopJ in the inhibition of chemoattractants expression. However, the recruitment of neutrophils to the lungs of mice infected with the mutant was still delayed and associated with rapid bacterial propagation and mortality. Interestingly, whereas KC, MIP-2 and G-CSF mRNA levels in the lungs were up-regulated early after infection with the mutant, their protein levels remained constant, suggesting that Y. pestis may employ additional mechanisms to suppress early chemoattractants induction in the lung. It therefore seems that prevention of the early influx of neutrophils to the lungs is of major importance for Y. pestis virulence. Indeed, pulmonary instillation of KC and MIP-2 to G-CSF-treated mice infected with Y. pestis led to rapid homing of neutrophils to the lung followed by a reduction in bacterial counts at 24 hr post-infection and improved survival rates. These observations shed new light on the virulence mechanisms of Y. pestis during pneumonic plague, and have implications for the development of novel

  12. TLR2-induced IL-10 production impairs neutrophil recruitment to infected tissues during neonatal bacterial sepsis.

    PubMed

    Andrade, Elva B; Alves, Joana; Madureira, Pedro; Oliveira, Liliana; Ribeiro, Adília; Cordeiro-da-Silva, Anabela; Correia-Neves, Margarida; Trieu-Cuot, Patrick; Ferreira, Paula

    2013-11-01

    Sepsis is the third most common cause of neonatal death, with Group B Streptococcus (GBS) being the leading bacterial agent. The pathogenesis of neonatal septicemia is still unsolved. We described previously that host susceptibility to GBS infection is due to early IL-10 production. In this study, we investigated whether triggering TLR2 to produce IL-10 is a risk factor for neonatal bacterial sepsis. We observed that, in contrast to wild-type (WT) pups, neonatal TLR2-deficient mice were resistant to GBS-induced sepsis. Moreover, if IL-10 signaling were blocked in WT mice, they also were resistant to sepsis. This increased survival rate was due to an efficient recruitment of neutrophils to infected tissues that leads to bacterial clearance, thus preventing the development of sepsis. To confirm that IL-10 produced through TLR2 activation prevents neutrophil recruitment, WT pups were treated with the TLR2 agonist Pam3CSK4 prior to nebulization with the neutrophil chemotactic agent LTB4. Neutrophil recruitment into the neonatal lungs was inhibited in pups treated with Pam3CSK4. However, the migration was restored in Pam3CSK4-treated pups when IL-10 signaling was blocked (either by anti-IL-10R mAb treatment or by using IL-10-deficient mice). Our findings highlight that TLR2-induced IL-10 production is a key event in neonatal susceptibility to bacterial sepsis.

  13. Intravascular clearance of disseminating Cryptococcus neoformans in the brain can be improved by enhancing neutrophil recruitment in mice.

    PubMed

    Sun, Donglei; Zhang, Mingshun; Liu, Gongguan; Wu, Hui; Li, Chang; Zhou, Hong; Zhang, Xiquan; Shi, Meiqing

    2016-07-01

    Extrapulmonary dissemination of Cryptococcus neoformans (C. neoformans) is one of the most critical steps in the development of meningoencephalitis. Here, we report that clearance of the disseminating C. neoformans occurs within the brain microvasculature. Interestingly, the efficiency of the intravascular clearance in the brain is reduced compared to that in the lung. Intravascular clearance is mainly mediated by neutrophils, and complement C5a receptor signaling is crucial for mediating neutrophil recruitment in the vasculature. C. neoformans stimulated actin polymerization of neutrophils is critically involved in their recruitment to the lung, which is associated with the unique vascular structure detected in the lung. The relatively lower efficiency of fungal clearance in the brain vasculature correlates with less efficient recruitment of neutrophils. Accordingly, intravascular clearance of C. neoformans in the brain could be remarkably improved by increasing the recruitment of neutrophils. We conclude that neutrophils have the ability to eliminate C. neoformans arrested in the vasculature. However, insufficient recruitment of neutrophils limited the optimal clearance of this microorganism in the brain. These results imply that a therapeutic strategy aimed at enhancing the accumulation of neutrophils could help prevent cryptococcal meningoencephalitis. PMID:27109176

  14. Annexin A1 and the Resolution of Inflammation: Modulation of Neutrophil Recruitment, Apoptosis, and Clearance

    PubMed Central

    Sugimoto, Michelle Amantéa; Vago, Juliana Priscila; Teixeira, Mauro Martins; Sousa, Lirlândia Pires

    2016-01-01

    Neutrophils (also named polymorphonuclear leukocytes or PMN) are essential components of the immune system, rapidly recruited to sites of inflammation, providing the first line of defense against invading pathogens. Since neutrophils can also cause tissue damage, their fine-tuned regulation at the inflammatory site is required for proper resolution of inflammation. Annexin A1 (AnxA1), also known as lipocortin-1, is an endogenous glucocorticoid-regulated protein, which is able to counterregulate the inflammatory events restoring homeostasis. AnxA1 and its mimetic peptides inhibit neutrophil tissue accumulation by reducing leukocyte infiltration and activating neutrophil apoptosis. AnxA1 also promotes monocyte recruitment and clearance of apoptotic leukocytes by macrophages. More recently, some evidence has suggested the ability of AnxA1 to induce macrophage reprogramming toward a resolving phenotype, resulting in reduced production of proinflammatory cytokines and increased release of immunosuppressive and proresolving molecules. The combination of these mechanisms results in an effective resolution of inflammation, pointing to AnxA1 as a promising tool for the development of new therapeutic strategies to treat inflammatory diseases. PMID:26885535

  15. The time-of-day of myocardial infarction onset affects healing through oscillations in cardiac neutrophil recruitment.

    PubMed

    Schloss, Maximilian J; Horckmans, Michael; Nitz, Katrin; Duchene, Johan; Drechsler, Maik; Bidzhekov, Kiril; Scheiermann, Christoph; Weber, Christian; Soehnlein, Oliver; Steffens, Sabine

    2016-01-01

    Myocardial infarction (MI) is the leading cause of death in Western countries. Epidemiological studies show acute MI to be more prevalent in the morning and to be associated with a poorer outcome in terms of mortality and recovery. The mechanisms behind this association are not fully understood. Here, we report that circadian oscillations of neutrophil recruitment to the heart determine infarct size, healing, and cardiac function after MI Preferential cardiac neutrophil recruitment during the active phase (Zeitgeber time, ZT13) was paralleled by enhanced myeloid progenitor production, increased circulating numbers of CXCR2(hi) neutrophils as well as upregulated cardiac adhesion molecule and chemokine expression. MI at ZT13 resulted in significantly higher cardiac neutrophil infiltration compared to ZT5, which was inhibited by CXCR2 antagonism or neutrophil-specific CXCR2 knockout. Limiting exaggerated neutrophilic inflammation at this time point significantly reduced the infarct size and improved cardiac function. PMID:27226028

  16. Nicorandil inhibits neutrophil recruitment in carrageenan-induced experimental pleurisy in mice.

    PubMed

    Matsui, Tamires C; Coura, Giovanna M E; Melo, Ivo S F; Batista, Carla R A; Augusto, Paulo Sérgio A; Godin, Adriana M; Araújo, Débora P; César, Isabela C; Ribeiro, Lucas S; Souza, Danielle G; Klein, André; de Fátima, Ângelo; Machado, Renes R; Coelho, Márcio M

    2015-12-15

    Nicorandil is a drug characterized by the coupling of a nitric oxide (NO) donor to nicotinamide. We have previously demonstrated that nicotinamide exhibits activity in different models of pain and inflammation. Now, we investigated the effects induced by per os (p.o.) administration of nicorandil (25, 50 or 100mg/Kg) on neutrophil recruitment in a carrageenan-induced model of pleurisy in mice. Effects induced by nicorandil (100mg/kg) were compared with those induced by equimolar doses of nicotinamide (58mg/kg) and N-(2-hydroxyethyl)-nicotinamide (NHN; 79mg/kg). We also investigated whether effects on the production of inflammatory mediators play a role in the activity of nicorandil. P.o. nicorandil, 0.5h before and 1h after the i.pl. injection of carrageenan, reduced neutrophil recruitment. However, equimolar doses of nicotinamide or NHN failed to induce such effect. Single treatment (previous or late) with nicorandil (100mg/Kg, p.o.) also reduced neutrophils recruitment, although to a lesser extent when compared to the double treatment. Nicorandil reduced the concentrations of interleukin-1β, CXCL-1 and prostaglandin E2 in the pleural exudate. Concluding, we demonstrated the activity of nicorandil in a model of pleurisy induced by carrageenan. This activity was characterized by reduction of the neutrophil accumulation and inhibition of production of inflammatory mediators. The effects induced by nicorandil on the leukocytes recruitment and production of inflammatory mediators contribute to a better understanding of its clinical benefits and indicate that these benefits may be due to its vasodilating and anti-inflammatory activities. PMID:26607465

  17. Plasticity in Tumor-Promoting Inflammation: Impairment of Macrophage Recruitment Evokes a Compensatory Neutrophil Response12

    PubMed Central

    Pahler, Jessica C; Tazzyman, Simon; Erez, Neta; Chen, Yung-Yi; Murdoch, Craig; Nozawa, Hiroaki; Lewis, Claire E; Hanahan, Douglas

    2008-01-01

    Previous studies in the K14-HPV/E2 mouse model of cervical carcinogenesis demonstrated that infiltrating macrophages are the major source of matrix metalloproteinase 9 (MMP-9), a metalloprotease important for tumor angiogenesis and progression. We observed increased expression of the macrophage chemoattractant, CCL2, and its receptor, CCR2, concomitant with macrophage influx and MMP-9 expression. To study the role of CCL2-CCR2 signaling in cervical tumorigenesis, we generated CCR2-deficient K14-HPV/E2 mice. Cervixes of CCR2-null mice contained significantly fewer macrophages. Surprisingly, there was only a modest delay in time to progression from dysplasia to carcinoma in the CCR2-deficient mice, and no difference in end-stage tumor incidence or burden. Moreover, there was an unexpected persistence of MMP-9 activity, associated with increased abundance of MMP-9+ neutrophils in tumors from CCR2-null mice. In vitro bioassays revealed that macrophages produce soluble factor(s) that can suppress neutrophil dynamics, as evidenced by reduced chemotaxis in response to CXCL8, and impaired invasion into three-dimensional tumor masses grown in vitro. Our data suggest a mechanism whereby CCL2 attracts proangiogenic CCR2+ macrophages with the ancillary capability to limit infiltration by neutrophils. If such tumor-promoting macrophages are suppressed, MMP-9+ neutrophils are then recruited, providing alternative paracrine support for tumor angiogenesis and progression. PMID:18392134

  18. Platelet-Derived CCL5 Regulates CXC Chemokine Formation and Neutrophil Recruitment in Acute Experimental Colitis.

    PubMed

    Yu, Changhui; Zhang, Songen; Wang, Yongzhi; Zhang, Su; Luo, Lingtao; Thorlacius, Henrik

    2016-02-01

    Accumulating data suggest that platelets not only regulate thrombosis and haemostasis but also inflammatory processes. Platelets contain numerous potent pro-inflammatory compounds, including the chemokines CCL5 and CXCL4, although their role in acute colitis remains elusive. The aim of this study is to examine the role of platelets and platelet-derived chemokines in acute colitis. Acute colitis is induced in female Balb/c mice by administration of 5% dextran sodium sulfate (DSS) for 5 days. Animals receive a platelet-depleting, anti-CCL5, anti-CXCL4, or a control antibody prior to DSS challenge. Colonic tissue is collected for quantification of myeloperoxidase (MPO) activity, CXCL5, CXCL2, interleukin-6 (IL-6), and CCL5 levels as well as morphological analyses. Platelet depletion reduce tissue damage and clinical disease activity index in DSS-exposed animals. Platelet depletion not only reduces levels of CXCL2 and CXCL5 but also levels of CCL5 in the inflamed colon. Immunoneutralization of CCL5 but not CXCL4 reduces tissue damage, CXC chemokine expression, and neutrophil recruitment in DSS-treated animals. These findings show that platelets play a key role in acute colitis by regulating CXC chemokine generation, neutrophil infiltration, and tissue damage in the colon. Moreover, our results suggest that platelet-derived CCL5 is an important link between platelet activation and neutrophil recruitment in acute colitis.

  19. Mast cell activation and neutrophil recruitment promotes early and robust inflammation in the meninges in EAE.

    PubMed

    Christy, Alison L; Walker, Margaret E; Hessner, Martin J; Brown, Melissa A

    2013-05-01

    The meninges are often considered inert tissues that house the CSF and provide protection for the brain and spinal cord. Yet emerging data demonstrates that they are also active sites of immune responses. Furthermore, the blood-CSF barrier surrounding meningeal blood vessels, together with the blood-brain barrier (BBB), is postulated to serve as a gateway for the pathological infiltration of immune cells into the CNS in multiple sclerosis (MS). Our previous studies using mast cell-deficient (Kit(W/Wv)) mice demonstrated that mast cells resident in the dura mater and pia mater exacerbate experimental autoimmune encephalomyelitis (EAE), a rodent model of MS, by facilitating CNS inflammatory cell influx. Here we examined the underlying mechanisms that mediate these effects. We demonstrate that there are dramatic alterations in immune associated gene expression in the meninges in pre-clinical disease, including those associated with mast cell and neutrophil function. Meningeal mast cells are activated within 24 h of disease induction, but do not directly compromise CNS vascular integrity. Rather, through production of TNF, mast cells elicit an early influx of neutrophils, cells known to alter vascular permeability, into the meninges. These data add to the growing evidence that inflammation in the meninges precedes CNS immune cell infiltration and establish that mast cells are among the earliest participants in these disease-initiating events. We hypothesize that mast cell-dependent neutrophil recruitment and activation in the meninges promotes early breakdown of the local BBB and CSF-blood barrier allowing initial immune cell access to the CNS.

  20. CD43 deficiency has no impact in competitive in vivo assays of neutrophil or activated T cell recruitment efficiency.

    PubMed

    Carlow, Douglas A; Ziltener, Hermann J

    2006-11-01

    Using noncompetitive methodologies comparing CD43(+/+) and CD43(-/-) mice, it has been reported that CD43(-/-) leukocytes exhibit reduced recruitment efficiency to sites of inflammation. More recent analyses demonstrate that CD43 on activated T cells can function as an E-selectin ligand (E-SelL) in vitro, suggesting that CD43 might promote rolling interactions during recruitment of leukocytes and account for the reported recruitment deficits in CD43(-/-) T cells and neutrophils in vivo. Internally controlled competitive in vivo methods using fluorescent tracking dyes were applied to compare recruitment efficiency of CD43(+/+) vs CD43(-/-) activated T cells to inflamed skin and of peripheral blood neutrophils to inflamed peritoneum. A simple CFSE perfusion method was developed to distinguish arterial/venous vasculature and confirm appropriate extravasation through venules in a Con A-induced cutaneous inflammation model. In vivo recruitment of peripheral blood neutrophils to inflamed peritoneum was core 2 GlcNAcT-I dependent, but recruitment efficiency was not influenced by absence of CD43. There were also no significant differences in core 2 GlcNAcT-I-dependent, selectin-dependent, cutaneous recruitment of activated T cells from CD43(+/+) and congenic CD43(-/-) mice in either B6 or P-selectin(-/-) recipients despite biochemical confirmation that a CD43-specific E-SelL was present on activated T cells. We conclude that recruitment of neutrophils and activated T cells in these in vivo models is not influenced by CD43 expression and that if CD43 on activated T cells performs an E-SelL function in vivo, it contributes in a limited physiological context.

  1. Schistosoma japonicum egg specific protein SjE16.7 recruits neutrophils and induces inflammatory hepatic granuloma initiation.

    PubMed

    Wu, Chenyun; Chen, Qing; Fang, Yan; Wu, Jianhua; Han, Yanyan; Wang, Ying; Yang, Yang; Chu, Min; Feng, Yan; Tan, Linping; Guo, Xiaokui; Hu, Wei; Wang, Zhaojun

    2014-02-01

    Neutrophils are known to play a major role in the egg granulomatous lesions caused by Schistosoma japonicum, but the precise mechanism by which eggs recruit or active neutrophil is unknown. Here we report S. japonicum egg specific EF-hand protein-SjE16.7 is a potent neutrophil recruiter and initiates the egg associated inflammatory granuloma in schistosomiasis. We show that the expression of SjE16.7 at level of both mRNA and protein is restricted to the egg stage. It locates in the miracidium and subshell area of the egg and can be secreted by the egg. The antigenic properties of SjE16.7 strongly suggest a role for SjE16.7 as an egg-derived molecule involved in host-parasite interactions. To study SjE16.7 functions in vivo, we challenged murine air pouch with recombinant SjE16.7. The results showed SjE16.7 trigged more inflammatory cell infiltration than vehicle or control protein. Using peritoneal exudate neutrophils from mice, we found that SjE16.7 significantly induced neutrophil chemotaxis in vitro, and the observed phenotypes were associated with enhanced Rac GTPase activation in SjE16.7 treated cells. Finally, in vivo hepatic granuloma formation model showed SjE16.7 coupled beads recruited more inflammatory cell infiltration than control beads. Our findings suggest SjE16.7 is an important pathogenic factor derived from egg. By recruiting neutrophils and inducing local inflammation, SjE16.7 facilitates eggs to be excreted through gut tissues and also initiates pathology in the liver; therefore SjE16.7 is a possible target for the prevention and treatment of schistosomiasis. PMID:24551263

  2. Neutrophil-Derived CCL3 Is Essential for the Rapid Recruitment of Dendritic Cells to the Site of Leishmania major Inoculation in Resistant Mice

    PubMed Central

    Charmoy, Mélanie; Brunner-Agten, Saskia; Aebischer, David; Auderset, Floriane; Launois, Pascal; Milon, Geneviève; Proudfoot, Amanda E. I.; Tacchini-Cottier, Fabienne

    2010-01-01

    Neutrophils are rapidly and massively recruited to sites of microbial infection, where they can influence the recruitment of dendritic cells. Here, we have analyzed the role of neutrophil released chemokines in the early recruitment of dendritic cells (DCs) in an experimental model of Leishmania major infection. We show in vitro, as well as during infection, that the parasite induced the expression of CCL3 selectively in neutrophils from L. major resistant mice. Neutrophil-secreted CCL3 was critical in chemotaxis of immature DCs, an effect lost upon CCL3 neutralisation. Depletion of neutrophils prior to infection, as well as pharmacological or genetic inhibition of CCL3, resulted in a significant decrease in DC recruitment at the site of parasite inoculation. Decreased DC recruitment in CCL3−/− mice was corrected by the transfer of wild type neutrophils at the time of infection. The early release of CCL3 by neutrophils was further shown to have a transient impact on the development of a protective immune response. Altogether, we identified a novel role for neutrophil-secreted CCL3 in the first wave of DC recruitment to the site of infection with L. major, suggesting that the selective release of neutrophil-secreted chemokines may regulate the development of immune response to pathogens. PMID:20140197

  3. Neutrophil Recruitment and Activation in Decidua with Intra-Amniotic IL-1beta in the Preterm Rhesus Macaque1

    PubMed Central

    Presicce, Pietro; Senthamaraikannan, Paranthaman; Alvarez, Manuel; Rueda, Cesar M.; Cappelletti, Monica; Miller, Lisa A.; Jobe, Alan H.; Chougnet, Claire A.; Kallapur, Suhas G.

    2014-01-01

    ABSTRACT Chorioamnionitis, an infection/inflammation of the fetomaternal membranes, is frequently associated with preterm delivery. The mechanisms of inflammation in chorioamnionitis are poorly understood. We hypothesized that neutrophils recruited to the decidua would be the major producers of proinflammatory cytokines. We injected intra-amniotic (IA) interleukin 1beta (IL-1beta) at ∼80% gestation in rhesus macaque monkeys, Macaca mulatta, delivered the fetuses surgically 24 h or 72 h after IA injections, and investigated the role of immune cells in the chorion-amnion decidua. IA IL-1beta induced a robust infiltration of neutrophils and significant increases of proinflammatory cytokines in the chorioamnion decidua at 24 h after exposure, with a subsequent decrease at 72 h. Neutrophils in the decidua were the major source of tumor necrosis factor alpha (TNFalpha) and IL-8. Interestingly, IA IL-1beta also induced a significant increase in anti-inflammatory indoleamine 2,3-dioxygenase (IDO) expression in the decidua neutrophils. The frequency of regulatory T cells (Tregs) and FOXP3 mRNA expression in the decidua did not change after IA IL-1beta injection. Collectively, our data demonstrate that in this model of sterile chorioamnionitis, the decidua neutrophils cause the inflammation in the gestational tissues but may also act as regulators to dampen the inflammation. These results help to understand the contribution of neutrophils to the pathogenesis of chorioamnionitis-induced preterm labor. PMID:25537373

  4. Tir Triggers Expression of CXCL1 in Enterocytes and Neutrophil Recruitment during Citrobacter rodentium Infection

    PubMed Central

    Habibzay, Maryam; Glegola-Madejska, Izabela; Guenot, Marianne; Collins, James W.

    2015-01-01

    The hallmarks of enteropathogenic Escherichia coli (EPEC) infection are formation of attaching and effacing (A/E) lesions on mucosal surfaces and actin-rich pedestals on cultured cells, both of which are dependent on the type III secretion system effector Tir. Following translocation into cultured cells and clustering by intimin, Tir Y474 is phosphorylated, leading to recruitment of Nck, activation of N-WASP, and actin polymerization via the Arp2/3 complex. A secondary, weak, actin polymerization pathway is triggered via an NPY motif (Y454). Importantly, Y454 and Y474 play no role in A/E lesion formation on mucosal surfaces following infection with the EPEC-like mouse pathogen Citrobacter rodentium. In this study, we investigated the roles of Tir segments located upstream of Y451 and downstream of Y471 in C. rodentium colonization and A/E lesion formation. We also tested the role that Tir residues Y451 and Y471 play in host immune responses to C. rodentium infection. We found that deletion of amino acids 382 to 462 or 478 to 547 had no impact on the ability of Tir to mediate A/E lesion formation, although deletion of amino acids 478 to 547 affected Tir translocation. Examination of enterocytes isolated from infected mice revealed that a C. rodentium strain expressing Tir_Y451A/Y471A recruited significantly fewer neutrophils to the colon and triggered less colonic hyperplasia on day 14 postinfection than the wild-type strain. Consistently, enterocytes isolated from mice infected with C. rodentium expressing Tir_Y451A/Y471A expressed significantly less CXCL1. These result show that Tir-induced actin remodeling plays a direct role in modulation of immune responses to C. rodentium infection. PMID:26077760

  5. Tir Triggers Expression of CXCL1 in Enterocytes and Neutrophil Recruitment during Citrobacter rodentium Infection.

    PubMed

    Crepin, Valerie F; Habibzay, Maryam; Glegola-Madejska, Izabela; Guenot, Marianne; Collins, James W; Frankel, Gad

    2015-09-01

    The hallmarks of enteropathogenic Escherichia coli (EPEC) infection are formation of attaching and effacing (A/E) lesions on mucosal surfaces and actin-rich pedestals on cultured cells, both of which are dependent on the type III secretion system effector Tir. Following translocation into cultured cells and clustering by intimin, Tir Y474 is phosphorylated, leading to recruitment of Nck, activation of N-WASP, and actin polymerization via the Arp2/3 complex. A secondary, weak, actin polymerization pathway is triggered via an NPY motif (Y454). Importantly, Y454 and Y474 play no role in A/E lesion formation on mucosal surfaces following infection with the EPEC-like mouse pathogen Citrobacter rodentium. In this study, we investigated the roles of Tir segments located upstream of Y451 and downstream of Y471 in C. rodentium colonization and A/E lesion formation. We also tested the role that Tir residues Y451 and Y471 play in host immune responses to C. rodentium infection. We found that deletion of amino acids 382 to 462 or 478 to 547 had no impact on the ability of Tir to mediate A/E lesion formation, although deletion of amino acids 478 to 547 affected Tir translocation. Examination of enterocytes isolated from infected mice revealed that a C. rodentium strain expressing Tir_Y451A/Y471A recruited significantly fewer neutrophils to the colon and triggered less colonic hyperplasia on day 14 postinfection than the wild-type strain. Consistently, enterocytes isolated from mice infected with C. rodentium expressing Tir_Y451A/Y471A expressed significantly less CXCL1. These result show that Tir-induced actin remodeling plays a direct role in modulation of immune responses to C. rodentium infection.

  6. Role of platelet-activating factor in polymorphonuclear neutrophil recruitment in reperfused ischemic rabbit heart.

    PubMed

    Montrucchio, G; Alloatti, G; Mariano, F; Comino, A; Cacace, G; Polloni, R; De Filippi, P G; Emanuelli, G; Camussi, G

    1993-02-01

    This study investigated the role of platelet-activating factor in the recruitment of polymorphonuclear neutrophils (PMN) in a rabbit model of cardiac ischemia and reperfusion. The accumulation of PMN was evaluated 2 and 24 hours after removal of 40 minutes of coronary occlusion by morphometric analysis and 111In-labeled PMN infiltration. The administration of two structurally unrelated platelet-activating factor-receptor antagonists (SDZ 63-675, 5 mg/kg body weight, and WEB 2170, 5 mg/kg body weight) before reperfusion significantly reduced the accumulation of PMN, as well as the hemodynamic alterations and the size of necrotic area. Two hours after reperfusion, the percentage of increase of 111In-labeled PMN in transmural central ischemic zone was significantly reduced in rabbits pretreated with SDZ 63-675 (51.4 +/- 7.9) or WEB 2170 (32.4 +/- 8.8) with respect to untreated rabbits (107.6 +/- 13.5). The morphometric analysis of myocardial sections confirmed the reduction of PMN infiltration at 2 hours and demonstrated that at 24 hours the phenomenon was even more significant. In addition, SDZ 63-675 and WEB 2170 prevented early transient bradycardia and hypotension and reduced the infarct size, judged by staining with tetrazolium at 2 and 24 hours after reperfusion, and by histological examination at 24 hours. These results suggest that platelet-activating factor is involved in the accumulation of PMN in the reperfused ischemic myocardium and contributes to the evolution of myocardial injury.

  7. VAP-1 blockade prevents subarachnoid hemorrhage-associated cerebrovascular dilating dysfunction via repression of a neutrophil recruitment-related mechanism.

    PubMed

    Xu, Haoliang; Testai, Fernando D; Valyi-Nagy, Tibor; N Pavuluri, Mani; Zhai, Fengguo; Nanegrungsunk, Danop; Paisansathan, Chanannait; Pelligrino, Dale A

    2015-04-01

    Our previous findings indicated that in rats subjected to subarachnoid hemorrhage (SAH), suppression of post-SAH neuroinflammation via vascular adhesion protein-1 (VAP-1) blockade provides significant neuroprotection. We and others have reported that neuroinflammation contributes to cerebral microvascular impairment. Thus, in the present study, we tested the hypotheses that: (1) treatment with LJP-1586, a selective VAP-1 blocker, prevents SAH-associated pial arteriolar dilating dysfunction; and (2) the vasculoprotective effect of LJP-1586 arises from inhibiting SAH-elicited neutrophil recruitment. We utilized an endovascular perforation model of SAH. Rats subjected to SAH were either treated with LJP-1586 or rendered neutropenic via anti-neutrophil-antibody treatment. Findings from these groups were compared to their respective control groups. At 48 h post-SAH, rats were evaluated for neurobehavioral function, pial venular leukocyte trafficking, and pial arteriolar reactivity to topically-applied acetylcholine (ACh) and S-nitroso-N-acetyl penicillamine (SNAP). Pial arteriolar responses decreased at 48 h post-SAH. However, in the presence of LJP-1586, those responses were significantly preserved. Neutrophil-depletion yielded a substantial suppression of SAH-associated leukocyte adhesion and infiltration. This was accompanied by a significant preservation of pial arteriolar dilating function, suggesting a direct link between neutrophil recruitment and the loss of cerebral microvascular reactivity. Moreover, neutrophil depletion also was associated with significant protection of neurobehavioral function. The present findings suggest that attenuating SAH-linked elevation in neutrophil trafficking will protect against the development of microvascular dysfunction and subsequent neurological impairment.

  8. Neutrophil Recruitment by Tumor Necrosis Factor from Mast Cells in Immune Complex Peritonitis

    NASA Astrophysics Data System (ADS)

    Zhang, Yan; Ramos, Bernard F.; Jakschik, Barbara A.

    1992-12-01

    During generalized immune complex-induced inflammation of the peritoneal cavity, two peaks of tumor necrosis factor (TNF) were observed in the peritoneal exudate of normal mice. In mast cell-deficient mice, the first peak was undetected, and the second peak of TNF and neutrophil influx were significantly reduced. Antibody to TNF significantly inhibited neutrophil infiltration in normal but not in mast cell-deficient mice. Mast cell repletion of the latter normalized TNF, neutrophil mobilization, and the effect of the antibody to TNF. Thus, in vivo, mast cells produce the TNF that augments neutrophil emigration.

  9. Optineurin deficiency in mice contributes to impaired cytokine secretion and neutrophil recruitment in bacteria-driven colitis.

    PubMed

    Chew, Thean S; O'Shea, Nuala R; Sewell, Gavin W; Oehlers, Stefan H; Mulvey, Claire M; Crosier, Philip S; Godovac-Zimmermann, Jasminka; Bloom, Stuart L; Smith, Andrew M; Segal, Anthony W

    2015-08-01

    Crohn's disease (CD) is associated with delayed neutrophil recruitment and bacterial clearance at sites of acute inflammation as a result of impaired secretion of proinflammatory cytokines by macrophages. To investigate the impaired cytokine secretion and confirm our previous findings, we performed transcriptomic analysis in macrophages and identified a subgroup of individuals with CD who had low expression of the autophagy receptor optineurin (OPTN). We then clarified the role of OPTN deficiency in: macrophage cytokine secretion; mouse models of bacteria-driven colitis and peritonitis; and zebrafish Salmonella infection. OPTN-deficient bone-marrow-derived macrophages (BMDMs) stimulated with heat-killed Escherichia coli secreted less proinflammatory TNFα and IL6 cytokines despite similar gene transcription, which normalised with lysosomal and autophagy inhibitors, suggesting that TNFα is mis-trafficked to lysosomes via bafilomycin-A-dependent pathways in the absence of OPTN. OPTN-deficient mice were more susceptible to Citrobacter colitis and E. coli peritonitis, and showed reduced levels of proinflammatory TNFα in serum, diminished neutrophil recruitment to sites of acute inflammation and greater mortality, compared with wild-type mice. Optn-knockdown zebrafish infected with Salmonella also had higher mortality. OPTN plays a role in acute inflammation and neutrophil recruitment, potentially via defective macrophage proinflammatory cytokine secretion, which suggests that diminished OPTN expression in humans might increase the risk of developing CD. PMID:26044960

  10. Optineurin deficiency in mice contributes to impaired cytokine secretion and neutrophil recruitment in bacteria-driven colitis

    PubMed Central

    Chew, Thean S.; O'Shea, Nuala R.; Sewell, Gavin W.; Oehlers, Stefan H.; Mulvey, Claire M.; Crosier, Philip S.; Godovac-Zimmermann, Jasminka; Bloom, Stuart L.; Smith, Andrew M.; Segal, Anthony W.

    2015-01-01

    ABSTRACT Crohn's disease (CD) is associated with delayed neutrophil recruitment and bacterial clearance at sites of acute inflammation as a result of impaired secretion of proinflammatory cytokines by macrophages. To investigate the impaired cytokine secretion and confirm our previous findings, we performed transcriptomic analysis in macrophages and identified a subgroup of individuals with CD who had low expression of the autophagy receptor optineurin (OPTN). We then clarified the role of OPTN deficiency in: macrophage cytokine secretion; mouse models of bacteria-driven colitis and peritonitis; and zebrafish Salmonella infection. OPTN-deficient bone-marrow-derived macrophages (BMDMs) stimulated with heat-killed Escherichia coli secreted less proinflammatory TNFα and IL6 cytokines despite similar gene transcription, which normalised with lysosomal and autophagy inhibitors, suggesting that TNFα is mis-trafficked to lysosomes via bafilomycin-A-dependent pathways in the absence of OPTN. OPTN-deficient mice were more susceptible to Citrobacter colitis and E. coli peritonitis, and showed reduced levels of proinflammatory TNFα in serum, diminished neutrophil recruitment to sites of acute inflammation and greater mortality, compared with wild-type mice. Optn-knockdown zebrafish infected with Salmonella also had higher mortality. OPTN plays a role in acute inflammation and neutrophil recruitment, potentially via defective macrophage proinflammatory cytokine secretion, which suggests that diminished OPTN expression in humans might increase the risk of developing CD. PMID:26044960

  11. Role of platelet-activating factor in polymorphonuclear neutrophil recruitment in reperfused ischemic rabbit heart.

    PubMed Central

    Montrucchio, G.; Alloatti, G.; Mariano, F.; Comino, A.; Cacace, G.; Polloni, R.; De Filippi, P. G.; Emanuelli, G.; Camussi, G.

    1993-01-01

    This study investigated the role of platelet-activating factor in the recruitment of polymorphonuclear neutrophils (PMN) in a rabbit model of cardiac ischemia and reperfusion. The accumulation of PMN was evaluated 2 and 24 hours after removal of 40 minutes of coronary occlusion by morphometric analysis and 111In-labeled PMN infiltration. The administration of two structurally unrelated platelet-activating factor-receptor antagonists (SDZ 63-675, 5 mg/kg body weight, and WEB 2170, 5 mg/kg body weight) before reperfusion significantly reduced the accumulation of PMN, as well as the hemodynamic alterations and the size of necrotic area. Two hours after reperfusion, the percentage of increase of 111In-labeled PMN in transmural central ischemic zone was significantly reduced in rabbits pretreated with SDZ 63-675 (51.4 +/- 7.9) or WEB 2170 (32.4 +/- 8.8) with respect to untreated rabbits (107.6 +/- 13.5). The morphometric analysis of myocardial sections confirmed the reduction of PMN infiltration at 2 hours and demonstrated that at 24 hours the phenomenon was even more significant. In addition, SDZ 63-675 and WEB 2170 prevented early transient bradycardia and hypotension and reduced the infarct size, judged by staining with tetrazolium at 2 and 24 hours after reperfusion, and by histological examination at 24 hours. These results suggest that platelet-activating factor is involved in the accumulation of PMN in the reperfused ischemic myocardium and contributes to the evolution of myocardial injury. Images Figure 5 Figure 6 PMID:8434642

  12. MET is required for the recruitment of anti-tumoural neutrophils

    PubMed Central

    Finisguerra, Veronica; Di Conza, Giusy; Di Matteo, Mario; Serneels, Jens; Costa, Sandra; Thompson, A.A. Roger; Wauters, Els; Walmsley, Sarah; Prenen, Hans; Granot, Zvi; Casazza, Andrea; Mazzone, Massimiliano

    2015-01-01

    Mutations or amplification of the MET proto-oncogene are involved in the pathogenesis of several tumours1-4, which rely on the constitutive engagement of this pathway for their growth and survival1,5. However, MET is expressed not only by cancer cells but also by tumour-associated stromal cells although its precise role in this compartment is not well characterized6-11. Here, we show that MET is required for neutrophil chemoattraction and cytotoxicity in response to its ligand HGF. Met deletion in neutrophils enhances tumour growth and metastasis. This phenotype correlates with reduced neutrophil infiltration to both primary tumour and metastatic site. Similarly, Met is necessary for neutrophil transudation during colitis, skin rash or peritonitis. Mechanistically, Met is induced by tumour-derived TNF-α or other inflammatory stimuli in both mouse and human neutrophils. This induction is instrumental for neutrophil transmigration across an activated endothelium and iNOS production upon HGF stimulation. Consequently, HGF/MET-dependent nitric oxide release by neutrophils promotes cancer cell killing, which abates tumour growth and metastasis. Following systemic administration of a MET kinase inhibitor, we prove that the therapeutic benefit of MET targeting in cancer cells is partly countered by the pro-tumoural effect rising from MET blockade in neutrophils. Our work identifies an unprecedented role of MET in neutrophils, suggests a potential “Achilles’ heel” of MET-targeted therapies in cancer, and supports the rationale for evaluating anti-MET drugs in certain inflammatory diseases. PMID:25985180

  13. Crucial role of SLP-76 and ADAP for neutrophil recruitment in mouse kidney ischemia-reperfusion injury

    PubMed Central

    Block, Helena; Herter, Jan M.; Rossaint, Jan; Stadtmann, Anika; Kliche, Stefanie; Lowell, Clifford A.

    2012-01-01

    Neutrophils trigger inflammation-induced acute kidney injury (AKI), a frequent and potentially lethal occurrence in humans. Molecular mechanisms underlying neutrophil recruitment to sites of inflammation have proved elusive. In this study, we demonstrate that SLP-76 (SH2 domain–containing leukocyte phosphoprotein of 76 kD) and ADAP (adhesion and degranulation promoting adaptor protein) are involved in E-selectin–mediated integrin activation and slow leukocyte rolling, which promotes ischemia-reperfusion–induced AKI in mice. By using genetically engineered mice and transduced Slp76−/− primary leukocytes, we demonstrate that ADAP as well as two N-terminal–located tyrosines and the SH2 domain of SLP-76 are required for downstream signaling and slow leukocyte rolling. The Tec family kinase Bruton tyrosine kinase is downstream of SLP-76 and, together with ADAP, regulates PI3Kγ (phosphoinositide 3-kinase–γ)- and PLCγ2 (phospholipase Cγ2)-dependent pathways. Blocking both pathways completely abolishes integrin affinity and avidity regulation. Thus, SLP-76 and ADAP are involved in E-selectin–mediated integrin activation and neutrophil recruitment to inflamed kidneys, which may underlie the development of life-threatening ischemia-reperfusion–induced AKI in humans. PMID:22291096

  14. GDF-15 inhibits integrin activation and mouse neutrophil recruitment through the ALK-5/TGF-βRII heterodimer.

    PubMed

    Artz, Annette; Butz, Stefan; Vestweber, Dietmar

    2016-07-28

    Growth differentiation factor 15 (GDF-15) is the first cytokine known to counteract chemokine-induced activation of leukocyte integrins. We showed recently that this activity dampens neutrophil recruitment into inflamed tissue and is required for survival of myocardial infarction in mice. The receptor responsible for this GDF-15-triggered anti-inflammatory mechanism on myeloid cells is not known. Here, we identify this receptor as transforming growth factor β receptor I (TGF-βRI) (activin receptor-like kinase 5 [ALK-5]) and TGF-β receptor II (TGF-βRII). We show that interference with these receptors by small-molecule inhibitors, antibodies, or small interfering RNA, blocked the GDF-15 effect on leukocyte integrin activation. Likewise, gene inactivation of each of the 2 receptors in neutrophils isolated from conditional gene-deficient mice abolished the inhibitory effect of GDF-15 on CXCL1-induced β2-integrin activation and neutrophil diapedesis. Rapid neutrophil arrest induced by CXCL1 in vivo was inhibited by GDF-15 in an ALK-5 and TGF-βRII dependent way. As for GDF-15 gene-deficient mice, we found that extravasation of neutrophils deficient for ALK-5 or TGF-βRII was strongly increased in the interleukin-1β inflamed cremaster. The inhibitory effects of GDF-15 on neutrophil integrin activation and in vivo neutrophil arrest were also found for TGF-β1. Mechanistically, GDF-15 and TGF-β1 interfered with integrin activation by inhibiting the activation of Ras-related protein 1 (Rap-1), an effect that depended on CalDAG- guanine nucleotide exchange factor 1 (GEF1) and cell division control protein 42 homolog. We conclude that both GDF-15 and TGF-β1 counteract chemokine-induced integrin activation on neutrophils via the ALK-5/TGF-βRII heterodimer. This represents a novel, rapid anti-inflammatory activity of the 2 TGF-β receptors and of TGF-β1. PMID:27235139

  15. Neutrophil recruitment limited by high-affinity bent β2 integrin binding ligand in cis

    PubMed Central

    Fan, Zhichao; McArdle, Sara; Marki, Alex; Mikulski, Zbigniew; Gutierrez, Edgar; Engelhardt, Britta; Deutsch, Urban; Ginsberg, Mark; Groisman, Alex; Ley, Klaus

    2016-01-01

    Neutrophils are essential for innate immunity and inflammation and many neutrophil functions are β2 integrin-dependent. Integrins can extend (E+) and acquire a high-affinity conformation with an ‘open' headpiece (H+). The canonical switchblade model of integrin activation proposes that the E+ conformation precedes H+, and the two are believed to be structurally linked. Here we show, using high-resolution quantitative dynamic footprinting (qDF) microscopy combined with a homogenous conformation-reporter binding assay in a microfluidic device, that a substantial fraction of β2 integrins on human neutrophils acquire an unexpected E−H+ conformation. E−H+ β2 integrins bind intercellular adhesion molecules (ICAMs) in cis, which inhibits leukocyte adhesion in vitro and in vivo. This endogenous anti-inflammatory mechanism inhibits neutrophil aggregation, accumulation and inflammation. PMID:27578049

  16. Interleukin-33 facilitates neutrophil recruitment and bacterial clearance in S. aureus-caused peritonitis.

    PubMed

    Lan, Fang; Yuan, Baohong; Liu, Tao; Luo, Xiaochun; Huang, Ping; Liu, Yunjun; Dai, Liangcheng; Yin, Hui

    2016-04-01

    Interleukin (IL)-33, a newly recognized member of IL-1 family of cytokines, plays an important role in polarizing Th2-associated immunity. Recently growing evidence indicates that IL-33 also represents a crucial mediator of antimicrobial infection. In this study, we investigated the effect of IL-33 on antibacterial response using an acute Staphylococcus aureus peritoneal infection model. Our results showed that IL-33 administration induced a rapid bacterial clearance and markedly reduced the S. aureus infection-related mortality. IL-33-treated mice displayed increased neutrophil influx into the focus of infection and higher concentrations of chemokine CXCL2 in the peritoneum than untreated mice. The beneficial effect of IL-33 priming was related to reversal of the S. aureus-induced reduction of CXCR2 expression on the surface of neutrophils. Furthermore, conditioning of neutrophils by IL-33 led to the enhancement of complement receptor 3 expression induced by S. aureus, which in turn facilitates the phagocytosis of opsonized S. aureus. Finally, neutrophils primed by IL-33 upregulated the production of reactive oxygen species and the subsequent killing activity for S. aureus. All together, these findings suggest that IL-33, through regulating multiple steps of neutrophil-mediated bactericidal function, provides a profound effect in host antimicrobial defense response. PMID:26991049

  17. Interleukin-33 facilitates neutrophil recruitment and bacterial clearance in S. aureus-caused peritonitis.

    PubMed

    Lan, Fang; Yuan, Baohong; Liu, Tao; Luo, Xiaochun; Huang, Ping; Liu, Yunjun; Dai, Liangcheng; Yin, Hui

    2016-04-01

    Interleukin (IL)-33, a newly recognized member of IL-1 family of cytokines, plays an important role in polarizing Th2-associated immunity. Recently growing evidence indicates that IL-33 also represents a crucial mediator of antimicrobial infection. In this study, we investigated the effect of IL-33 on antibacterial response using an acute Staphylococcus aureus peritoneal infection model. Our results showed that IL-33 administration induced a rapid bacterial clearance and markedly reduced the S. aureus infection-related mortality. IL-33-treated mice displayed increased neutrophil influx into the focus of infection and higher concentrations of chemokine CXCL2 in the peritoneum than untreated mice. The beneficial effect of IL-33 priming was related to reversal of the S. aureus-induced reduction of CXCR2 expression on the surface of neutrophils. Furthermore, conditioning of neutrophils by IL-33 led to the enhancement of complement receptor 3 expression induced by S. aureus, which in turn facilitates the phagocytosis of opsonized S. aureus. Finally, neutrophils primed by IL-33 upregulated the production of reactive oxygen species and the subsequent killing activity for S. aureus. All together, these findings suggest that IL-33, through regulating multiple steps of neutrophil-mediated bactericidal function, provides a profound effect in host antimicrobial defense response.

  18. Enhanced angiogenesis, hypoxia and neutrophil recruitment during Myc-induced liver tumorigenesis in zebrafish

    PubMed Central

    Zhao, Ye; Huang, Xiaoqian; Ding, Tony Weixi; Gong, Zhiyuan

    2016-01-01

    Angiogenesis, hypoxia and immune cells are important components in tumor microenvironment affecting tumor growth. Here we employed a zebrafish liver tumor model to investigate the effect of Myc expression on angiogenesis, hypoxia and tumor-infiltrated neutrophils during the tumor initiation stage. We found that induced Myc expression in the liver caused a dramatic increase of liver size with neoplastic features. The tumorigenic liver was accompanied by enhanced angiogenesis and inhibition of angiogenesis by an inhibitor (SU5416 or sunitinib) hindered the tumorigenic growth, suggesting an essential role of angiogenesis in tumorigenic growth of liver tumor in this zebrafish model. Myc induction also caused hypoxia, which could be further enhanced by hypoxia activator, ML228, to lead to a further enlargement of tumorigenic liver. Furthermore, Myc overexpression incurred an increase of liver-infiltrated neutrophils and the increase could be suppressed by angiogenesis inhibitors or by morpholino knockdown inhibition of neutrophil differentiation, leading to a suppression of growth of tumorigenic livers. Finally, the enhanced angiogenesis, hypoxia and tumor-infiltrated neutrophils by Myc overexpression were validated by RT-qPCR examination of expression of relevant biomarker genes. In sum, the current study demonstrated that the Myc-induced liver tumor model in zebrafish provides an excellent platform for study of tumor microenvironment. PMID:27549025

  19. NKT cells mediate the recruitment of neutrophils by stimulating epithelial chemokine secretion during colitis.

    PubMed

    Huang, Enyu; Liu, Ronghua; Lu, Zhou; Liu, Jiajing; Liu, Xiaoming; Zhang, Dan; Chu, Yiwei

    2016-05-27

    Ulcerative colitis (UC) is a kind of inflammatory bowel diseases characterized by chronic inflammation and ulcer in colon, and UC patients have increased risk of getting colorectal cancer. NKT cells are cells that express both NK cell markers and semi-invariant CD1d-restricted TCRs, can regulate immune responses via secreting a variety of cytokines upon activation. In our research, we found that the NKT cell-deficient CD1d(-/-) mice had relieved colitis in the DSS-induced colitis model. Further investigations revealed that the colon of CD1d(-/-) mice expressed less neutrophil-attracting chemokine CXCL 1, 2 and 3, and had decreased neutrophil infiltration. Infiltrated neutrophils also produced less reactive oxygen species (ROS) and TNF-α, indicating they may cause less epithelial damage. In addition, colitis-associated colorectal cancer was also relieved in CD1d(-/-) mice. During colitis, NKT cells strongly expressed TNF-α, which could stimulate CXCL 1, 2, 3 expressions by the epithelium. In conclusion, NKT cells can regulate colitis via the NKT cell-epithelium-neutrophil axis. Targeting this mechanism may help to improve the therapy of UC and prevent colitis-associated colorectal cancer. PMID:27063801

  20. Vaccine adjuvants alum and MF59 induce rapid recruitment of neutrophils and monocytes that participate in antigen transport to draining lymph nodes.

    PubMed

    Calabro, Samuele; Tortoli, Marco; Baudner, Barbara C; Pacitto, Alessandra; Cortese, Mario; O'Hagan, Derek T; De Gregorio, Ennio; Seubert, Anja; Wack, Andreas

    2011-02-17

    Vaccine adjuvants such as alum and the oil-in-water emulsion MF59 are used to enhance immune responses towards pure soluble antigens, but their mechanism of action is still largely unclear. Since most adjuvanted vaccines are administered intramuscularly, we studied immune responses in the mouse muscle and found that both adjuvants were potent inducers of chemokine production and promoted rapid recruitment of CD11b(+) cells. The earliest and most abundantly recruited cell type are neutrophils, followed by monocytes, eosinophils and later dendritic cells (DCs) and macrophages. Using fluorescent forms of MF59 and ovalbumin (OVA) antigen, we show that all recruited cell types take up both adjuvant and antigen to transport them to the draining lymph nodes (LNs). There, we found antigen-positive neutrophils and monocytes within hours of injection, later followed by B cells and DCs. Compared to alum, MF59-injection lead to a more prominent neutrophil recruitment and a more efficient antigen re-localization from the injection site to the LN. As antigen-transporting neutrophils were observed in draining LNs, we asked whether these cells play an essential role in MF59-mediated adjuvanticity. However, antibody-mediated neutrophil ablation left MF59-adjuvanticity unaltered. Further studies will reveal whether other single cell types are crucial or whether the different recruited cell populations are redundant with overlapping functions.

  1. Reversible Demyelination, Blood-Brain Barrier Breakdown, and Pronounced Neutrophil Recruitment Induced by Chronic IL-1 Expression in the Brain

    PubMed Central

    Ferrari, Carina C.; Depino, Amaicha M.; Prada, Federico; Muraro, Nara; Campbell, Sandra; Podhajcer, Osvaldo; Perry, V. Hugh; Anthony, Daniel C.; Pitossi, Fernando J.

    2004-01-01

    Interleukin-1β (IL-1) expression is associated with a spectrum of neuroinflammatory processes related to chronic neurodegenerative diseases. The single-bolus microinjection of IL-1 into the central nervous system (CNS) parenchyma gives rise to delayed and localized neutrophil recruitment, transient blood-brain barrier (BBB) breakdown, but no overt damage to CNS integrity. However, acute microinjections of IL-1 do not mimic the chronic IL-1 expression, which is a feature of many CNS diseases. To investigate the response of the CNS to chronic IL-1 expression, we injected a recombinant adenovirus expressing IL-1 into the striatum. At the peak of IL-1 expression (days 8 and 14 post-injection), there was a marked recruitment of neutrophils, vasodilatation, and breakdown of the BBB. Microglia and astrocyte activation was evident during the first 14 days post-injection. At days 8 and 14, extensive demyelination was observed but the number of neurons was not affected by any treatment. Finally, at 30 days, signs of inflammation were no longer present, there was evidence of tissue reorganization, the BBB was intact, and the process of remyelination was noticeable. In summary, our data show that chronic expression of IL-1, in contrast to its acute delivery, can reversibly damage CNS integrity and implicates this cytokine or downstream components as major mediators of demyelination in chronic inflammatory and demyelinating diseases. PMID:15509551

  2. Activated prostaglandin D2 receptors on macrophages enhance neutrophil recruitment into the lung

    PubMed Central

    Jandl, Katharina; Stacher, Elvira; Bálint, Zoltán; Sturm, Eva Maria; Maric, Jovana; Peinhaupt, Miriam; Luschnig, Petra; Aringer, Ida; Fauland, Alexander; Konya, Viktoria; Dahlen, Sven-Erik; Wheelock, Craig E.; Kratky, Dagmar; Olschewski, Andrea; Marsche, Gunther; Schuligoi, Rufina; Heinemann, Akos

    2016-01-01

    Background Prostaglandin (PG) D2 is an early-phase mediator in inflammation, but its action and the roles of the 2 D-type prostanoid receptors (DPs) DP1 and DP2 (also called chemoattractant receptor–homologous molecule expressed on TH2 cells) in regulating macrophages have not been elucidated to date. Objective We investigated the role of PGD2 receptors on primary human macrophages, as well as primary murine lung macrophages, and their ability to influence neutrophil action in vitro and in vivo. Methods In vitro studies, including migration, Ca2+ flux, and cytokine secretion, were conducted with primary human monocyte-derived macrophages and neutrophils and freshly isolated murine alveolar and pulmonary interstitial macrophages. In vivo pulmonary inflammation was assessed in male BALB/c mice. Results Activation of DP1, DP2, or both receptors on human macrophages induced strong intracellular Ca2+ flux, cytokine release, and migration of macrophages. In a murine model of LPS-induced pulmonary inflammation, activation of each PGD2 receptor resulted in aggravated airway neutrophilia, tissue myeloperoxidase activity, cytokine contents, and decreased lung compliance. Selective depletion of alveolar macrophages abolished the PGD2-enhanced inflammatory response. Activation of PGD2 receptors on human macrophages enhanced the migratory capacity and prolonged the survival of neutrophils in vitro. In human lung tissue specimens both DP1 and DP2 receptors were located on alveolar macrophages along with hematopoietic PGD synthase, the rate-limiting enzyme of PGD2 synthesis. Conclusion For the first time, our results show that PGD2 markedly augments disease activity through its ability to enhance the proinflammatory actions of macrophages and subsequent neutrophil activation. PMID:26792210

  3. CEACAM1 negatively regulates IL-1β production in LPS activated neutrophils by recruiting SHP-1 to a SYK-TLR4-CEACAM1 complex.

    PubMed

    Lu, Rongze; Pan, Hao; Shively, John E

    2012-01-01

    LPS-activated neutrophils secrete IL-1β by activation of TLR-4. Based on studies in macrophages, it is likely that ROS and lysosomal destabilization regulated by Syk activation may also be involved. Since neutrophils have abundant expression of the ITIM-containing co-receptor CEACAM1 and Gram-negative bacteria such as Neisseria utilize CEACAM1 as a receptor that inhibits inflammation, we hypothesized that the overall production of IL-1β in LPS treated neutrophils may be negatively regulated by CEACAM1. We found that LPS treated neutrophils induced phosphorylation of Syk resulting in the formation of a complex including TLR4, p-Syk, and p-CEACAM1, which in turn, recruited the inhibitory phosphatase SHP-1. LPS treatment leads to ROS production, lysosomal damage, caspase-1 activation and IL-1β secretion in neutrophils. The absence of this regulation in Ceacam1⁻/⁻ neutrophils led to hyper production of IL-1β in response to LPS. The hyper production of IL-1β was abrogated by in vivo reconstitution of wild type but not ITIM-mutated CEACAM1 bone marrow stem cells. Blocking Syk activation by kinase inhibitors or RNAi reduced Syk phosphorylation, lysosomal destabilization, ROS production, and caspase-1 activation in Ceacam1⁻/⁻ neutrophils. We conclude that LPS treatment of neutrophils triggers formation of a complex of TLR4 with pSyk and pCEACAM1, which upon recruitment of SHP-1 to the ITIMs of pCEACAM1, inhibits IL-1β production by the inflammasome. Thus, CEACAM1 fine-tunes IL-1β production in LPS treated neutrophils, explaining why the additional utilization of CEACAM1 as a pathogen receptor would further inhibit inflammation.

  4. Cardiosphere-Derived Cells Facilitate Heart Repair by Modulating M1/M2 Macrophage Polarization and Neutrophil Recruitment

    PubMed Central

    Hasan, Al Shaimaa; Luo, Lan; Yan, Chen; Zhang, Tian-Xia; Urata, Yoshishige; Goto, Shinji; Mangoura, Safwat A.; Abdel-Raheem, Mahmoud H.; Zhang, Shouhua; Li, Tao-Sheng

    2016-01-01

    Cardiosphere-derived cells (CDCs), one of the promising stem cell sources for myocardial repair, have been tested in clinical trials and resulted in beneficial effects; however, the relevant mechanisms are not fully understood. In this study, we examined the hypothesis that CDCs favor heart repair by switching the macrophages from a pro-inflammatory phenotype (M1) into a regulatory anti-inflammatory phenotype (M2). Macrophages from mice were cultured with CDCs-conditioned medium or with fibroblasts-conditioned medium as a control. Immunostaining showed that CDCs-conditioned medium significantly enhanced the expression of CD206 (a marker for M2 macrophages), but decreased the expression of CD86 (a marker for M1 macrophages) 3 days after culture. For animal studies, we used an acute myocardial infarction model of mice. We injected CDCs, fibroblasts, or saline only into the border zone of infarction. Then we collected the heart tissues for histological analysis 5 and 14 days after treatment. Compared with control animals, CDCs treatment significantly decreased M1 macrophages and neutrophils but increased M2 macrophages in the infarcted heart. Furthermore, CDCs-treated mice had reduced infarct size and fewer apoptotic cells compared to the controls. Our data suggest that CDCs facilitate heart repair by modulating M1/M2 macrophage polarization and neutrophil recruitment, which may provide a new insight into the mechanisms of stem cell-based myocardial repair. PMID:27764217

  5. Interleukin-10 plays a key role in the modulation of neutrophils recruitment and lung inflammation during infection by Streptococcus pneumoniae.

    PubMed

    Peñaloza, Hernán F; Nieto, Pamela A; Muñoz-Durango, Natalia; Salazar-Echegarai, Francisco J; Torres, Javiera; Parga, María J; Alvarez-Lobos, Manuel; Riedel, Claudia A; Kalergis, Alexis M; Bueno, Susan M

    2015-09-01

    Streptococcus pneumoniae is a major aetiological agent of pneumonia worldwide, as well as otitis media, sinusitis, meningitis and sepsis. Recent reports have suggested that inflammation of lungs due to S. pneumoniae infection promotes bacterial dissemination and severe disease. However, the contribution of anti-inflammatory molecules to the pathogenesis of S. pneumoniae remains unknown. To elucidate whether the production of the anti-inflammatory cytokine interleukin-10 (IL-10) is beneficial or detrimental for the host during pneumococcal pneumonia, we performed S. pneumoniae infections in mice lacking IL-10 (IL-10(-/-) mice). The IL-10(-/-) mice showed increased mortality, higher expression of pro-inflammatory cytokines, and an exacerbated recruitment of neutrophils into the lungs after S. pneumoniae infection. However, IL-10(-/-) mice showed significantly lower bacterial loads in lungs, spleen, brain and blood, when compared with mice that produced this cytokine. Our results support the notion that production of IL-10 during S. pneumoniae infection modulates the expression of pro-inflammatory cytokines and the infiltration of neutrophils into the lungs. This feature of IL-10 is important to avoid excessive inflammation of tissues and to improve host survival, even though bacterial dissemination is less efficient in the absence of this cytokine.

  6. Interleukin-10 plays a key role in the modulation of neutrophils recruitment and lung inflammation during infection by Streptococcus pneumoniae

    PubMed Central

    Peñaloza, Hernán F; Nieto, Pamela A; Muñoz-Durango, Natalia; Salazar-Echegarai, Francisco J; Torres, Javiera; Parga, María J; Alvarez-Lobos, Manuel; Riedel, Claudia A; Kalergis, Alexis M; Bueno, Susan M

    2015-01-01

    Streptococcus pneumoniae is a major aetiological agent of pneumonia worldwide, as well as otitis media, sinusitis, meningitis and sepsis. Recent reports have suggested that inflammation of lungs due to S. pneumoniae infection promotes bacterial dissemination and severe disease. However, the contribution of anti-inflammatory molecules to the pathogenesis of S. pneumoniae remains unknown. To elucidate whether the production of the anti-inflammatory cytokine interleukin-10 (IL-10) is beneficial or detrimental for the host during pneumococcal pneumonia, we performed S. pneumoniae infections in mice lacking IL-10 (IL-10−/− mice). The IL-10−/− mice showed increased mortality, higher expression of pro-inflammatory cytokines, and an exacerbated recruitment of neutrophils into the lungs after S. pneumoniae infection. However, IL-10−/− mice showed significantly lower bacterial loads in lungs, spleen, brain and blood, when compared with mice that produced this cytokine. Our results support the notion that production of IL-10 during S. pneumoniae infection modulates the expression of pro-inflammatory cytokines and the infiltration of neutrophils into the lungs. This feature of IL-10 is important to avoid excessive inflammation of tissues and to improve host survival, even though bacterial dissemination is less efficient in the absence of this cytokine. PMID:26032199

  7. Endogenous osteopontin promotes ozone-induced neutrophil recruitment to the lungs and airway hyperresponsiveness to methacholine

    PubMed Central

    Barreno, Ramon X.; Richards, Jeremy B.; Schneider, Daniel J.; Cromar, Kevin R.; Nadas, Arthur J.; Hernandez, Christopher B.; Hallberg, Lance M.; Price, Roger E.; Hashmi, Syed S.; Blackburn, Michael R.; Haque, Ikram U.

    2013-01-01

    Inhalation of ozone (O3), a common environmental pollutant, causes pulmonary injury, pulmonary inflammation, and airway hyperresponsiveness (AHR) in healthy individuals and exacerbates many of these same sequelae in individuals with preexisting lung disease. However, the mechanisms underlying these phenomena are poorly understood. Consequently, we sought to determine the contribution of osteopontin (OPN), a hormone and a pleiotropic cytokine, to the development of O3-induced pulmonary injury, pulmonary inflammation, and AHR. To that end, we examined indices of these aforementioned sequelae in mice genetically deficient in OPN and in wild-type, C57BL/6 mice 24 h following the cessation of an acute (3 h) exposure to filtered room air (air) or O3 (2 parts/million). In wild-type mice, O3 exposure increased bronchoalveolar lavage fluid (BALF) OPN, whereas immunohistochemical analysis demonstrated that there were no differences in the number of OPN-positive alveolar macrophages between air- and O3-exposed wild-type mice. O3 exposure also increased BALF epithelial cells, protein, and neutrophils in wild-type and OPN-deficient mice compared with genotype-matched, air-exposed controls. However, following O3 exposure, BALF neutrophils were significantly reduced in OPN-deficient compared with wild-type mice. When airway responsiveness to inhaled acetyl-β-methylcholine chloride (methacholine) was assessed using the forced oscillation technique, O3 exposure caused hyperresponsiveness to methacholine in the airways and lung parenchyma of wild-type mice, but not OPN-deficient mice. These results demonstrate that OPN is increased in the air spaces following acute exposure to O3 and functionally contributes to the development of O3-induced pulmonary inflammation and airway and lung parenchymal hyperresponsiveness to methacholine. PMID:23666750

  8. FGF23 signaling impairs neutrophil recruitment and host defense during CKD

    PubMed Central

    Rossaint, Jan; Oehmichen, Jessica; Van Aken, Hugo; Reuter, Stefan; Pavenstädt, Hermann J.; Meersch, Melanie; Unruh, Mark

    2016-01-01

    Chronic kidney disease (CKD) has been associated with impaired host response and increased susceptibility to infections. Leukocyte recruitment during inflammation must be tightly regulated to protect the host against pathogens. FGF23 levels are increased in blood during CKD, and levels of this hormone have been associated with a variety of adverse effects in CKD patients. Here, we have shown that CKD impairs leukocyte recruitment into inflamed tissue and host defense in mice and humans. FGF23 neutralization during CKD in murine models restored leukocyte recruitment and host defense. Intravital microscopy of animals with chronic kidney failure showed that FGF23 inhibits chemokine-activated leukocyte arrest on the endothelium, and downregulation of FGF receptor 2 (FGFR2) on PMNs rescued host defense in these mice. In vitro, FGF23 inhibited PMN adhesion, arrest under flow, and transendothelial migration. Mechanistically, FGF23 binding to FGFR2 counteracted selectin- and chemokine-triggered β2 integrin activation on PMNs by activating protein kinase A (PKA) and inhibiting activation of the small GTPase Rap1. Moreover, knockdown of PKA abolished the inhibitory effect of FGF23 on integrin activation. Together, our data reveal that FGF23 acts directly on PMNs and dampens host defense by direct interference with chemokine signaling and integrin activation. PMID:26878171

  9. Neutrophil Recruitment to the Lung in both C5a and CXCL1-Induced Alveolitis is Impaired in Vitamin D Binding Protein Deficient Mice

    PubMed Central

    Trujillo, Glenda; Habiel, David M.; Ge, Lingyin; Ramadass, Mahalakshmi; Cooke, Nancy E.; Kew, Richard R.

    2013-01-01

    Knowledge of how neutrophils respond to chemotactic signals in a complex inflammatory environment is not completely understood. Moreover, even less is known about factors in physiological fluids that regulate the activity of chemoattractants. The vitamin D binding protein (DBP) has been shown to significantly enhance chemotaxis to complement activation peptide C5a using purified proteins in vitro, and by ex vivo depletion of DBP in physiological fluids, but this function has not been determined in vivo. DBP null (-/-) mice were used to investigate how a systemic absence of this plasma protein affects leukocyte recruitment in alveolitis models of lung inflammation. DBP-/- mice had significantly reduced (~50%) neutrophil recruitment to the lungs compared to their wild-type DBP+/+ counterparts in three different alveolitis models, two acute and one chronic. The histology of DBP-/- mouse lungs also showed significantly less injury than wild-type animals. The chemotactic cofactor function of DBP appears to be selective for neutrophil recruitment, but in contrast to previous in vitro results, in vivo DBP can enhance the activity of other chemoattractants including CXCL1. The reduced neutrophil response in DBP-/- mice could be rescued to wild-type levels by administering exogenous DBP. Finally, in inflammatory fluids DBP binds to G-actin released from damaged cells and this complex may be the active chemotactic cofactor. Results show for the first time that DBP is a significant chemotactic cofactor in vivo and not specific for C5a, suggesting that this ubiquitous plasma protein may have a more significant role in neutrophil recruitment than previously recognized. PMID:23752613

  10. IL-17RA in Non-Hematopoietic Cells Controls CXCL-1 and 5 Critical to Recruit Neutrophils to the Lung of Mycobacteria-Infected Mice during the Adaptive Immune Response.

    PubMed

    Lombard, Robin; Doz, Emilie; Carreras, Florence; Epardaud, Mathieu; Le Vern, Yves; Buzoni-Gatel, Dominique; Winter, Nathalie

    2016-01-01

    During chronic infection with Mycobacterium tuberculosis (Mtb), bacilli multiplication is constrained within lung granulomas until excessive inflammation destroys the lung. Neutrophils are recruited early and participate in granuloma formation, but excessive neutrophilia exacerbates the tuberculosis disease. Neutrophils thus appear as potential targets for therapeutic interventions, especially in patients for whom no antibiotic treatment is possible. Signals that regulate neutrophil recruitment to the lung during mycobacterial infection need to be better understood. We demonstrated here, in the mouse model, that neutrophils were recruited to the lung in two waves after intranasal infection with virulent Mtb or the live attenuated vaccine strain Bacillus Calmette Guérin (BCG). A first wave of neutrophils was swiftly recruited, followed by a subsequent adaptive wave that reached the lung together with IFN-γ- and IL-17A-producing T cells. Interestingly, the second neutrophil wave did not participate to mycobacteria control in the lung and established contacts with T cells. The adaptive wave was critically dependent on the expression of IL-17RA, the receptor for IL-17A, expressed in non-hematopoietic cells. In absence of this receptor, curtailed CXCL-1 and 5 production in the lung restrained neutrophil recruitment. CXCL-1 and 5 instillation reconstituted lung neutrophil recruitment in BCG-infected IL17RA-/- mice. PMID:26871571

  11. The Nlrp3 inflammasome, IL-1β, and neutrophil recruitment are required for susceptibility to a nonhealing strain of Leishmania major in C57BL/6 mice.

    PubMed

    Charmoy, Melanie; Hurrell, Benjamin P; Romano, Audrey; Lee, Sang Hun; Ribeiro-Gomes, Flavia; Riteau, Nicolas; Mayer-Barber, Katrin; Tacchini-Cottier, Fabienne; Sacks, David L

    2016-04-01

    Infection of C57BL/6 mice with most Leishmania major strains results in a healing lesion and clearance of parasites from the skin. Infection of C57BL/6 mice with the L. major Seidman strain (LmSd), isolated from a patient with chronic lesions, despite eliciting a strong Th1 response, results in a nonhealing lesion, poor parasite clearance, and complete destruction of the ear dermis. We show here that in comparison to a healing strain, LmSd elicited early upregulation of IL-1β mRNA and IL-1β-producing dermal cells and prominent neutrophil recruitment to the infected skin. Mice deficient in Nlrp3, apoptosis-associated speck-like protein containing a caspase recruitment domain, or caspase-1/11, or lacking IL-1β or IL-1 receptor signaling, developed healing lesions and cleared LmSd from the infection site. Mice resistant to LmSd had a stronger antigen-specific Th1 response. The possibility that IL-1β might act through neutrophil recruitment to locally suppress immunity was supported by the healing observed in neutropenic Genista mice. Secretion of mature IL-1β by LmSd-infected macrophages in vitro was dependent on activation of the Nlrp3 inflammasome and caspase-1. These data reveal that Nlrp3 inflammasome-dependent IL-1β, associated with localized neutrophil recruitment, plays a crucial role in the development of a nonhealing form of cutaneous leishmaniasis in conventionally resistant mice.

  12. Interferon Regulatory Factor 8-Deficiency Determines Massive Neutrophil Recruitment but T Cell Defect in Fast Growing Granulomas during Tuberculosis

    PubMed Central

    Sali, Michela; Anfossi, Antonio Giovanni; Abalsamo, Laura; Palucci, Ivana; Mattei, Fabrizio; Sanchez, Massimo; Giagu, Anna; Antuofermo, Elisabetta; Fadda, Giovanni; Belardelli, Filippo; Delogu, Giovanni; Gabriele, Lucia

    2013-01-01

    Following Mycobacterium tuberculosis (Mtb) infection, immune cell recruitment in lungs is pivotal in establishing protective immunity through granuloma formation and neogenesis of lymphoid structures (LS). Interferon regulatory factor-8 (IRF-8) plays an important role in host defense against Mtb, although the mechanisms driving anti-mycobacterial immunity remain unclear. In this study, IRF-8 deficient mice (IRF-8−/−) were aerogenously infected with a low-dose Mtb Erdman virulent strain and the course of infection was compared with that induced in wild-type (WT-B6) counterparts. Tuberculosis (TB) progression was examined in both groups using pathological, microbiological and immunological parameters. Following Mtb exposure, the bacterial load in lungs and spleens progressed comparably in the two groups for two weeks, after which IRF-8−/− mice developed a fatal acute TB whereas in WT-B6 the disease reached a chronic stage. In lungs of IRF-8−/−, uncontrolled growth of pulmonary granulomas and impaired development of LS were observed, associated with unbalanced homeostatic chemokines, progressive loss of infiltrating T lymphocytes and massive prevalence of neutrophils at late infection stages. Our data define IRF-8 as an essential factor for the maintenance of proper immune cell recruitment in granulomas and LS required to restrain Mtb infection. Moreover, IRF-8−/− mice, relying on a common human and mouse genetic mutation linked to susceptibility/severity of mycobacterial diseases, represent a valuable model of acute TB for comparative studies with chronically-infected congenic WT-B6 for dissecting protective and pathological immune reactions. PMID:23717393

  13. Interferon regulatory factor 8-deficiency determines massive neutrophil recruitment but T cell defect in fast growing granulomas during tuberculosis.

    PubMed

    Rocca, Stefano; Schiavoni, Giovanna; Sali, Michela; Anfossi, Antonio Giovanni; Abalsamo, Laura; Palucci, Ivana; Mattei, Fabrizio; Sanchez, Massimo; Giagu, Anna; Antuofermo, Elisabetta; Fadda, Giovanni; Belardelli, Filippo; Delogu, Giovanni; Gabriele, Lucia

    2013-01-01

    Following Mycobacterium tuberculosis (Mtb) infection, immune cell recruitment in lungs is pivotal in establishing protective immunity through granuloma formation and neogenesis of lymphoid structures (LS). Interferon regulatory factor-8 (IRF-8) plays an important role in host defense against Mtb, although the mechanisms driving anti-mycobacterial immunity remain unclear. In this study, IRF-8 deficient mice (IRF-8⁻/⁻) were aerogenously infected with a low-dose Mtb Erdman virulent strain and the course of infection was compared with that induced in wild-type (WT-B6) counterparts. Tuberculosis (TB) progression was examined in both groups using pathological, microbiological and immunological parameters. Following Mtb exposure, the bacterial load in lungs and spleens progressed comparably in the two groups for two weeks, after which IRF-8⁻/⁻ mice developed a fatal acute TB whereas in WT-B6 the disease reached a chronic stage. In lungs of IRF-8⁻/⁻, uncontrolled growth of pulmonary granulomas and impaired development of LS were observed, associated with unbalanced homeostatic chemokines, progressive loss of infiltrating T lymphocytes and massive prevalence of neutrophils at late infection stages. Our data define IRF-8 as an essential factor for the maintenance of proper immune cell recruitment in granulomas and LS required to restrain Mtb infection. Moreover, IRF-8⁻/⁻ mice, relying on a common human and mouse genetic mutation linked to susceptibility/severity of mycobacterial diseases, represent a valuable model of acute TB for comparative studies with chronically-infected congenic WT-B6 for dissecting protective and pathological immune reactions. PMID:23717393

  14. Interleukin-23 (IL-23), independent of IL-17 and IL-22, drives neutrophil recruitment and innate inflammation during Clostridium difficile colitis in mice.

    PubMed

    McDermott, Andrew J; Falkowski, Nicole R; McDonald, Roderick A; Pandit, Chinmay R; Young, Vincent B; Huffnagle, Gary B

    2016-01-01

    Our objective was to determine the role of the inflammatory cytokine interleukin-23 (IL-23) in promoting neutrophil recruitment, inflammatory cytokine expression and intestinal histopathology in response to Clostridium difficile infection. Wild-type (WT) and p19(-/-) (IL-23KO) mice were pre-treated with cefoperazone in their drinking water for 5 days, and after a 2-day recovery period were challenged with spores from C. difficile strain VPI 10463. Interleukin-23 deficiency was associated with significant defects in both the recruitment of CD11b(High) Ly6G(H) (igh) neutrophils to the colon and the expression of neutrophil chemoattractants and stabilization factors including Cxcl1, Cxcl2, Ccl3 and Csf3 within the colonic mucosa as compared with WT animals. Furthermore, the expression of inflammatory cytokines including Il33, Tnf and Il6 was significantly reduced in IL-23-deficient animals. There was also a trend towards less severe colonic histopathology in the absence of IL-23. The induction of Il17a and Il22 was also significantly abrogated in IL-23KO mice. Inflammatory cytokine expression and neutrophilic inflammation were not reduced in IL-17a-deficient mice or in mice treated with anti-IL-22 depleting monoclonal antibody. However, induction of RegIIIg was significantly reduced in animals treated with anti-IL-22 antibody. Taken together, these data indicate that IL-23, but not IL-17a or IL-22, promotes neutrophil recruitment and inflammatory cytokine and chemokine expression in the colon in response to C. difficile infection.

  15. The α-Tocopherol Form of Vitamin E Reverses Age-Associated Susceptibility to Streptococcus pneumoniae Lung Infection by Modulating Pulmonary Neutrophil Recruitment

    PubMed Central

    Ghanem, Elsa N. Bou; Clark, Stacie; Du, Xiaogang; Wu, Dayong; Camilli, Andrew; Leong, John M.; Meydani, Simin N.

    2016-01-01

    Streptococcus pneumoniae infections are an important cause of morbidity and mortality in older patients. Uncontrolled neutrophil-driven pulmonary inflammation exacerbates this disease. To test whether the α-tocopherol (α-Toc) form of vitamin E, a regulator of immunity, can modulate neutrophil responses as a preventive strategy to mitigate the age-associated decline in resistance to S. pneumoniae, young (4 mo) and old (22–24 mo) C57BL/6 mice were fed a diet containing 30-PPM (control) or 500-PPM (supplemented) α-Toc for 4 wk and intratracheally infected with S. pneumoniae. Aged mice fed a control diet were exquisitely more susceptible to S. pneumoniae than young mice. At 2 d postinfection, aged mice suffered 1000-fold higher pulmonary bacterial burden, 2.2-fold higher levels of neutrophil recruitment to the lung, and a 2.25-fold higher rate of lethal septicemia. Strikingly, α-Toc supplementation of aged mice resulted in a 1000-fold lower bacterial lung burden and full control of infection. This α-Toc–induced resistance to pneumococcal challenge was associated with a 2-fold fewer pulmonary neutrophils, a level comparable to S. pneumoniae–challenged, conventionally fed young mice. α-Toc directly inhibited neutrophil egress across epithelial cell monolayers in vitro in response to pneumococci or hepoxilin-A3, an eicosanoid required for pneumococcus-elicited neutrophil trans-epithelial migration. α-Toc altered expression of multiple epithelial and neutrophil adhesion molecules involved in migration, including CD55, CD47, CD18/CD11b, and ICAM-1. These findings suggest that α-Toc enhances resistance of aged mice to bacterial pneumonia by modulating the innate immune response, a finding that has potential clinical significance in combating infection in aged individuals through nutritional intervention. PMID:25512603

  16. Selectin blocking activity of a fucosylated chondroitin sulfate glycosaminoglycan from sea cucumber. Effect on tumor metastasis and neutrophil recruitment.

    PubMed

    Borsig, Lubor; Wang, Lianchun; Cavalcante, Moises C M; Cardilo-Reis, Larissa; Ferreira, Paola L; Mourão, Paulo A S; Esko, Jeffrey D; Pavão, Mauro S G

    2007-05-18

    Heparin is an excellent inhibitor of P- and L-selectin binding to the carbohydrate determinant, sialyl Lewis(x). As a consequence of its anti-selectin activity, heparin attenuates metastasis and inflammation. Here we show that fucosylated chondroitin sulfate (FucCS), a polysaccharide isolated from sea cucumber composed of a chondroitin sulfate backbone substituted at the 3-position of the beta-D-glucuronic acid residues with 2,4-disulfated alpha-L-fucopyranosyl branches, is a potent inhibitor of P- and L-selectin binding to immobilized sialyl Lewis(x) and LS180 carcinoma cell attachment to immobilized P- and L-selectins. Inhibition occurs in a concentration-dependent manner. Furthermore, FucCS was 4-8-fold more potent than heparin in the inhibition of the P- and L-selectin-sialyl Lewis(x) interactions. No inhibition of E-selectin was observed. FucCS also inhibited lung colonization by adenocarcinoma MC-38 cells in an experimental metastasis model in mice, as well as neutrophil recruitment in two models of inflammation (thioglycollate-induced peritonitis and lipopolysaccharide-induced lung inflammation). Inhibition occurred at a dose that produces no significant change in plasma activated partial thromboplastin time. Removal of the sulfated fucose branches on the FucCS abolished the inhibitory effect in vitro and in vivo. Overall, the results suggest that invertebrate FucCS may be a potential alternative to heparin for blocking metastasis and inflammatory reactions without the undesirable side effects of anticoagulant heparin.

  17. IL-17 contributes to neutrophil recruitment but not to control of viral replication during acute mouse adenovirus type 1 respiratory infection.

    PubMed

    McCarthy, Mary K; Zhu, Lingqiao; Procario, Megan C; Weinberg, Jason B

    2014-05-01

    IL-17-producing CD4(+) helper T cells (Th17 cells) promote inflammatory responses to many pathogens. We used mouse adenovirus type 1 (MAV-1) to determine contributions of IL-17 to adenovirus pathogenesis. MAV-1 infection of C57BL/6 mice upregulated lung expression of IL-17 and the Th17-associated factors IL-23 and RORγt. Only CD4(+)T cells were associated with virus-specific IL-17 production. Fewer neutrophils were recruited to airways of IL-17(-/-) mice following MAV-1 infection, but there were no other differences in pulmonary inflammation between IL-17(+/+) and IL-17(-/-) mice. Mice depleted of neutrophils using anti-Gr-1 antibody had greater lung viral loads than controls. Despite impaired neutrophil recruitment, there were no differences between IL-17(+/+) and IL-17(-/-) mice in peak lung viral loads, clearance of virus from the lungs, or establishment of protective immunity. We demonstrate robust Th17 responses during MAV-1 respiratory infection, but these responses are not essential for control of virus infection or for virus-induced pulmonary inflammation. PMID:24889245

  18. Atrial natriuretic peptide down-regulates neutrophil recruitment on inflamed endothelium by reducing cell deformability and resistance to detachment force

    PubMed Central

    Morikis, Vasilios A.; Radecke, Chris; Jiang, Yanyan; Heinrich, Volkmar; Curry, Fitz-Roy; Simon, Scott I.

    2016-01-01

    BACKGROUND Recombinant atrial natriuretic peptide (ANP) is administered in patients with acute heart failure in Japan to improve renal function and hemodynamics, but its anti-inflammatory effect on activated leukocytes may also contribute to its therapeutic efficacy. OBJECTIVE Examine unconventional role of ANP in neutrophil adhesion to inflamed endothelium. METHODS Human neutrophils were perfused over endothelial monolayers in a microfluidic lab-chip assay. Cell rheology was assessed by micropipette aspiration to assess changes in cortical tension and viscosity. Fluorescence microscopy was applied to measure adhesive contact area and β2-integrin focal bond formation. RESULTS ANP inhibited neutrophil rolling and firm adhesion without influencing the upregulation of cellular adhesion molecules on endothelium or the regulation of high affinity CD18 and shedding of L-selectin during neutrophil activation. Exposed to fluid shear, integrin mediated arrest was disrupted with ANP treatment, which elicited formation of long tethers and diminished cell spreading and contact. This correlated with a ~40% increase in neutrophil viscosity and a reduction in the adhesive footprint. CONCLUSIONS A decrease in cell deformation and neutrophil flattening with ANP results in fewer integrin bond clusters, which translates to higher tensile forces and impaired adhesion strengthening and cell detachment. PMID:26639357

  19. Kaposi's sarcoma-associated herpesvirus infection of endothelial cells inhibits neutrophil recruitment through an interleukin-6-dependent mechanism: a new paradigm for viral immune evasion.

    PubMed

    Butler, L M; Jeffery, H C; Wheat, R L; Rae, P C; Townsend, K; Alkharsah, K R; Schulz, T F; Nash, G B; Blackbourn, D J

    2011-07-01

    Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma (KS), an endothelial cell (EC) neoplasm characterized by dysregulated angiogenesis and inflammation. KSHV infection of EC causes production of proinflammatory mediators, regarded as possible initiators of the substantial mononuclear leukocyte recruitment seen in KS. Conversely, KSHV immune evasion strategies exist, such as degradation of EC leukocyte adhesion receptors by viral proteins. Here, we report the effects of KSHV infection of primary EC on recruitment of flowing leukocytes. Infection did not initiate adhesion of any leukocyte subset per se. However, on cytokine-stimulated EC, KSHV specifically inhibited neutrophil, but not PBL or monocyte, transmigration, an observation consistent with the inflammatory cell profile found in KS lesions in vivo. This inhibition could be recapitulated on uninfected EC using supernatant from infected cultures. These supernatants contained elevated levels of human interleukin 6 (hIL-6), and both the KSHV- and the supernatant-induced inhibitions of neutrophil transmigration were abrogated in the presence of a hIL-6 neutralizing antibody. Furthermore, preconditioning of EC with hIL-6 mimicked the effect of KSHV. Using RNA interference (RNAi), we show that upregulation of suppressor of cytokine signaling 3 (SOCS3) was necessary for this effect of hIL-6. These studies reveal a novel paracrine mode of KSHV immune evasion, resulting in reduced recruitment of neutrophils, a cell type whose antiviral and antitumor roles are becoming increasingly appreciated. Moreover, the findings have implications for our understanding of the contribution of hIL-6 to the pathogenesis of other inflammatory disorders and tumors in which this cytokine is abundant.

  20. Vinpocetine reduces lipopolysaccharide-induced inflammatory pain and neutrophil recruitment in mice by targeting oxidative stress, cytokines and NF-κB.

    PubMed

    Ruiz-Miyazawa, Kenji W; Pinho-Ribeiro, Felipe A; Zarpelon, Ana C; Staurengo-Ferrari, Larissa; Silva, Rangel L; Alves-Filho, Jose C; Cunha, Thiago M; Cunha, Fernando Q; Casagrande, Rubia; Verri, Waldiceu A

    2015-07-25

    In response to lipopolysaccharide (LPS), tissue resident macrophages and recruited neutrophils produce inflammatory mediators through activation of Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) signaling pathway. These mediators include inflammatory cytokines and reactive oxygen species that, in turn, sensitize nociceptors and lead to inflammatory pain. Vinpocetine is a nootropic drug widely used to treat cognitive and neurovascular disorders, and more recently its anti-inflammatory properties through inhibition of NF-κB activation have been described. In the present study, we used the intraplantar and intraperitoneal LPS stimulus in mice to investigate the effects of vinpocetine pre-treatment (3, 10, or 30mg/kg by gavage) in hyperalgesia, leukocyte recruitment, oxidative stress, and pro-inflammatory cytokine production (TNF-α, IL-1β, and IL-33). LPS-induced NF-κB activation and cytokine production were investigated using RAW 264.7 macrophage cell in vitro. Vinpocetine (30mg/kg) significantly reduces hyperalgesia to mechanical and thermal stimuli, and myeloperoxidase (MPO) activity (a neutrophil marker) in the plantar paw skin, and also inhibits neutrophil and mononuclear cell recruitment, superoxide anion and nitric oxide production, oxidative stress, and cytokine production (TNF-α, IL-1β and IL-33) in the peritoneal cavity. At least in part, these effects seem to be mediated by direct effects of vinpocetine on macrophages, since it inhibited the production of the same cytokines (TNF-α, IL-1β and IL-33) and the NF-κB activation in LPS-stimulated RAW 264.7 macrophages. Our results suggest that vinpocetine represents an important therapeutic approach to treat inflammation and pain induced by a gram-negative bacterial component by targeting NF-κB activation and NF-κB-related cytokine production in macrophages.

  1. Effects of leukotriene B4 in the human lung. Recruitment of neutrophils into the alveolar spaces without a change in protein permeability.

    PubMed Central

    Martin, T R; Pistorese, B P; Chi, E Y; Goodman, R B; Matthay, M A

    1989-01-01

    Leukotriene B4 (LTB4) is a major product of human alveolar macrophages and has potent chemotactic activity for neutrophils (PMN) in vitro. To evaluate the effects of LTB4 in the normal human lung, we instilled LTB4 (5 X 10(-7)M, 10 ml) into a subsegment of the right middle lobe and 0.9% NaCl (10 ml) into a subsegment of the lingula using a fiberoptic bronchoscope in 12 healthy human volunteers. 4 h later, we performed bronchoalveolar lavage of the same subsegments. Compared with the NaCl instillation, LTB4 caused a large increase in lavage total cells (NaCl = 6.8 +/- 1.0 X 10(6) vs. LTB4 = 26.4 +/- 5.0 X 10(6), P less than 0.01), most of which were PMN (NaCl = 12.2 +/- 4.6% vs. LTB4 = 55.7 +/- 6.0%, P less than 0.001). In contrast, there was only a small increase in lavage total protein, and the lavage total protein correlated weakly with lavage total cells and PMN. The production of superoxide anion by the lavage PMN in response to phorbol myristate acetate was similar to that of peripheral blood PMN. The migration of lavage PMN was normal toward the chemotactic peptide FMLP, but reduced toward LTB4 and zymosan-activated human serum. Morphometric analysis using transmission electron microscopy indicated a selective loss of small granules in the lung neutrophils as compared with peripheral blood neutrophils. The data indicate that in the normal human lung, LTB4 can recruit active PMN into the airspaces without causing a significant change in the protein permeability of the epithelial barrier. Images PMID:2553777

  2. Polymorphonuclear Neutrophils Are Necessary for the Recruitment of CD8+ T Cells in the Liver in a Pregnant Mouse Model of Chlamydophila abortus (Chlamydia psittaci Serotype 1) Infection

    PubMed Central

    de Oca, Roberto Montes; Buendía, Antonio J.; Del Río, Laura; Sánchez, Joaquín; Salinas, Jesús; Navarro, Jose A.

    2000-01-01

    The role of polymorphonuclear neutrophils (PMNs) in the development of the specific immune response against Chlamydophila abortus (Chlamydia psittaci serotype 1) infection was studied in a pregnant mouse model involving treatment with RB6-8C5 monoclonal antibody. PMN depletion significantly affected the immune response in the liver, in which the T-lymphocyte and F4/80+ cell populations decreased, particularly the CD8+ T-cell population. A Th1-like response, characterized by high levels of gamma interferon without detectable levels of interleukin 4 (IL-4) in serum, was observed in both depleted and nondepleted mice, although an increased production of IL-10 was detected in the depleted group. Our results suggest that PMNs play a very important role in the recruitment of other leukocyte populations to the inflammatory foci but have little influence in the polarization of the immune specific response toward a Th1-like response. PMID:10679002

  3. Polymorphonuclear neutrophils are necessary for the recruitment of CD8(+) T cells in the liver in a pregnant mouse model of Chlamydophila abortus (Chlamydia psittaci serotype 1) infection.

    PubMed

    de Oca, R M; Buendía, A J; Del Río, L; Sánchez, J; Salinas, J; Navarro, J A

    2000-03-01

    The role of polymorphonuclear neutrophils (PMNs) in the development of the specific immune response against Chlamydophila abortus (Chlamydia psittaci serotype 1) infection was studied in a pregnant mouse model involving treatment with RB6-8C5 monoclonal antibody. PMN depletion significantly affected the immune response in the liver, in which the T-lymphocyte and F4/80(+) cell populations decreased, particularly the CD8(+) T-cell population. A Th1-like response, characterized by high levels of gamma interferon without detectable levels of interleukin 4 (IL-4) in serum, was observed in both depleted and nondepleted mice, although an increased production of IL-10 was detected in the depleted group. Our results suggest that PMNs play a very important role in the recruitment of other leukocyte populations to the inflammatory foci but have little influence in the polarization of the immune specific response toward a Th1-like response.

  4. Lipopolysaccharide modulates neutrophil recruitment and macrophage polarization on lymphatic vessels and impairs lymphatic function in rat mesentery.

    PubMed

    Chakraborty, Sanjukta; Zawieja, Scott D; Wang, Wei; Lee, Yang; Wang, Yuan J; von der Weid, Pierre-Yves; Zawieja, David C; Muthuchamy, Mariappan

    2015-12-15

    Impairment of the lymphatic system is apparent in multiple inflammatory pathologies connected to elevated endotoxins such as LPS. However, the direct mechanisms by which LPS influences the lymphatic contractility are not well understood. We hypothesized that a dynamic modulation of innate immune cell populations in mesentery under inflammatory conditions perturbs tissue cytokine/chemokine homeostasis and subsequently influences lymphatic function. We used rats that were intraperitoneally injected with LPS (10 mg/kg) to determine the changes in the profiles of innate immune cells in the mesentery and in the stretch-mediated contractile responses of isolated lymphatic preparations. Results demonstrated a reduction in the phasic contractile activity of mesenteric lymphatic vessels from LPS-injected rats and a severe impairment of lymphatic pump function and flow. There was a significant reduction in the number of neutrophils and an increase in monocytes/macrophages present on the lymphatic vessels and in the clear mesentery of the LPS group. This population of monocytes and macrophages established a robust M2 phenotype, with the majority showing high expression of CD163 and CD206. Several cytokines and chemoattractants for neutrophils and macrophages were significantly changed in the mesentery of LPS-injected rats. Treatment of lymphatic muscle cells (LMCs) with LPS showed significant changes in the expression of adhesion molecules, VCAM1, ICAM1, CXCR2, and galectin-9. LPS-TLR4-mediated regulation of pAKT, pERK pI-κB, and pMLC20 in LMCs promoted both contractile and inflammatory pathways. Thus, our data provide the first evidence connecting the dynamic changes in innate immune cells on or near the lymphatics and complex cytokine milieu during inflammation with lymphatic dysfunction.

  5. The Neutrophil Response Induced by an Agonist for Free Fatty Acid Receptor 2 (GPR43) Is Primed by Tumor Necrosis Factor Alpha and by Receptor Uncoupling from the Cytoskeleton but Attenuated by Tissue Recruitment.

    PubMed

    Björkman, Lena; Mårtensson, Jonas; Winther, Malene; Gabl, Michael; Holdfeldt, André; Uhrbom, Martin; Bylund, Johan; Højgaard Hansen, Anders; Pandey, Sunil K; Ulven, Trond; Forsman, Huamei; Dahlgren, Claes

    2016-10-15

    Ligands with improved potency and selectivity for free fatty acid receptor 2 (FFA2R) have become available, and we here characterize the neutrophil responses induced by one such agonist (Cmp1) and one antagonist (CATPB). Cmp1 triggered an increase in the cytosolic concentration of Ca(2+), and the neutrophils were then desensitized to Cmp1 and to acetate, a naturally occurring FFA2R agonist. The antagonist CATPB selectively inhibited responses induced by Cmp1 or acetate. The activated FFA2R induced superoxide anion secretion at a low level in naive blood neutrophils. This response was largely increased by tumor necrosis factor alpha (TNF-α) in a process associated with a recruitment of easily mobilizable granules, but neutrophils recruited to an aseptic inflammation in vivo were nonresponding. Superoxide production induced by Cmp1 was increased in latrunculin A-treated neutrophils, but no reactivation of desensitized FFA2R was induced by this drug, suggesting that the cytoskeleton is not directly involved in terminating the response. The functional and regulatory differences between the receptors that recognize short-chain fatty acids and formylated peptides, respectively, imply different roles of these receptors in the orchestration of inflammation and confirm the usefulness of a selective FFA2R agonist and antagonist as tools for the exploration of the precise role of the FFA2R. PMID:27503855

  6. The lymph node neutrophil.

    PubMed

    Hampton, Henry R; Chtanova, Tatyana

    2016-04-01

    Secondary lymphoid organs provide a specialized microenvironment tailored to foster communication between cells of the innate and adaptive immune systems. These interactions allow immune cells to coordinate multilayered defense against pathogens. Until recently dendritic cells and macrophages were thought to comprise the main innate immune cell subsets responsible for delivering signals that drive the adaptive immune response, while the function of neutrophils was largely confined to the innate immune system. However, the discovery of neutrophils in lymph nodes has raised the question of whether neutrophils might play a more extensive role not only in innate immunity per se, but also in coordinating the interactions between innate and adaptive immune responses. In this review we discuss the mechanisms and consequences of neutrophil recruitment to lymph nodes and how this recruitment influences subsequent immune responses both in situ and at distant sites. PMID:27025975

  7. Neutrophil swarming: an essential process of the neutrophil tissue response.

    PubMed

    Kienle, Korbinian; Lämmermann, Tim

    2016-09-01

    Neutrophil infiltration into inflamed and infected tissues is a fundamental process of the innate immune response. While neutrophil interactions with the blood vessel wall have been intensely studied over the last decades, neutrophil dynamics beyond the vasculature have for a long time remained poorly investigated. Recent intravital microscopy studies of neutrophil populations directly at the site of tissue damage or microbial invasion have changed our perspective on neutrophil responses within tissues. Swarm-like migration patterns of neutrophils, referred to as 'neutrophil swarming', have been detected in diverse tissues under conditions of sterile inflammation and infection with various pathogens, including bacteria, fungi, and parasites. Current work has begun to unravel the molecular pathways choreographing the sequential phases of highly coordinated chemotaxis followed by neutrophil accumulation and the formation of substantial neutrophil clusters. It is now clear that intercellular communication among neutrophils amplifies their recruitment in a feed-forward manner, which provides them with a level of self-organization during neutrophil swarming. This review will summarize recent developments and current concepts on neutrophil swarming, an important process of the neutrophil tissue response with a critical role in maintaining the balance between host protection and inflammation-driven tissue destruction. PMID:27558329

  8. Interleukin-17 contributes to generation of Th1 immunity and neutrophil recruitment during Chlamydia muridarum genital tract infection but is not required for macrophage influx or normal resolution of infection.

    PubMed

    Scurlock, Amy M; Frazer, Lauren C; Andrews, Charles W; O'Connell, Catherine M; Foote, Isaac P; Bailey, Sarabeth L; Chandra-Kuntal, Kumar; Kolls, Jay K; Darville, Toni

    2011-03-01

    Interleukin 17 (IL-17) contributes to development of Th1 immunity and neutrophil influx during Chlamydia muridarum pulmonary infection, but its role during C. muridarum genital tract infection has not been described. We detected similar numbers of Chlamydia-specific Th17 and Th1 cells in iliac nodes of wild-type mice early during genital C. muridarum infection, while Th1 cells predominated later. il17ra(-/-) mice exhibited a reduced chlamydia-specific Th1 response in draining iliac nodes and decreased local IFN-γ production. Neutrophil influx into the genital tract was also decreased. However, il17ra(-/-) mice resolved infection normally, and no difference in pathology was observed compared to the wild type. Macrophage influx and tumor necrosis factor alpha (TNF-α) production were increased in il17ra(-/-) mice, providing a compensatory mechanism to effectively control chlamydial genital tract infection despite a reduced Th1 response. In ifnγ(-/-) mice, a marked increase in cellular infiltrates and chronic pathology was associated with an increased Th17 response. Although neutralization of IL-17 in ifnγ(-/-) mice decreased neutrophil influx, macrophage infiltration remained intact and the bacterial burden was not increased. Collectively, these results indicate that IL-17 contributes to the generation of Th1 immunity and neutrophil recruitment but is not required for macrophage influx or normal resolution of C. muridarum genital infection. These data highlight the redundant immune mechanisms operative at this mucosal site and the importance of examining site-specific responses to mucosal pathogens. PMID:21149587

  9. Interleukin-17 Contributes to Generation of Th1 Immunity and Neutrophil Recruitment during Chlamydia muridarum Genital Tract Infection but Is Not Required for Macrophage Influx or Normal Resolution of Infection ▿

    PubMed Central

    Scurlock, Amy M.; Frazer, Lauren C.; Andrews, Charles W.; O'Connell, Catherine M.; Foote, Isaac P.; Bailey, Sarabeth L.; Chandra-Kuntal, Kumar; Kolls, Jay K.; Darville, Toni

    2011-01-01

    Interleukin 17 (IL-17) contributes to development of Th1 immunity and neutrophil influx during Chlamydia muridarum pulmonary infection, but its role during C. muridarum genital tract infection has not been described. We detected similar numbers of Chlamydia-specific Th17 and Th1 cells in iliac nodes of wild-type mice early during genital C. muridarum infection, while Th1 cells predominated later. il17ra−/− mice exhibited a reduced chlamydia-specific Th1 response in draining iliac nodes and decreased local IFN-γ production. Neutrophil influx into the genital tract was also decreased. However, il17ra−/− mice resolved infection normally, and no difference in pathology was observed compared to the wild type. Macrophage influx and tumor necrosis factor alpha (TNF-α) production were increased in il17ra−/− mice, providing a compensatory mechanism to effectively control chlamydial genital tract infection despite a reduced Th1 response. In ifnγ−/− mice, a marked increase in cellular infiltrates and chronic pathology was associated with an increased Th17 response. Although neutralization of IL-17 in ifnγ−/− mice decreased neutrophil influx, macrophage infiltration remained intact and the bacterial burden was not increased. Collectively, these results indicate that IL-17 contributes to the generation of Th1 immunity and neutrophil recruitment but is not required for macrophage influx or normal resolution of C. muridarum genital infection. These data highlight the redundant immune mechanisms operative at this mucosal site and the importance of examining site-specific responses to mucosal pathogens. PMID:21149587

  10. Lower Respiratory Tract Infection of the Ferret by 2009 H1N1 Pandemic Influenza A Virus Triggers Biphasic, Systemic, and Local Recruitment of Neutrophils

    PubMed Central

    Camp, Jeremy V.; Bagci, Ulas; Chu, Yong-Kyu; Squier, Brendan; Fraig, Mostafa; Uriarte, Silvia M.; Guo, Haixun; Mollura, Daniel J.

    2015-01-01

    ABSTRACT Infection of the lower respiratory tract by influenza A viruses results in increases in inflammation and immune cell infiltration in the lung. The dynamic relationships among the lung microenvironments, the lung, and systemic host responses during infection remain poorly understood. Here we used extensive systematic histological analysis coupled with live imaging to gain access to these relationships in ferrets infected with the 2009 H1N1 pandemic influenza A virus (H1N1pdm virus). Neutrophil levels rose in the lungs of H1N1pdm virus-infected ferrets 6 h postinfection and became concentrated at areas of the H1N1pdm virus-infected bronchiolar epithelium by 1 day postinfection (dpi). In addition, neutrophil levels were increased throughout the alveolar spaces during the first 3 dpi and returned to baseline by 6 dpi. Histochemical staining revealed that neutrophil infiltration in the lungs occurred in two waves, at 1 and 3 dpi, and gene expression within microenvironments suggested two types of neutrophils. Specifically, CCL3 levels, but not CXCL8/interleukin 8 (IL-8) levels, were higher within discrete lung microenvironments and coincided with increased infiltration of neutrophils into the lung. We used live imaging of ferrets to monitor host responses within the lung over time with [18F]fluorodeoxyglucose (FDG). Sites in the H1N1pdm virus-infected ferret lung with high FDG uptake had high levels of proliferative epithelium. In summary, neutrophils invaded the H1N1pdm virus-infected ferret lung globally and focally at sites of infection. Increased neutrophil levels in microenvironments did not correlate with increased FDG uptake; hence, FDG uptake may reflect prior infection and inflammation of lungs that have experienced damage, as evidenced by bronchial regeneration of tissues in the lungs at sites with high FDG levels. IMPORTANCE Severe influenza disease is characterized by an acute infection of the lower airways that may progress rapidly to organ failure

  11. STK11/LKB1 Deficiency Promotes Neutrophil Recruitment and Proinflammatory Cytokine Production to Suppress T-cell Activity in the Lung Tumor Microenvironment.

    PubMed

    Koyama, Shohei; Akbay, Esra A; Li, Yvonne Y; Aref, Amir R; Skoulidis, Ferdinandos; Herter-Sprie, Grit S; Buczkowski, Kevin A; Liu, Yan; Awad, Mark M; Denning, Warren L; Diao, Lixia; Wang, Jing; Parra-Cuentas, Edwin R; Wistuba, Ignacio I; Soucheray, Margaret; Thai, Tran; Asahina, Hajime; Kitajima, Shunsuke; Altabef, Abigail; Cavanaugh, Jillian D; Rhee, Kevin; Gao, Peng; Zhang, Haikuo; Fecci, Peter E; Shimamura, Takeshi; Hellmann, Matthew D; Heymach, John V; Hodi, F Stephen; Freeman, Gordon J; Barbie, David A; Dranoff, Glenn; Hammerman, Peter S; Wong, Kwok-Kin

    2016-03-01

    STK11/LKB1 is among the most commonly inactivated tumor suppressors in non-small cell lung cancer (NSCLC), especially in tumors harboring KRAS mutations. Many oncogenes promote immune escape, undermining the effectiveness of immunotherapies, but it is unclear whether the inactivation of tumor suppressor genes, such as STK11/LKB1, exerts similar effects. In this study, we investigated the consequences of STK11/LKB1 loss on the immune microenvironment in a mouse model of KRAS-driven NSCLC. Genetic ablation of STK11/LKB1 resulted in accumulation of neutrophils with T-cell-suppressive effects, along with a corresponding increase in the expression of T-cell exhaustion markers and tumor-promoting cytokines. The number of tumor-infiltrating lymphocytes was also reduced in LKB1-deficient mouse and human tumors. Furthermore, STK11/LKB1-inactivating mutations were associated with reduced expression of PD-1 ligand PD-L1 in mouse and patient tumors as well as in tumor-derived cell lines. Consistent with these results, PD-1-targeting antibodies were ineffective against Lkb1-deficient tumors. In contrast, treating Lkb1-deficient mice with an IL6-neutralizing antibody or a neutrophil-depleting antibody yielded therapeutic benefits associated with reduced neutrophil accumulation and proinflammatory cytokine expression. Our findings illustrate how tumor suppressor mutations can modulate the immune milieu of the tumor microenvironment, and they offer specific implications for addressing STK11/LKB1-mutated tumors with PD-1-targeting antibody therapies. PMID:26833127

  12. Neutrophils in Cancer: Two Sides of the Same Coin

    PubMed Central

    Uribe-Querol, Eileen; Rosales, Carlos

    2015-01-01

    Neutrophils are the most abundant leukocytes in blood and are considered to be the first line of defense during inflammation and infections. In addition, neutrophils are also found infiltrating many types of tumors. Tumor-associated neutrophils (TANs) have relevant roles in malignant disease. Indeed neutrophils may be potent antitumor effector cells. However, increasing clinical evidence shows TANs correlate with poor prognosis. The tumor microenvironment controls neutrophil recruitment and in turn TANs help tumor progression. Hence, TANs can be beneficial or detrimental to the host. It is the purpose of this review to highlight these two sides of the neutrophil coin in cancer and to describe recent studies that provide some light on the mechanisms for neutrophil recruitment to the tumor, for neutrophils supporting tumor progression, and for neutrophil activation to enhance their antitumor functions. PMID:26819959

  13. Anti-inflammatory Effect of Methyl Gallate on Experimental Arthritis: Inhibition of Neutrophil Recruitment, Production of Inflammatory Mediators, and Activation of Macrophages.

    PubMed

    Correa, Luana Barbosa; Pádua, Tatiana Almeida; Seito, Leonardo Noboru; Costa, Thadeu Estevam Moreira Maramaldo; Silva, Magaiver Andrade; Candéa, André Luis Peixoto; Rosas, Elaine Cruz; Henriques, Maria G

    2016-06-24

    Methyl gallate (MG) is a prevalent phenolic acid in the plant kingdom, and its presence in herbal medicines might be related to its remarkable biological effects, such as its antioxidant, antitumor, and antimicrobial activities. Although some indirect evidence suggests anti-inflammatory activity for MG, there are no studies demonstrating this effect in animal models. Herein, we demonstrated that MG (0.7-70 mg/kg) inhibited zymosan-induced experimental arthritis in a dose-dependent manner. The oral administration of MG (7 mg/kg) attenuates arthritis induced by zymosan, affecting edema formation, leukocyte migration, and the production of inflammatory mediators (IL-1β, IL-6, TNF-α, CXCL-1, LTB4, and PGE2). Pretreatment with MG inhibited in vitro neutrophil chemotaxis elicited by CXCL-1, as well as the adhesion of these cells to TNF-α-primed endothelial cells. MG also impaired zymosan-stimulated macrophages by inhibiting IL-6 and NO production, COX-2 and iNOS expression, and intracellular calcium mobilization. Thus, MG is likely to present an anti-inflammatory effect by targeting multiple cellular events such as the production of various inflammatory mediators, as well as leukocyte activation and migration. PMID:27227459

  14. A comparison of the pulmonary defenses against streptococcal infection in rats and mice following O3 exposure: Differences in disease susceptibility and neutrophil recruitment

    SciTech Connect

    Gilmour, M.I.; Selgrade, M.K. )

    1993-12-01

    Ozone (O3) exposure reduces alveolar macrophage (AM) phagocytosis in mice and increases their susceptibility to Streptococcus zooepidemicus. O3 exposure also decreases AM phagocytosis in rats but does not result in mortality to infection. To investigate the mechanism of disease protection in rats, antibacterial defenses of two strains of mice and F344 rats were compared. O3 exposure (3 hr, 0.4 or 0.8 ppm) and infection with S. zooepidemicus resulted in a dose-dependent proliferation of bacteria in the lungs of mice and high mortality. Polymorphonuclear leukocytes (PMNs) were observed in severely affected individuals 2 or more days postinfection and did not alter the fatal infection. In contrast, microbial inactivation was only impaired in O3-exposed rat lungs during the first 48 hr after infection. In these animals PMNs could be isolated from bronchoalveolar lavage fluid between 6 and 48 hr postinfection with the peak response occurring at 24 hr. Pretreatment with anti-PMN serum eliminated the neutrophil influx and impaired further the bactericidal activity in ozone-exposed rats. The results suggest that inhaled streptococci are cleared normally from the mouse lung by AMs. Following exposure to O3, AM phagocytosis is reduced and the mice develop a fatal infection. The persistence of bacteria in the lungs of O3-exposed rats triggers a transient influx of PMNs whose appearance corresponds with elimination of the bacteria. Differences in antimicrobial defenses between various experimental species and humans need to be better understood in order to predict effects of air pollutants on susceptibility to infection in man.

  15. Pulmonary neutrophil recruitment and bronchial reactivity in formaldehyde-exposed rats are modulated by mast cells and differentially by neuropeptides and nitric oxide

    SciTech Connect

    Lino dos Santos Franco, Adriana; Damazo, Amilcar Sabino; Beraldo de Souza, Hyula Regines; Domingos, Helory Vanni; Oliveira-Filho, Ricardo Martins; Oliani, Sonia Maria; Costa, Soraia Katia Pereira; Tavares de Lima, Wothan . E-mail: wtdelima@icb.usp.br

    2006-07-01

    We have used a pharmacological approach to study the mechanisms underlying the rat lung injury and the airway reactivity changes induced by inhalation of formaldehyde (FA) (1% formalin solution, 90 min once a day, 4 days). The reactivity of isolated tracheae and intrapulmonary bronchi were assessed in dose-response curves to methacholine (MCh). Local and systemic inflammatory phenomena were evaluated in terms of leukocyte countings in bronchoalveolar lavage (BAL) fluid, blood, bone marrow lavage and spleen. Whereas the tracheal reactivity to MCh did not change, a significant bronchial hyporesponsiveness (BHR) was found after FA inhalation as compared with naive rats. Also, FA exposure significantly increased the total cell numbers in BAL, in peripheral blood and in the spleen, but did not modify the counts in bone marrow. Capsaicin hindered the increase of leukocyte number recovered in BAL fluid after FA exposure. Both compound 48/80 and indomethacin were able to prevent the lung neutrophil influx after FA, but indomethacin had no effect on that of mononuclear cells. Following FA inhalation, the treatment with sodium cromoglycate (SCG), but not with the nitric oxide (NO) synthase inhibitor L-NAME, significantly reduced the total cell number in BAL. Compound 48/80, L-NAME and SCG significantly prevented BHR to MCh after FA inhalation, whereas capsaicin was inactive in this regard. On the other hand, indomethacin exacerbated BHR. These data suggest that after FA inhalation, the resulting lung leukocyte influx and BHR may involve nitric oxide, airway sensory fibers and mast cell-derived mediators. The effect of NO seemed to be largely restricted to the bronchial tonus, whereas neuropeptides appeared to be linked to the inflammatory response, therefore indicating that the mechanisms responsible for the changes of airway responsiveness caused by FA may be separate from those underlying its inflammatory lung effects.

  16. Neutrophil Functions in Periodontal Homeostasis

    PubMed Central

    Cortés-Vieyra, Ricarda; Rosales, Carlos

    2016-01-01

    Oral tissues are constantly exposed to damage from the mechanical effort of eating and to microorganisms, mostly bacteria. In healthy gingiva tissue remodeling and a balance between bacteria and innate immune cells are maintained. However, excess of bacteria biofilm (plaque) creates an inflammation state that recruits more immune cells, mainly neutrophils to the gingiva. Neutrophils create a barrier for bacteria to reach inside tissues. When neutrophils are insufficient, bacteria thrive causing more inflammation that has been associated with systemic effects on other conditions such as atherosclerosis, diabetes, and cancer. But paradoxically when neutrophils persist, they can also promote a chronic inflammatory state that leads to periodontitis, a condition that leads to damage of the bone-supporting tissues. In periodontitis, bone loss is a serious complication. How a neutrophil balance is needed for maintaining healthy oral tissues is the focus of this review. We present recent evidence on how alterations in neutrophil number and function can lead to inflammatory bone loss, and how some oral bacteria signal neutrophils to block their antimicrobial functions and promote an inflammatory state. Also, based on this new information, novel therapeutic approaches are discussed. PMID:27019855

  17. Exposure to Leishmania braziliensis Triggers Neutrophil Activation and Apoptosis

    PubMed Central

    Hurrell, Benjamin P.; Celes, Fabiana S.; Curvelo, Rebecca P.; Prates, Deboraci B.; Barral, Aldina; Borges, Valeria M.; Tacchini-Cottier, Fabienne; de Oliveira, Camila I.

    2015-01-01

    Background Neutrophils are the first line of defense against invading pathogens and are rapidly recruited to the sites of Leishmania inoculation. During Leishmania braziliensis infection, depletion of inflammatory cells significantly increases the parasite load whereas co-inoculation of neutrophils plus L. braziliensis had an opposite effect. Moreover, the co-culture of infected macrophages and neutrophils also induced parasite killing leading us to ask how neutrophils alone respond to an L. braziliensis exposure. Herein we focused on understanding the interaction between neutrophils and L. braziliensis, exploring cell activation and apoptotic fate. Methods and Findings Inoculation of serum-opsonized L. braziliensis promastigotes in mice induced neutrophil accumulation in vivo, peaking at 24 h. In vitro, exposure of thyoglycollate-elicited inflammatory or bone marrow neutrophils to L. braziliensis modulated the expression of surface molecules such as CD18 and CD62L, and induced the oxidative burst. Using mCherry-expressing L. braziliensis, we determined that such effects were mainly observed in infected and not in bystander cells. Neutrophil activation following contact with L. braziliensis was also confirmed by the release of TNF-α and neutrophil elastase. Lastly, neutrophils infected with L. braziliensis but not with L. major displayed markers of early apoptosis. Conclusions We show that L. braziliensis induces neutrophil recruitment in vivo and that neutrophils exposed to the parasite in vitro respond through activation and release of inflammatory mediators. This outcome may impact on parasite elimination, particularly at the early stages of infection. PMID:25756874

  18. Microbe-dependent lymphatic migration of neutrophils modulates lymphocyte proliferation in lymph nodes

    PubMed Central

    Hampton, Henry R.; Bailey, Jacqueline; Tomura, Michio; Brink, Robert; Chtanova, Tatyana

    2015-01-01

    Neutrophil recruitment to the site of injury is an essential first step of an anti-bacterial response. However, little is known about the basis for and relevance of neutrophil migration from inflamed tissue into lymphoid organs. We established a photoconversion-based system to monitor the fate of neutrophils recruited to inflamed skin. While neutrophils are efficiently recruited to sites of both microbial and sterile lesions, subsequent re-localization to draining lymph nodes happens only when bacteria are present in the primary lesion. Skin egress of neutrophils occurs via lymphatic vessels and is dependent on CD11b and CXCR4 but not CCR7. Neutrophils are the predominant immune cell to migrate from inflamed skin into lymph nodes where they augment lymphocyte proliferation. Furthermore, inhibition of neutrophil migration from skin reduces T-cell proliferation in draining lymph nodes. Thus neutrophils mediate rapid cellular communication between the initial injury site and secondary lymphoid organs and modulate immune responsiveness. PMID:25972253

  19. Intergrin-dependent neutrophil migration in the injured mouse cornea

    Technology Transfer Automated Retrieval System (TEKTRAN)

    As an early responder to an inflammatory stimulus, neutrophils must exit the vasculature and migrate through the extravascular tissue to the site of insult, which is often remote from the point of extravasation. Following a central epithelial corneal abrasion, neutrophils recruited from the peripher...

  20. Major neutrophil functions subverted by Porphyromonas gingivalis.

    PubMed

    Olsen, Ingar; Hajishengallis, George

    2016-01-01

    Polymorphonuclear leukocytes (neutrophils) constitute an integrated component of the innate host defense in the gingival sulcus/periodontal pocket. However, the keystone periodontal pathogen Porphyromonas gingivalis has in the course of evolution developed a number of capacities to subvert this defense to its own advantage. The present review describes the major mechanisms that P. gingivalis uses to subvert neutrophil homeostasis, such as impaired recruitment and chemotaxis, resistance to granule-derived antimicrobial agents and to the oxidative burst, inhibition of phagocytic killing while promoting a nutritionally favorable inflammatory response, and delay of neutrophil apoptosis. Studies in animal models have shown that at least some of these mechanisms promote the dysbiotic transformation of the periodontal polymicrobial community, thereby leading to inflammation and bone loss. It is apparent that neutrophil-P. gingivalis interactions and subversion of innate immunity are key contributing factors to the pathogenesis of periodontal disease. PMID:26993626

  1. Neutrophils: Between Host Defence, Immune Modulation, and Tissue Injury

    PubMed Central

    Kruger, Philipp; Saffarzadeh, Mona; Weber, Alexander N. R.; Rieber, Nikolaus; Radsak, Markus; von Bernuth, Horst; Benarafa, Charaf; Roos, Dirk; Skokowa, Julia; Hartl, Dominik

    2015-01-01

    Neutrophils, the most abundant human immune cells, are rapidly recruited to sites of infection, where they fulfill their life-saving antimicrobial functions. While traditionally regarded as short-lived phagocytes, recent findings on long-term survival, neutrophil extracellular trap (NET) formation, heterogeneity and plasticity, suppressive functions, and tissue injury have expanded our understanding of their diverse role in infection and inflammation. This review summarises our current understanding of neutrophils in host-pathogen interactions and disease involvement, illustrating the versatility and plasticity of the neutrophil, moving between host defence, immune modulation, and tissue damage. PMID:25764063

  2. Exosomes Mediate LTB4 Release during Neutrophil Chemotaxis

    PubMed Central

    Majumdar, Ritankar; Tavakoli Tameh, Aidin; Parent, Carole A.

    2016-01-01

    Leukotriene B4 (LTB4) is secreted by chemotactic neutrophils, forming a secondary gradient that amplifies the reach of primary chemoattractants. This strategy increases the recruitment range for neutrophils and is important during inflammation. Here, we show that LTB4 and its synthesizing enzymes localize to intracellular multivesicular bodies that, upon stimulation, release their content as exosomes. Purified exosomes can activate resting neutrophils and elicit chemotactic activity in a LTB4 receptor-dependent manner. Inhibition of exosome release leads to loss of directional motility with concomitant loss of LTB4 release. Our findings establish that the exosomal pool of LTB4 acts in an autocrine fashion to sensitize neutrophils towards the primary chemoattractant, and in a paracrine fashion to mediate the recruitment of neighboring neutrophils in trans. We envision that this mechanism is used by other signals to foster communication between cells in harsh extracellular environments. PMID:26741884

  3. Human filarial Wolbachia lipopeptide directly activates human neutrophils in vitro.

    PubMed

    Tamarozzi, F; Wright, H L; Johnston, K L; Edwards, S W; Turner, J D; Taylor, M J

    2014-10-01

    The host inflammatory response to the Onchocerca volvulus endosymbiont, Wolbachia, is a major contributing factor in the development of chronic pathology in humans (onchocerciasis/river blindness). Recently, the toll-like pattern recognition receptor motif of the major inflammatory ligands of filarial Wolbachia, membrane-associated diacylated lipoproteins, was functionally defined in murine models of pathology, including mediation of neutrophil recruitment to the cornea. However, the extent to which human neutrophils can be activated in response to this Wolbachia pattern recognition motif is not known. Therefore, the responses of purified peripheral blood human neutrophils to a synthetic N-terminal diacylated lipopeptide (WoLP) of filarial Wolbachia peptidoglycan-associated lipoprotein (PAL) were characterized. WoLP exposure led to a dose-dependent activation of healthy, human neutrophils that included gross morphological alterations and modulation of surface expressed integrins involved in tethering, rolling and extravasation. WoLP exposure induced chemotaxis but not chemokinesis of neutrophils, and secretion of the major neutrophil chemokine, interleukin 8. WoLP also induced and primed the respiratory burst, and enhanced neutrophil survival by delay of apoptosis. These results indicate that the major inflammatory motif of filarial Wolbachia lipoproteins directly activates human neutrophils in vitro and promotes a molecular pathway by which human neutrophils are recruited to sites of Onchocerca parasitism. PMID:24909063

  4. Interleukin-1 (IL-1) signaling in intestinal stromal cells controls KC/ CXCL1 secretion, which correlates with recruitment of IL-22- secreting neutrophils at early stages of Citrobacter rodentium infection.

    PubMed

    Lee, Yong-Soo; Yang, Hyungjun; Yang, Jin-Young; Kim, Yeji; Lee, Su-Hyun; Kim, Ji Heui; Jang, Yong Ju; Vallance, Bruce A; Kweon, Mi-Na

    2015-08-01

    Attaching and effacing pathogens, including enterohemorrhagic Escherichia coli in humans and Citrobacter rodentium in mice, raise serious public health concerns. Here we demonstrate that interleukin-1 receptor (IL-1R) signaling is indispensable for protection against C. rodentium infection in mice. Four days after infection with C. rodentium, there were significantly fewer neutrophils (CD11b+ Ly6C+ Ly6G+) in the colons of IL-1R−/− mice than in wild-type mice. Levels of mRNA and protein of KC/CXCL1 were also significantly reduced in colon homogenates of infected IL-1R−/− mice relative to wild-type mice. Of note, infiltrated CD11b+ Ly6C+ Ly6G+ neutrophils were the main source of IL-22 secretion after C. rodentium infection. Interestingly, intestinal stromal cells isolated from IL-1R−/− mice secreted lower levels of KC/CXCL1 than stromal cells from wild-type mice during C. rodentium infection. Similar effects were found when mouse intestinal stromal cells and human nasal polyp stromal cells were treated with IL-1R antagonists (i.e., anakinra) in vitro. These results suggest that IL-1 signaling plays a pivotal role in activating mucosal stromal cells to secrete KC/CXCL1, which is essential for infiltration of IL-22-secreting neutrophils upon bacterial infection.

  5. Rac1 signaling regulates neutrophil-dependent tissue damage in experimental colitis.

    PubMed

    Yu, Changhui; Zhang, Su; Song, Lei; Wang, Yusheng; Hwaiz, Rundk; Luo, Lingtao; Thorlacius, Henrik

    2014-10-15

    Excessive neutrophil recruitment in the colon is a major feature in acute colitis although the signaling mechanisms behind colonic recruitment of neutrophils remain elusive. Herein, we hypothesized that Rac1 activity might play an important role in neutrophil infiltration in the inflamed colon. Female Balb/c mice were treated with the Rac1 inhibitor NSC23766 (0.5 and 5mg/kg) before and daily after administration of 5% dextran sodium sulfate (DSS). Colonic tissue was collected for quantification of neutrophil recruitment, interleukin-6 (IL-6) and CXC chemokine formation as well as histological damage score five days after challenge with DSS. Rac1 activity was determined by western blot and Mac-1 expression by flow cytometry in neutrophils. Administration of NSC23766 decreased DSS-induced neutrophil recruitment and tissue damage in the colon. Rac1 inhibition decreased colonic formation of IL-6 and CXC chemokines in experimental colitis. Chemokine challenge increased Rac1 activity in neutrophils and NSC23766 markedly reduced this neutrophil activity of Rac1. Inhibition of Rac1 abolished CXC chemokine-induced neutrophil chemotaxis and up-regulation of Mac-1 in vitro. Taken together, Rac1 signaling plays a significant role in controlling accumulation of neutrophils and tissue injury in experimental colitis. Thus, our novel results suggest that targeting Rac1 signaling might be a useful way to protect against neutrophil-mediated tissue injury in acute colitis.

  6. How neutrophils kill fungi.

    PubMed

    Gazendam, Roel P; van de Geer, Annemarie; Roos, Dirk; van den Berg, Timo K; Kuijpers, Taco W

    2016-09-01

    Neutrophils play a critical role in the prevention of invasive fungal infections. Whereas mouse studies have demonstrated the role of various neutrophil pathogen recognition receptors (PRRs), signal transduction pathways, and cytotoxicity in the murine antifungal immune response, much less is known about the killing of fungi by human neutrophils. Recently, novel primary immunodeficiencies have been identified in patients with a susceptibility to fungal infections. These human 'knock-out' neutrophils expand our knowledge to understand the role of PRRs and signaling in human fungal killing. From the studies with these patients it is becoming clear that neutrophils employ fundamentally distinct mechanisms to kill Candida albicans or Aspergillus fumigatus. PMID:27558342

  7. The Neutrophil Btk Signalosome Regulates Integrin Activation during Sterile Inflammation

    PubMed Central

    Volmering, Stephanie; Block, Helena; Boras, Mark; Lowell, Clifford A.; Zarbock, Alexander

    2016-01-01

    SUMMARY Neutrophils are recruited from the blood to sites of sterile inflammation, where they are involved in wound healing but can also cause tissue damage. During sterile inflammation, necrotic cells release pro-inflammatory molecules including formylated peptides. However, the signaling pathway triggered by formylated peptides to integrin activation and leukocyte recruitment is unknown. By using spinning-disk confocal intravital microscopy, we examined the molecular mechanisms of leukocyte recruitment to sites of focal hepatic necrosis in vivo. We demonstrated that the Bruton’s tyrosine kinase (Btk) was required for multiple Mac-1 activation events involved in neutrophil recruitment and functions during sterile inflammation triggered by fMLF. The Src family kinase Hck, Wiskott-Aldrich-syndrome protein, and phospholipase Cγ2 were also involved in this pathway required for fMLF-triggered Mac-1 activation and neutrophil recruitment. Thus, we have identified a neutrophil Btk signalosome that is involved in a signaling pathway triggered by formylated peptides leading to the selective activation of Mac-1 and neutrophil recruitment during sterile inflammation. PMID:26777396

  8. Dimethylfumarate Impairs Neutrophil Functions.

    PubMed

    Müller, Susen; Behnen, Martina; Bieber, Katja; Möller, Sonja; Hellberg, Lars; Witte, Mareike; Hänsel, Martin; Zillikens, Detlef; Solbach, Werner; Laskay, Tamás; Ludwig, Ralf J

    2016-01-01

    Host defense against pathogens relies on neutrophil activation. Inadequate neutrophil activation is often associated with chronic inflammatory diseases. Neutrophils also constitute a significant portion of infiltrating cells in chronic inflammatory diseases, for example, psoriasis and multiple sclerosis. Fumarates improve the latter diseases, which so far has been attributed to the effects on lymphocytes and dendritic cells. Here, we focused on the effects of dimethylfumarate (DMF) on neutrophils. In vitro, DMF inhibited neutrophil activation, including changes in surface marker expression, reactive oxygen species production, formation of neutrophil extracellular traps, and migration. Phagocytic ability and autoantibody-induced, neutrophil-dependent tissue injury ex vivo was also impaired by DMF. Regarding the mode of action, DMF modulates-in a stimulus-dependent manner-neutrophil activation using the phosphoinositide 3-kinase/Akt-p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 pathways. For in vivo validation, mouse models of epidermolysis bullosa acquisita, an organ-specific autoimmune disease caused by autoantibodies to type VII collagen, were employed. In the presence of DMF, blistering induced by injection of anti-type VII collagen antibodies into mice was significantly impaired. DMF treatment of mice with clinically already-manifested epidermolysis bullosa acquisita led to disease improvement. Collectively, we demonstrate a profound inhibitory activity of DMF on neutrophil functions. These findings encourage wider use of DMF in patients with neutrophil-mediated diseases. PMID:26763431

  9. ISOLATION OF MOUSE NEUTROPHILS

    PubMed Central

    Swamydas, Muthulekha; Luo, Yi; Dorf, Martin E.; Lionakis, Michail S.

    2015-01-01

    Neutrophils represent the first line of defense against bacterial and fungal pathogens. Indeed, patients with inherited and acquired qualitative and quantitative neutrophil defects are at high risk for developing bacterial and fungal infections and suffering adverse outcomes from these infections. Therefore, research aiming at defining the molecular factors that modulate neutrophil effector function under homeostatic conditions and during infection is essential for devising strategies to augment neutrophil function and improve the outcome of infected individuals. This unit describes a reproducible density gradient centrifugation-based protocol that can be applied in any laboratory to harvest large numbers of highly enriched and highly viable neutrophils from the bone marrow of mice both at the steady state and following infection with Candida albicans as described in UNIT 19.6. In another protocol, we also present a method that combines gentle enzymatic tissue digestion with a positive immunomagnetic selection technique or Fluorescence-activated cell sorting (FACS) to harvest highly pure and highly viable preparations of neutrophils directly from mouse tissues such as the kidney, the liver or the spleen. Finally, methods for isolating neutrophils from mouse peritoneal fluid and peripheral blood are included. Mouse neutrophils isolated by these protocols can be used for examining several aspects of cellular function ex vivo including pathogen binding, phagocytosis and killing, neutrophil chemotaxis, oxidative burst, degranulation and cytokine production, and for performing neutrophil adoptive transfer experiments. PMID:26237011

  10. Review of the neutrophil response to Bordetella pertussis infection.

    PubMed

    Eby, Joshua C; Hoffman, Casandra L; Gonyar, Laura A; Hewlett, Erik L

    2015-12-01

    The nature and timing of the neutrophil response to infection with Bordetella pertussis is influenced by multiple virulence factors expressed by the bacterium. After inoculation of the host airway, the recruitment of neutrophils signaled by B. pertussis lipooligosaccharide (LOS) is suppressed by pertussis toxin (PTX). Over the next week, the combined activities of PTX, LOS and adenylate cyclase toxin (ACT) result in production of cytokines that generate an IL-17 response, promoting neutrophil recruitment which peaks at 10-14 days after inoculation in mice. Arriving at the site of infection, neutrophils encounter the powerful local inhibitory activity of ACT, in conjunction with filamentous hemagglutinin. With the help of antibodies, neutrophils contribute to clearance of B. pertussis, but only after 28-35 days in a naïve mouse. Studies of the lasting, antigen-specific IL-17 response to infection in mice and baboons has led to progress in vaccine development and understanding of pathogenesis. Questions remain about the mediators that coordinate neutrophil recruitment and the mechanisms by which neutrophils overcome B. pertussis virulence factors.

  11. Major neutrophil functions subverted by Porphyromonas gingivalis

    PubMed Central

    Olsen, Ingar; Hajishengallis, George

    2016-01-01

    Polymorphonuclear leukocytes (neutrophils) constitute an integrated component of the innate host defense in the gingival sulcus/periodontal pocket. However, the keystone periodontal pathogen Porphyromonas gingivalis has in the course of evolution developed a number of capacities to subvert this defense to its own advantage. The present review describes the major mechanisms that P. gingivalis uses to subvert neutrophil homeostasis, such as impaired recruitment and chemotaxis, resistance to granule-derived antimicrobial agents and to the oxidative burst, inhibition of phagocytic killing while promoting a nutritionally favorable inflammatory response, and delay of neutrophil apoptosis. Studies in animal models have shown that at least some of these mechanisms promote the dysbiotic transformation of the periodontal polymicrobial community, thereby leading to inflammation and bone loss. It is apparent that neutrophil–P. gingivalis interactions and subversion of innate immunity are key contributing factors to the pathogenesis of periodontal disease. PMID:26993626

  12. Blocking neutrophil diapedesis prevents hemorrhage during thrombocytopenia

    PubMed Central

    Hillgruber, Carina; Pöppelmann, Birgit; Weishaupt, Carsten; Steingräber, Annika Kathrin; Wessel, Florian; Berdel, Wolfgang E.; Gessner, J. Engelbert; Ho-Tin-Noé, Benoît

    2015-01-01

    Spontaneous organ hemorrhage is the major complication in thrombocytopenia with a potential fatal outcome. However, the exact mechanisms regulating vascular integrity are still unknown. Here, we demonstrate that neutrophils recruited to inflammatory sites are the cellular culprits inducing thrombocytopenic tissue hemorrhage. Exposure of thrombocytopenic mice to UVB light provokes cutaneous petechial bleeding. This phenomenon is also observed in immune-thrombocytopenic patients when tested for UVB tolerance. Mechanistically, we show, analyzing several inflammatory models, that it is neutrophil diapedesis through the endothelial barrier that is responsible for the bleeding defect. First, bleeding is triggered by neutrophil-mediated mechanisms, which act downstream of capturing, adhesion, and crawling on the blood vessel wall and require Gαi signaling in neutrophils. Second, mutating Y731 in the cytoplasmic tail of VE-cadherin, known to selectively affect leukocyte diapedesis, but not the induction of vascular permeability, attenuates bleeding. Third, and in line with this, simply destabilizing endothelial junctions by histamine did not trigger bleeding. We conclude that specifically targeting neutrophil diapedesis through the endothelial barrier may represent a new therapeutic avenue to prevent fatal bleeding in immune-thrombocytopenic patients. PMID:26169941

  13. Neutrophils in cancer.

    PubMed

    Treffers, Louise W; Hiemstra, Ida H; Kuijpers, Taco W; van den Berg, Timo K; Matlung, Hanke L

    2016-09-01

    Neutrophils play an important role in cancer. This does not only relate to the well-established prognostic value of the presence of neutrophils, either in the blood or in tumor tissue, in the context of cancer progression or for the monitoring of therapy, but also to their active role in the progression of cancer. In the current review, we describe what is known in general about the role of neutrophils in cancer. What is emerging is a complex, rather heterogeneous picture with both pro- and anti-tumorigenic roles, which apparently differs with cancer type and disease stage. Furthermore, we will discuss the well-known role of neutrophils as myeloid-derived suppressor cells (MDSC), and also on the role of neutrophils as important effector cells during antibody therapy in cancer. It is clear that neutrophils contribute substantially to cancer progression in multiple ways, and this includes both direct effects on the cancer cells and indirect effect on the tumor microenvironment. While in many cases neutrophils have been shown to promote tumor progression, for instance by acting as MDSC, there are also protective effects, particularly when antibody immunotherapy is performed. A better understanding of the role of neutrophils is likely to provide opportunities for immunomodulation and for improving the treatment of cancer patients. PMID:27558343

  14. Myeloperoxidase Stimulates Neutrophil Degranulation.

    PubMed

    Grigorieva, D V; Gorudko, I V; Sokolov, A V; Kostevich, V A; Vasilyev, V B; Cherenkevich, S N; Panasenko, O M

    2016-08-01

    Myeloperoxidase, heme enzyme of azurophilic granules in neutrophils, is released into the extracellular space in the inflammation foci. In neutrophils, it stimulates a dose-dependent release of lactoferrin (a protein of specific granules), lysozyme (a protein of specific and azurophilic granules), and elastase (a protein of azurophilic granules). 4-Aminobenzoic acid hydrazide, a potent inhibitor of peroxidase activity of myeloperoxidase, produced no effect on neutrophil degranulation. Using signal transduction inhibitors (genistein, methoxyverapamil, wortmannin, and NiCl2), we demonstrated that myeloperoxidase-induced degranulation of neutrophils resulted from enzyme interaction with the plasma membrane and depends on activation of tyrosine kinases, phosphatidylinositol 3-kinases (PI3K), and calcium signaling. Myeloperoxidase modified by oxidative/halogenation stress (chlorinated and monomeric forms of the enzyme) lost the potency to activate neutrophil degranulation. PMID:27597056

  15. ATF3 is a novel regulator of mouse neutrophil migration

    PubMed Central

    Boespflug, Nicholas D.; Kumar, Sachin; McAlees, Jaclyn W.; Phelan, James D.; Grimes, H. Leighton; Hoebe, Kasper; Hai, Tsonwin; Karp, Christopher L.

    2014-01-01

    Expression of the activating transcription factor 3 (ATF3) gene is induced by Toll-like receptor (TLR) signaling. In turn, ATF3 protein inhibits the expression of various TLR-driven proinflammatory genes. Given its counter-regulatory role in diverse innate immune responses, we defined the effects of ATF3 on neutrophilic airway inflammation in mice. ATF3 deletion was associated with increased lipopolysaccharide (LPS)-driven airway epithelia production of CXCL1, but not CXCL2, findings concordant with a consensus ATF3-binding site identified solely in the Cxcl1 promoter. Unexpectedly, ATF3-deficient mice did not exhibit increased airway neutrophilia after LPS challenge. Bone marrow chimeras revealed a specific reduction in ATF3−/− neutrophil recruitment to wild-type lungs. In vitro, ATF3−/− neutrophils exhibited a profound chemotaxis defect. Global gene expression analysis identified ablated Tiam2 expression in ATF3−/− neutrophils. TIAM2 regulates cellular motility by activating Rac1-mediated focal adhesion disassembly. Notably, ATF3−/− and ATF3-sufficient TIAM2 knockdown neutrophils, both lacking TIAM2, exhibited increased focal complex area, along with excessive CD11b-mediated F-actin polymerization. Together, our data describe a dichotomous role for ATF3-mediated regulation of neutrophilic responses: inhibition of neutrophil chemokine production but promotion of neutrophil chemotaxis. PMID:24470589

  16. Patrolling monocytes promote intravascular neutrophil activation and glomerular injury in the acutely inflamed glomerulus.

    PubMed

    Finsterbusch, Michaela; Hall, Pam; Li, Anqi; Devi, Sapna; Westhorpe, Clare L V; Kitching, A Richard; Hickey, Michael J

    2016-08-30

    Nonclassical monocytes undergo intravascular patrolling in blood vessels, positioning them ideally to coordinate responses to inflammatory stimuli. Under some circumstances, the actions of monocytes have been shown to involve promotion of neutrophil recruitment. However, the mechanisms whereby patrolling monocytes control the actions of neutrophils in the circulation are unclear. Here, we examined the contributions of monocytes to antibody- and neutrophil-dependent inflammation in a model of in situ immune complex-mediated glomerulonephritis. Multiphoton and spinning disk confocal intravital microscopy revealed that monocytes patrol both uninflamed and inflamed glomeruli using β2 and α4 integrins and CX3CR1. Monocyte depletion reduced glomerular injury, demonstrating that these cells promote inappropriate inflammation in this setting. Monocyte depletion also resulted in reductions in neutrophil recruitment and dwell time in glomerular capillaries and in reactive oxygen species (ROS) generation by neutrophils, suggesting a role for cross-talk between monocytes and neutrophils in induction of glomerulonephritis. Consistent with this hypothesis, patrolling monocytes and neutrophils underwent prolonged interactions in glomerular capillaries, with the duration of these interactions increasing during inflammation. Moreover, neutrophils that interacted with monocytes showed increased retention and a greater propensity for ROS generation in the glomerulus. Also, renal patrolling monocytes, but not neutrophils, produced TNF during inflammation, and TNF inhibition reduced neutrophil dwell time and ROS production, as well as renal injury. These findings show that monocytes and neutrophils undergo interactions within the glomerular microvasculature. Moreover, evidence indicates that, in response to an inflammatory stimulus, these interactions allow monocytes to promote neutrophil recruitment and activation within the glomerular microvasculature, leading to neutrophil

  17. Recruiter's Manual.

    ERIC Educational Resources Information Center

    Reed, Michael; Recio, Manuel

    The manual assists both experienced and inexperienced personnel in defining and completing the entire range of tasks associated with the position of Pennsylvania Migrant Education Recruiter. The recruiter's primary responsibilities are to identify migrant children in the area and enroll those children eligible under Title I ESEA (Elementary and…

  18. Vestibular recruitment

    NASA Technical Reports Server (NTRS)

    Tsemakhov, S. G.

    1980-01-01

    Vestibular recruitment is defined through the analysis of several references. It is concluded that vestibular recruitment is an objective phenomenon which manifests itself during the affection of the vestibular receptor and thus serves as a diagnostic tool during affection of the vestibular system.

  19. Resident recruitment.

    PubMed

    Longmaid, H Esterbrook

    2003-02-01

    This article has introduced the reader to the critical components of successful recruitment of radiology residents. With particular attention to the ACGME institutional and program requirements regarding resident recruitment, and an explanation of the support systems (ERAS and NRMP) currently available to those involved in applicant review and selection, the article has sought to delineate a sensible approach to recruitment. Successful recruiters have mastered the essentials of these programs and have learned to adapt the programs to their needs. As new program directors work with their departments' resident selection committees, they will identify the factors that faculty and current residents cite as most important in the successful selection of new residents. By structuring the application review process, exploiting the power of the ERAS, and crafting a purposeful and friendly interview process, radiology residency directors can find and recruit the residents who best match their programs. PMID:12585436

  20. Neutrophils scan for activated platelets to initiate inflammation

    PubMed Central

    Sreeramkumar, Vinatha; Adrover, José M.; Ballesteros, Ivan; Cuartero, Maria Isabel; Rossaint, Jan; Bilbao, Izaskun; Nácher, Maria; Pitaval, Christophe; Radovanovic, Irena; Fukui, Yoshinori; McEver, Rodger P.; Filippi, Marie-Dominique; Lizasoain, Ignacio; Ruiz-Cabello, Jesús; Zarbock, Alexander; Moro, María A.; Hidalgo, Andrés

    2014-01-01

    Immune and inflammatory responses require leukocytes to migrate within and through the vasculature, a process that is facilitated by their capacity to switch to a polarized morphology with asymmetric distribution of receptors. We report that neutrophil polarization within activated venules served to organize a protruding domain that engaged activated platelets present in the bloodstream. The selectin ligand PSGL-1 transduced signals emanating from these interactions, resulting in redistribution of receptors that drive neutrophil migration. Consequently, neutrophils unable to polarize or to transduce signals through PSGL-1 displayed aberrant crawling, and blockade of this domain protected mice against thrombo-inflammatory injury. These results reveal that recruited neutrophils scan for activated platelets, and suggest that their bipolarity allows integration of signals present at both the endothelium and the circulation before inflammation proceeds. PMID:25477463

  1. Identification and characterization of VEGF-A–responsive neutrophils expressing CD49d, VEGFR1, and CXCR4 in mice and humans

    PubMed Central

    Massena, Sara; Christoffersson, Gustaf; Vågesjö, Evelina; Seignez, Cédric; Gustafsson, Karin; Binet, François; Herrera Hidalgo, Carmen; Giraud, Antoine; Lomei, Jalal; Weström, Simone; Shibuya, Masabumi; Claesson-Welsh, Lena; Gerwins, Pär; Welsh, Michael; Kreuger, Johan

    2015-01-01

    Vascular endothelial growth factor A (VEGF-A) is upregulated during hypoxia and is the major regulator of angiogenesis. VEGF-A expression has also been found to recruit myeloid cells to ischemic tissues where they contribute to angiogenesis. This study investigates the mechanisms underlying neutrophil recruitment to VEGF-A as well as the characteristics of these neutrophils. A previously undefined circulating subset of neutrophils shown to be CD49d+VEGFR1highCXCR4high was identified in mice and humans. By using chimeric mice with impaired VEGF receptor 1 (VEGFR1) or VEGFR2 signaling (Flt-1tk−/−, tsad−/−), we found that parallel activation of VEGFR1 on neutrophils and VEGFR2 on endothelial cells was required for VEGF-A-induced recruitment of circulating neutrophils to tissue. Intravital microscopy of mouse microcirculation revealed that neutrophil recruitment by VEGF-A versus by the chemokine macrophage inflammatory protein 2 (MIP-2 [CXCL2]) involved the same steps of the recruitment cascade but that an additional neutrophil integrin (eg, VLA-4 [CD49d/CD29]) played a crucial role in neutrophil crawling and emigration to VEGF-A. Isolated CD49d+ neutrophils featured increased chemokinesis but not chemotaxis compared with CD49d– neutrophils in the presence of VEGF-A. Finally, by targeting the integrin α4 subunit (CD49d) in a transplantation-based angiogenesis model that used avascular pancreatic islets transplanted to striated muscle, we demonstrated that inhibiting the recruitment of circulating proangiogenic neutrophils to hypoxic tissue impairs vessel neoformation. Thus, angiogenesis can be modulated by targeting cell-surface receptors specifically involved in VEGF-A-dependent recruitment of proangiogenic neutrophils without compromising recruitment of the neutrophil population involved in the immune response to pathogens. PMID:26286848

  2. Immunofluorescence and Confocal Microscopy of Neutrophils

    PubMed Central

    Allen, Lee-Ann H.

    2015-01-01

    Rapid recruitment of neutrophils to sites of infection and their ability to phagocytose and kill microbes is an important aspect of the innate immune response. Challenges associated with imaging of these cells include their short lifespan and small size and the fact that unstimulated cells are nonadherent. In addition, although cytoplasmic granules are plentiful, the abundance of many other organelles is diminished. Here we reprise methods for analysis of resting and activated cells using immunofluorescence and confocal microscopy, including kinetic analysis of phagosome maturation and degranulation, and detection of intraphagosomal superoxide accumulation. We describe approaches for rapid cell fixation and permeabilization that maximize antigen detection and discuss other variables that also affect data interpretation and image quality (such as cell spreading, degranulation, and phagocytosis). Finally, we show that these methods are also applicable to studies of neutrophil interactions with the extracellular matrix. PMID:24504957

  3. Matters of life and death. How neutrophils die or survive along NET release and is "NETosis" = necroptosis?

    PubMed

    Desai, Jyaysi; Mulay, Shrikant R; Nakazawa, Daigo; Anders, Hans-Joachim

    2016-06-01

    Neutrophil extracellular trap (NET) formation is a hallmark of many disorders that involve neutrophil recruitment, tissue damage, and inflammation. As NET formation is often associated with neutrophil death, the term "NETosis" has become popular. Upon discovery that neutrophils may survive NET release, apparent misnomers, such as "vital NETosis," have been proposed. Meanwhile, it has become obvious that certain stimuli can trigger neutrophil necroptosis, a process associated with NET-like chromatin release. Here, we discuss the relationship between NET release and neutrophil death in view highlighting that many assays used in the field do not properly distinguish between the two. An updated nomenclature is needed replacing the term "NETosis" to meet the growing variety of settings leading to chromatin release with and without neutrophil death. Dissecting which triggers of NET release involve which signaling pathway will help to define drugable molecular targets that inhibit NET release and/or neutrophil necrosis in specific disorders.

  4. Lung inflammation promotes metastasis through neutrophil protease-mediated degradation of Tsp-1

    PubMed Central

    El Rayes, Tina; Catena, Raúl; Lee, Sharrell; Stawowczyk, Marcin; Joshi, Natasha; Fischbach, Claudia; Powell, Charles A.; Dannenberg, Andrew J.; Altorki, Nasser K.; Gao, Dingcheng; Mittal, Vivek

    2015-01-01

    Inflammation is inextricably associated with primary tumor progression. However, the contribution of inflammation to tumor outgrowth in metastatic organs has remained underexplored. Here, we show that extrinsic inflammation in the lungs leads to the recruitment of bone marrow-derived neutrophils, which degranulate azurophilic granules to release the Ser proteases, elastase and cathepsin G, resulting in the proteolytic destruction of the antitumorigenic factor thrombospondin-1 (Tsp-1). Genetic ablation of these neutrophil proteases protected Tsp-1 from degradation and suppressed lung metastasis. These results provide mechanistic insights into the contribution of inflammatory neutrophils to metastasis and highlight the unique neutrophil protease–Tsp-1 axis as a potential antimetastatic therapeutic target. PMID:26668367

  5. Platelets enhance neutrophil transendothelial migration

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Platelets are increasingly recognized as important mediators of inflammation in addition to thrombosis. While platelets have been shown to promote neutrophil (PMN) adhesion to endothelium in various inflammatory models, it is unclear whether platelets enhance neutrophil transmigration across inflame...

  6. Chemokine CXCL1-mediated neutrophil trafficking in the lung: Role of CXCR2 activation

    PubMed Central

    Sawant, Kirti V.; Xu, Renling; Cox, Robert; Hawkins, Hal; Sbrana, Elena; Kolli, Deepthi; Garofalo, Roberto P.; Rajarathnam, Krishna

    2015-01-01

    Chemokine CXCL1 and its receptor CXCR2 play a crucial role in host immune response by recruiting and activating neutrophils for microbial killing at the tissue site. Dysregulation in this process has been implicated in collateral tissue damage causing disease. CXCL1 reversibly exists as monomers and dimers, and it has been proposed that distinct monomer and dimer activities and monomer-dimer equilibrium regulate neutrophil function. However, the molecular mechanisms linking CXCL1/CXCR2 axis and neutrophil ‘beneficial’ and ‘destructive’ phenotypes are not known. In this study, we characterized neutrophil trafficking and its consequence in the mouse lung by CXCL1 WT (exists as monomer and dimer) and a non-dissociating dimer. Whereas WT, compared to dimer, was more active at low doses, both WT and dimer elicited large neutrophil efflux at high doses. Importantly, robust neutrophil recruitment elicited by WT or dimer was not detrimental to lung tissue integrity, and further, could not be correlated to surface CXCR2 levels. We conclude that CXCL1 monomer/dimer distribution and receptor interactions are highly coupled and regulate neutrophil trafficking, and that injury in the context of disease is a consequence of inappropriate CXCR2 activation at the target tissue and not due to mechanical forces exerted by neutrophils during recruitment. PMID:26138727

  7. Rapid Sequestration of Leishmania mexicana by Neutrophils Contributes to the Development of Chronic Lesion

    PubMed Central

    Hurrell, Benjamin P.; Schuster, Steffen; Grün, Eva; Coutaz, Manuel; Williams, Roderick A.; Held, Werner; Malissen, Bernard; Malissen, Marie; Yousefi, Shida; Simon, Hans-Uwe; Müller, Andreas J.; Tacchini-Cottier, Fabienne

    2015-01-01

    The protozoan Leishmania mexicana parasite causes chronic non-healing cutaneous lesions in humans and mice with poor parasite control. The mechanisms preventing the development of a protective immune response against this parasite are unclear. Here we provide data demonstrating that parasite sequestration by neutrophils is responsible for disease progression in mice. Within hours of infection L. mexicana induced the local recruitment of neutrophils, which ingested parasites and formed extracellular traps without markedly impairing parasite survival. We further showed that the L. mexicana-induced recruitment of neutrophils impaired the early recruitment of dendritic cells at the site of infection as observed by intravital 2-photon microscopy and flow cytometry analysis. Indeed, infection of neutropenic Genista mice and of mice depleted of neutrophils at the onset of infection demonstrated a prominent role for neutrophils in this process. Furthermore, an increase in monocyte-derived dendritic cells was also observed in draining lymph nodes of neutropenic mice, correlating with subsequent increased frequency of IFNγ-secreting T helper cells, and better parasite control leading ultimately to complete healing of the lesion. Altogether, these findings show that L. mexicana exploits neutrophils to block the induction of a protective immune response and impairs the control of lesion development. Our data thus demonstrate an unanticipated negative role for these innate immune cells in host defense, suggesting that in certain forms of cutaneous leishmaniasis, regulating neutrophil recruitment could be a strategy to promote lesion healing. PMID:26020515

  8. Hyperbaric oxygen treatment reduces neutrophil-endothelial adhesion in chronic wound conditions through S-nitrosation.

    PubMed

    Kendall, Alexandra C; Whatmore, Jacqueline L; Winyard, Paul G; Smerdon, Gary R; Eggleton, Paul

    2013-01-01

    Hyperbaric oxygen (HBO) therapy is an effective treatment for diabetic chronic wounds. HBO reduces inflammation and accelerates wound healing, by mechanisms that remain unclear. Here we examined a mechanism by which HBO may reduce neutrophil recruitment, through changes in endothelial and neutrophil adhesion molecule expression and function. Human umbilical vein endothelial cells and neutrophils were exposed to selected chronic wound conditions, comprising hypoxia in the presence of lipopolysaccharide and tumor necrosis factor-alpha, and then treated with HBO. We observed neutrophil adhesion to endothelial cells following treatment with chronic wound conditions, which was reversed by HBO treatment. This was partly explained by reduced expression of endothelial intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 by HBO. No changes in neutrophil adhesion molecule expression (CD18, CD11b, CD62L, CD31) were observed following HBO treatment. However, HBO decreased hydrogen peroxide generation by neutrophils, and induced nitrous oxide-related protein modifications. The transnitrosating agent S-nitroso-L-cysteine ethyl ester (600 μM) also reduced neutrophil adhesion to human umbilical vein endothelial cell monolayers, and the iNOS inhibitor 1400 W (10 μM) and HgCl2, which promotes the decomposition of S-nitrosothiols (1 mM), reversed the effect of HBO, suggesting that S-nitrosation may inhibit neutrophil-endothelial cell adhesion. This study indicates that HBO could reduce inflammation in wounds through reduced neutrophil recruitment, mediated by S-nitrosation. PMID:24134224

  9. Chemokine receptor Ccr1 drives neutrophil-mediated kidney immunopathology and mortality in invasive candidiasis.

    PubMed

    Lionakis, Michail S; Fischer, Brett G; Lim, Jean K; Swamydas, Muthulekha; Wan, Wuzhou; Richard Lee, Chyi-Chia; Cohen, Jeffrey I; Scheinberg, Phillip; Gao, Ji-Liang; Murphy, Philip M

    2012-01-01

    Invasive candidiasis is the 4(th) leading cause of nosocomial bloodstream infection in the US with mortality that exceeds 40% despite administration of antifungal therapy; neutropenia is a major risk factor for poor outcome after invasive candidiasis. In a fatal mouse model of invasive candidiasis that mimics human bloodstream-derived invasive candidiasis, the most highly infected organ is the kidney and neutrophils are the major cellular mediators of host defense; however, factors regulating neutrophil recruitment have not been previously defined. Here we show that mice lacking chemokine receptor Ccr1, which is widely expressed on leukocytes, had selectively impaired accumulation of neutrophils in the kidney limited to the late phase of the time course of the model; surprisingly, this was associated with improved renal function and survival without affecting tissue fungal burden. Consistent with this, neutrophils from wild-type mice in blood and kidney switched from Ccr1(lo) to Ccr1(high) at late time-points post-infection, when Ccr1 ligands were produced at high levels in the kidney and were chemotactic for kidney neutrophils ex vivo. Further, when a 1∶1 mixture of Ccr1(+/+) and Ccr1(-/-) donor neutrophils was adoptively transferred intravenously into Candida-infected Ccr1(+/+) recipient mice, neutrophil trafficking into the kidney was significantly skewed toward Ccr1(+/+) cells. Thus, neutrophil Ccr1 amplifies late renal immunopathology and increases mortality in invasive candidiasis by mediating excessive recruitment of neutrophils from the blood to the target organ.

  10. Complement factor H modulates the activation of human neutrophil granulocytes and the generation of neutrophil extracellular traps.

    PubMed

    Schneider, Andrea E; Sándor, Noémi; Kárpáti, Éva; Józsi, Mihály

    2016-04-01

    Factor H (FH) is a major inhibitor of the alternative pathway of complement activation in plasma and on certain host surfaces. In addition to being a complement regulator, FH can bind to various cells via specific receptors, including binding to neutrophil granulocytes through complement receptor type 3 (CR3; CD11b/CD18), and modulate their function. The cellular roles of FH are, however, poorly understood. Because neutrophils are important innate immune cells in inflammatory processes and the host defense against pathogens, we aimed at studying the effects of FH on various neutrophil functions, including the generation of extracellular traps. FH co-localized with CD11b on the surface of neutrophils isolated from peripheral blood of healthy individuals, and cell-bound FH retained its cofactor activity and enhanced C3b degradation. Soluble FH supported neutrophil migration and immobilized FH induced cell spreading. In addition, immobilized but not soluble FH enhanced IL-8 release from neutrophils. FH alone did not trigger the cells to produce neutrophil extracellular traps (NETs), but NET formation induced by PMA and by fibronectin plus fungal β-glucan were inhibited by immobilized, but not by soluble, FH. Moreover, in parallel with NET formation, immobilized FH also inhibited the production of reactive oxygen species induced by PMA and by fibronectin plus β-glucan. Altogether, these data indicate that FH has multiple regulatory roles on neutrophil functions. While it can support the recruitment of neutrophils, FH may also exert anti-inflammatory effects and influence local inflammatory and antimicrobial reactions, and reduce tissue damage by modulating NET formation. PMID:26938503

  11. The Neutrophil Nucleus and Its Role in Neutrophilic Function.

    PubMed

    Carvalho, Leonardo Olivieri; Aquino, Elaine Nascimento; Neves, Anne Caroline Dias; Fontes, Wagner

    2015-09-01

    The cell nucleus plays a key role in differentiation processes in eukaryotic cells. It is not the nucleus in particular, but the organization of the genes and their remodeling that provides the data for the adjustments to be made according to the medium. The neutrophil nucleus has a different morphology. It is a multi-lobed nucleus where some researchers argue no longer function. However, studies indicate that it is very probable the occurrence of chromatin remodeling during activation steps. It may be that the human neutrophil nucleus also contributes to the mobility of neutrophils through thin tissue spaces. Questions like these will be discussed in this small review. The topics include morphology of human neutrophil nucleus, maturation process and modifications of the neutrophil nucleus, neutrophil activation and chromatin modifications, causes and consequences of multi-lobulated segmented morphology, and importance of the nucleus in the formation of neutrophil extracellular traps (NETs).

  12. The Essential Role of Neutrophils during Infection with the Intracellular Bacterial Pathogen Listeria monocytogenes.

    PubMed

    Witter, Alexandra R; Okunnu, Busola M; Berg, Rance E

    2016-09-01

    Neutrophils have historically been characterized as first responder cells vital to host survival because of their ability to contain and eliminate bacterial and fungal pathogens. However, recent studies have shown that neutrophils participate in both protective and detrimental responses to a diverse array of inflammatory and infectious diseases. Although the contribution of neutrophils to extracellular infections has been investigated for decades, their specific role during intracellular bacterial infections has only recently been appreciated. During infection with the Gram-positive intracellular pathogen Listeria monocytogenes, neutrophils are recruited from the bone marrow to sites of infection where they use novel bacterial-sensing pathways leading to phagocytosis and production of bactericidal factors. This review summarizes the requirement of neutrophils during L. monocytogenes infection by examining both neutrophil trafficking and function during primary and secondary infection. PMID:27543669

  13. Neutrophils Interact with Adenovirus Vectors via Fc Receptors and Complement Receptor 1

    PubMed Central

    Cotter, Matthew J.; Zaiss, Anne K.; Muruve, Daniel A.

    2005-01-01

    Neutrophils are effectors of the innate immune response to adenovirus vectors. Following the systemic administration of Cy2-labeled AdLuc in mice, flow cytometry and PCR analysis of liver leukocytes revealed that 25% of recruited neutrophils interacted with adenovirus vectors. In vitro, flow cytometry of human neutrophils incubated with Cy2-labeled AdLuc also demonstrated a significant interaction with adenovirus vectors. Fluorescence and electron microscopy confirmed vector internalization by neutrophils. The AdLuc-neutrophil interaction reduced vector transduction efficiency by more than 50% in coincubation assays in epithelium-derived cells. Adenovirus vector uptake by neutrophils occurred independently of coxsackievirus adenovirus receptor (CAR) and capsid RGD motifs, since neutrophils do not express CAR and uptake of the RGD-deleted vector AdL.PB* was similar to that of AdLuc. Furthermore, both AdLuc and AdL.PB* activated neutrophils and induced similar degrees of L-selectin shedding. Neutrophil uptake of AdLuc was dependent on the presence of complement and antibodies, since the interaction between AdLuc and neutrophils was significantly reduced when they were incubated in immunoglobulin G-depleted or heat-inactivated human serum. Blocking of complement receptor 1 (CD35) but not complement receptor 3 (CD11b/CD18) significantly reduced neutrophil uptake of AdLuc. Blocking of FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16) individually or together also reduced neutrophil uptake of AdLuc, although less than blocking of CD35 alone. Combined CR1 and Fc receptor blockade synergistically inhibited neutrophil-AdLuc interactions close to baseline. These results demonstrate opsonin-dependent adenovirus vector interactions with neutrophils and their corresponding receptors. PMID:16282462

  14. Aquaporin-9-expressing neutrophils are required for the establishment of contact hypersensitivity

    PubMed Central

    Moniaga, Catharina Sagita; Watanabe, Sachiko; Honda, Tetsuya; Nielsen, Søren; Hara-Chikuma, Mariko

    2015-01-01

    Aquaporin-9 (AQP9), a water/glycerol channel protein, is expressed in several immune cells including neutrophils; however, its role in immune response remains unknown. Here we show the involvement of AQP9 in hapten-induced contact hypersensitivity (CHS), as a murine model of skin allergic contact dermatitis, using AQP9 knockout (AQP9−/−) mice. First, the CHS response to hapten dinitrofluorobenzene (DNFB) was impaired in AQP9−/− mice compared with wild-type (WT) mice. Adoptive transfer of sensitized AQP9−/− draining lymph node (dLN) cells into WT recipients resulted in a reduced CHS response, indicating impaired sensitization in AQP9−/− mice. Second, administration of WT neutrophils into AQP9−/− mice during sensitization rescued the impaired CHS response. Neutrophil recruitment to dLNs upon hapten application was attenuated by AQP9 deficiency. Coincidentally, AQP9−/− neutrophils showed a reduced CC-chemokine receptor 7 (CCR7) ligand-induced migration efficacy, which was attributed to the attenuated recruitment of neutrophils to dLNs. Furthermore, we found that neutrophil deficiency, observed in AQP9−/− or neutrophil-depleted mice, decreased IL-17A production by dLN cells, which might be responsible for T cell activation during a subsequent CHS response. Taken together, these findings suggest that AQP9 is required for the development of sensitization during cutaneous acquired immune responses via regulating neutrophil function. PMID:26489517

  15. Soluble CD40 ligand stimulates CD40-dependent activation of the β2 integrin Mac-1 and protein kinase C zeda (PKCζ) in neutrophils: implications for neutrophil-platelet interactions and neutrophil oxidative burst.

    PubMed

    Jin, Rong; Yu, Shiyong; Song, Zifang; Zhu, Xiaolei; Wang, Cuiping; Yan, Jinchuan; Wu, Fusheng; Nanda, Anil; Granger, D Neil; Li, Guohong

    2013-01-01

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

  16. Characterization of Neutrophil Function in Human Cutaneous Leishmaniasis Caused by Leishmania braziliensis.

    PubMed

    Conceição, Jacilara; Davis, Richard; Carneiro, Pedro Paulo; Giudice, Angela; Muniz, Aline C; Wilson, Mary E; Carvalho, Edgar M; Bacellar, Olívia

    2016-05-01

    Infection with different Leishmania spp. protozoa can lead to a variety of clinical syndromes associated in many cases with inflammatory responses in the skin. Although macrophages harbor the majority of parasites throughout chronic infection, neutrophils are the first inflammatory cells to migrate to the site of infection. Whether neutrophils promote parasite clearance or exacerbate disease in murine models varies depending on the susceptible or resistant status of the host. Based on the hypothesis that neutrophils contribute to a systemic inflammatory state in humans with symptomatic L. braziliensis infection, we evaluated the phenotype of neutrophils from patients with cutaneous leishmaniasis (CL) during the course of L. braziliensis infection. After in vitro infection with L. braziliensis, CL patient neutrophils produced more reactive oxygen species (ROS) and higher levels of CXCL8 and CXCL9, chemokines associated with recruitment of neutrophils and Th1-type cells, than neutrophils from control healthy subjects (HS). Despite this, CL patient and HS neutrophils were equally capable of phagocytosis of L. braziliensis. There was no difference between the degree of activation of neutrophils from CL versus healthy subjects, assessed by CD66b and CD62L expression using flow cytometry. Of interest, these studies revealed that both parasite-infected and bystander neutrophils became activated during incubation with L. braziliensis. The enhanced ROS and chemokine production in neutrophils from CL patients reverted to baseline after treatment of disease. These data suggest that the circulating neutrophils during CL are not necessarily more microbicidal, but they have a more pro-inflammatory profile after parasite restimulation than neutrophils from healthy subjects.

  17. Characterization of Neutrophil Function in Human Cutaneous Leishmaniasis Caused by Leishmania braziliensis

    PubMed Central

    Conceição, Jacilara; Davis, Richard; Carneiro, Pedro Paulo; Giudice, Angela; Muniz, Aline C.; Wilson, Mary E.; Carvalho, Edgar M.; Bacellar, Olívia

    2016-01-01

    Infection with different Leishmania spp. protozoa can lead to a variety of clinical syndromes associated in many cases with inflammatory responses in the skin. Although macrophages harbor the majority of parasites throughout chronic infection, neutrophils are the first inflammatory cells to migrate to the site of infection. Whether neutrophils promote parasite clearance or exacerbate disease in murine models varies depending on the susceptible or resistant status of the host. Based on the hypothesis that neutrophils contribute to a systemic inflammatory state in humans with symptomatic L. braziliensis infection, we evaluated the phenotype of neutrophils from patients with cutaneous leishmaniasis (CL) during the course of L. braziliensis infection. After in vitro infection with L. braziliensis, CL patient neutrophils produced more reactive oxygen species (ROS) and higher levels of CXCL8 and CXCL9, chemokines associated with recruitment of neutrophils and Th1-type cells, than neutrophils from control healthy subjects (HS). Despite this, CL patient and HS neutrophils were equally capable of phagocytosis of L. braziliensis. There was no difference between the degree of activation of neutrophils from CL versus healthy subjects, assessed by CD66b and CD62L expression using flow cytometry. Of interest, these studies revealed that both parasite-infected and bystander neutrophils became activated during incubation with L. braziliensis. The enhanced ROS and chemokine production in neutrophils from CL patients reverted to baseline after treatment of disease. These data suggest that the circulating neutrophils during CL are not necessarily more microbicidal, but they have a more pro-inflammatory profile after parasite restimulation than neutrophils from healthy subjects. PMID:27167379

  18. An L-selectin ligand distinct from P-selectin glycoprotein ligand-1 is expressed on endothelial cells and promotes neutrophil rolling in inflammation.

    PubMed

    Shigeta, Akiko; Matsumoto, Masanori; Tedder, Thomas F; Lowe, John B; Miyasaka, Masayuki; Hirata, Takako

    2008-12-15

    Neutrophils recruited from the blood are key players in the innate immune response. Selectins play critical roles in neutrophil recruitment by mediating their tethering and rolling in inflamed venules. While the roles of P- and E-selectin in this process are well established, the mechanisms of L-selectin-mediated neutrophil recruitment remain elusive. One proposal is that tethering is mediated by L-selectin on flowing neutrophils interacting with P-selectin glycoprotein ligand-1 (PSGL-1) on adherent neutrophils. To clarify whether L-selectin-mediated neutrophil recruitment depends entirely on PSGL-1, we examined the impact of L-selectin deficiency in mice with a PSGL-1-deficient background. L-selectin and PSGL-1 double-knockout mice exhibited a higher increase in their peripheral blood neutrophil count and a worse defect in neutrophil recruitment into the inflamed peritoneum than PSGL-1-deficient mice. Intravital microscopy of inflamed cremaster muscle venules showed that L-selectindeficiency or antibody blockade of L-selectin reduced the residual leukocyte rolling in PSGL-1-deficient mice. Flow cytometric analyses showed that the endothelial cells from the cremaster muscle bound L-selectin in a PSGL-1-independent manner. These results provide evidence for the existence of an L-selectin ligand distinct from PSGL-1 in inflammation and indicate that such a ligand is expressed on endothelial cells, promoting neutrophil rolling in vivo.

  19. An L-selectin ligand distinct from P-selectin glycoprotein ligand-1 is expressed on endothelial cells and promotes neutrophil rolling in inflammation

    PubMed Central

    Shigeta, Akiko; Matsumoto, Masanori; Tedder, Thomas F.; Lowe, John B.; Miyasaka, Masayuki

    2008-01-01

    Neutrophils recruited from the blood are key players in the innate immune response. Selectins play critical roles in neutrophil recruitment by mediating their tethering and rolling in inflamed venules. While the roles of P- and E-selectin in this process are well established, the mechanisms of L-selectin–mediated neutrophil recruitment remain elusive. One proposal is that tethering is mediated by L-selectin on flowing neutrophils interacting with P-selectin glycoprotein ligand-1 (PSGL-1) on adherent neutrophils. To clarify whether L-selectin–mediated neutrophil recruitment depends entirely on PSGL-1, we examined the impact of L-selectin deficiency in mice with a PSGL-1–deficient background. L-selectin and PSGL-1 double-knockout mice exhibited a higher increase in their peripheral blood neutrophil count and a worse defect in neutrophil recruitment into the inflamed peritoneum than PSGL-1–deficient mice. Intravital microscopy of inflamed cremaster muscle venules showed that L-selectindeficiency or antibody blockade of L-selectin reduced the residual leukocyte rolling in PSGL-1–deficient mice. Flow cytometric analyses showed that the endothelial cells from the cremaster muscle bound L-selectin in a PSGL-1–independent manner. These results provide evidence for the existence of an L-selectin ligand distinct from PSGL-1 in inflammation and indicate that such a ligand is expressed on endothelial cells, promoting neutrophil rolling in vivo. PMID:18818390

  20. Neutrophil kinetics in the dog.

    PubMed Central

    Deubelbeiss, K A; Dancey, J T; Harker, L A; Finch, C A

    1975-01-01

    The production of neutrophils in dogs has been estimated from the number of postmitotic neutrophils in the marrow and the transit time of a [3H]-thymidine pulse. The number of postmitotic neutrophils was derived from the erythron iron turnover measurement of erythroid number and the neutrophil-erythroid ratio in bone marrow sections. The mean value for marrow postmitotic neutrophils in dogs was 5.61 plus or minus 0.56 times 10-9 cells/kg. The mean transit time of these neutrophils was calculated to be 82.1 h. A marrow production of 1.65 times 10-9 neutrophils/kg/day was calculated from these data. The turnover of circulating neutrophils was measured by [3H]thymidine and [32P]diisopropylphospho-fluoridate (DF32P) labeling of blood neutrophils. [3H]-Thymidine labeling gave a calculated recovery of 65 per cent, a t1/2 disappearance time of 6.7 h, and a calculated turnover of 1.66 times 10-9 cells/kg/day. Corresponding results with DF32P tagging were 51 per cent, 5.4 h, and 2.89 times 10-9 cells/kg/day. The discrepancy between these two tags persisted in doubly tagged cells and was considered to be due to elution of DF32P. PMID:1120785

  1. Monocytic cell differentiation from band-stage neutrophils under inflammatory conditions via MKK6 activation

    PubMed Central

    Köffel, René; Meshcheryakova, Anastasia; Warszawska, Joanna; Hennig, Annika; Wagner, Karin; Jörgl, Almut; Gubi, Daniela; Moser, Doris; Hladik, Anastasiya; Hoffmann, Ulrike; Fischer, Michael B.; van den Berg, Wim; Koenders, Marije; Scheinecker, Clemens; Gesslbauer, Bernhard; Knapp, Sylvia

    2014-01-01

    During inflammation, neutrophils are rapidly mobilized from the bone marrow storage pool into peripheral blood (PB) to enter lesional sites, where most rapidly undergo apoptosis. Monocytes constitute a second wave of inflammatory immigrates, giving rise to long-lived macrophages and dendritic cell subsets. According to descriptive immunophenotypic and cell culture studies, neutrophils may directly “transdifferentiate” into monocytes/macrophages. We provide mechanistic data in human and murine models supporting the existence of this cellular pathway. First, the inflammatory signal–induced MKK6-p38MAPK cascade activates a monocyte differentiation program in human granulocyte colony-stimulating factor–dependent neutrophils. Second, adoptively transferred neutrophils isolated from G-CSF–pretreated mice rapidly acquired monocyte characteristics in response to inflammatory signals in vivo. Consistently, inflammatory signals led to the recruitment of osteoclast progenitor cell potential from ex vivo–isolated G-CSF–mobilized human blood neutrophils. Monocytic cell differentiation potential was retained in left-shifted band-stage neutrophils but lost in neutrophils from steady-state PB. MKK6-p38MAPK signaling in HL60 model cells led to diminishment of the transcription factor C/EBPα, which enabled the induction of a monocytic cell differentiation program. Gene profiling confirmed lineage conversion from band-stage neutrophils to monocytic cells. Therefore, inflammatory signals relayed by the MKK6-p38MAPK cascade induce monocytic cell differentiation from band-stage neutrophils. PMID:25214442

  2. Apoptotic cell clearance of Leishmania major-infected neutrophils by dendritic cells inhibits CD8+ T-cell priming in vitro by Mer tyrosine kinase-dependent signaling

    PubMed Central

    Ribeiro-Gomes, F L; Romano, A; Lee, S; Roffê, E; Peters, N C; Debrabant, A; Sacks, D

    2015-01-01

    Neutrophils are the predominant recruited and infected cells during the early stages of Leishmania major infection in the skin, and depletion of neutrophils promotes immunity to infection transmitted by sand fly bite. In order to better understand how the acute neutrophilic response suppresses immunity, we assessed the consequences of the interaction between neutrophils recovered from the skin-inoculation site and bone marrow-derived dendritic cells (DCs) in vitro. The capture of infected, apoptotic neutrophils by the DCs completely inhibited their cross-presentation function that was dependent on engagement of the receptor tyrosine kinase Mer on the DCs. The capture of uninfected neutrophils, or neutrophils infected with Toxoplasma gondii, had only slight immunomodulatory effects. These studies define the clearance of infected, apoptotic neutrophils by DCs and Mer receptor signaling as central to the early immune evasion strategies of L. major, with relevance to other vector-borne pathogens delivered by bite to the skin. PMID:26658192

  3. Neutrophil-mediated protection of cultured human vascular endothelial cells from damage by growing Candida albicans hyphae

    SciTech Connect

    Edwards, J.E. Jr.; Rotrosen, D.; Fontaine, J.W.; Haudenschild, C.C.; Diamond, R.D.

    1987-05-01

    Interactions were studied between human neutrophils and cultured human umbilical vein endothelial cells invaded by Candida albicans. In the absence of neutrophils, progressive Candida germination and hyphal growth extensively damaged endothelial cell monolayers over a period of 4 to 6 hours, as determined both by morphological changes and release of /sup 51/Cr from radiolabeled endothelial cells. Monolayers were completely destroyed and replaced by hyphae after 18 hours of incubation. In contrast, when added 2 hours after the monolayers had been infected with Candida, neutrophils selectively migrated toward and attached to hyphae at points of hyphal penetration into individual endothelial cells (observed by time-lapse video-microscopy). Attached neutrophils spread over hyphal surfaces both within and beneath the endothelial cells; neutrophil recruitment to initial sites of leukocyte-Candida-endothelial cell interactions continued throughout the first 60 minutes of observation. Neutrophil spreading and stasis were observed only along Candida hyphae and at sites of Candida-endothelial cell interactions. These events resulted in 58.0% killing of Candida at 2 hours and subsequent clearance of Candida from endothelial cell monolayers, as determined by microcolony counts and morphological observation. On introduction of additional neutrophils to yield higher ratios of neutrophils to endothelial cells (10 neutrophils:1 endothelial cell), neutrophil migration toward hyphal elements continued. Despite retraction or displacement of occasional endothelial cells by invading Candida and neutrophils, most endothelial cells remained intact, viable, and motile as verified both by morphological observations and measurement of /sup 51/Cr release from radiolabeled monolayers.

  4. Oral Neutrophil Transcriptome Changes Result in a Pro-Survival Phenotype in Periodontal Diseases

    PubMed Central

    Lakschevitz, Flavia S.; Aboodi, Guy M.; Glogauer, Michael

    2013-01-01

    Background Periodontal diseases are inflammatory processes that occur following the influx of neutrophils into the periodontal tissues in response to the subgingival bacterial biofilm. Current literature suggests that while neutrophils are protective and prevent bacterial infections, they also appear to contribute to damage of the periodontal tissues. In the present study we compare the gene expression profile changes in neutrophils as they migrate from the circulation into the oral tissues in patients with chronic periodontits and matched healthy subjects. We hypothesized that oral neutrophils in periodontal disease patients will display a disease specific transcriptome that differs from the oral neutrophil of healthy subjects. Methods Venous blood and oral rinse samples were obtained from healthy subjects and chronic periodontitis patients for neutrophil isolation. mRNA was isolated from the neutrophils, and gene expression microarray analysis was completed. Results were confirmed for specific genes of interest by qRT-PCR and Western Blot analysis. Results and Discussion Chronic periodontitis patients presented with increased recruitment of neutrophils to the oral cavity. Gene expression analysis revealed differences in the expression levels of genes from several biological pathways. Using hierarchical clustering analysis, we found that the apoptosis network was significantly altered in patients with chronic inflammation in the oral cavity, with up-regulation of pro-survival members of the Bcl-2 family and down-regulation of pro-apoptosis members in the same compartment. Additional functional analysis confirmed that the percentages of viable neutrophils are significantly increased in the oral cavity of chronic periodontitis patients. Conclusions Oral neutrophils from patients with periodontal disease displayed an altered transcriptome following migration into the oral tissues. This resulted in a pro-survival neutrophil phenotype in chronic periodontitis patients

  5. Marine Natural Product Inhibitors of Neutrophil-Associated Inflammation.

    PubMed

    Chen, Chun-Yu; Tsai, Yung-Fong; Chang, Wen-Yi; Yang, Shun-Chin; Hwang, Tsong-Long

    2016-01-01

    Neutrophils are widely recognized to play an important role in acute inflammatory responses, and recent evidence has expanded their role to modulating chronic inflammatory and autoimmune diseases. Reactive oxygen species (ROS) and microbicidal compounds released from neutrophils that are recruited to the site of inflammation contribute to the pathogenesis of multiple inflammation-associated diseases such as chronic obstructive pulmonary disease, atherosclerosis, and hepatitis. Marine organisms are a valuable source of bioactive compounds with potential for industrial and pharmaceutical application. Marine natural products that inhibit neutrophil activation could be used as drugs for the treatment of inflammatory diseases. Numerous studies investigating marine natural products have reported novel anti-inflammatory agents. Nevertheless, the detailed mechanisms underlying their actions, which could facilitate our understanding of the molecular events occurring in neutrophils, have not been reported in most of the associated research studies. Therefore, in this review, we will present marine products that inhibit neutrophil-associated inflammation. Furthermore, we will be limiting the detailed discussion to agents with well-investigated molecular targets. PMID:27472345

  6. Marine Natural Product Inhibitors of Neutrophil-Associated Inflammation

    PubMed Central

    Chen, Chun-Yu; Tsai, Yung-Fong; Chang, Wen-Yi; Yang, Shun-Chin; Hwang, Tsong-Long

    2016-01-01

    Neutrophils are widely recognized to play an important role in acute inflammatory responses, and recent evidence has expanded their role to modulating chronic inflammatory and autoimmune diseases. Reactive oxygen species (ROS) and microbicidal compounds released from neutrophils that are recruited to the site of inflammation contribute to the pathogenesis of multiple inflammation-associated diseases such as chronic obstructive pulmonary disease, atherosclerosis, and hepatitis. Marine organisms are a valuable source of bioactive compounds with potential for industrial and pharmaceutical application. Marine natural products that inhibit neutrophil activation could be used as drugs for the treatment of inflammatory diseases. Numerous studies investigating marine natural products have reported novel anti-inflammatory agents. Nevertheless, the detailed mechanisms underlying their actions, which could facilitate our understanding of the molecular events occurring in neutrophils, have not been reported in most of the associated research studies. Therefore, in this review, we will present marine products that inhibit neutrophil-associated inflammation. Furthermore, we will be limiting the detailed discussion to agents with well-investigated molecular targets. PMID:27472345

  7. Niacin inhibits carrageenan-induced neutrophil migration in mice.

    PubMed

    Ferreira, Raphael Gomes; Matsui, Tamires Cardoso; Gomides, Lindisley Ferreira; Godin, Adriana Martins; Menezes, Gustavo Batista; de Matos Coelho, Márcio; Klein, André

    2013-06-01

    Several emerging lines of evidence support an anti-inflammatory role for nicotinic acid (niacin); however, its role in the regulation of leukocyte migration in response to inflammatory stimuli has not been elucidated until now. Herein, we have examined the effect of nicotinic acid on neutrophil recruitment in experimentally induced inflammation. We demonstrated that nicotinic acid treatment inhibited interleukin (IL)-8-induced, leukotriene (LT)B4-induced, and carrageenan-induced neutrophil migration into the pleural cavity of BALB/c mice and reduced neutrophil rolling and adherence in a mouse cremaster muscle preparation. Surprisingly, nicotinic acid treatment increased the level of the neutrophil chemoattractant KC in response to carrageenan. These results suggest that nicotinic acid plays an important role in the regulation of inflammation due to its ability to inhibit the actions of the neutrophil chemoattractants IL-8 and LTB4. Further inhibition of chemoattractants leads to impairment of leukocyte rolling and adherence to the vascular endothelium in the microcirculation of inflamed tissues.

  8. The axonal repellent, Slit2, inhibits directional migration of circulating neutrophils.

    PubMed

    Tole, Soumitra; Mukovozov, Ilya M; Huang, Yi-Wei; Magalhaes, Marco A O; Yan, Ming; Crow, Min Rui; Liu, Guang Ying; Sun, Chun Xiang; Durocher, Yves; Glogauer, Michael; Robinson, Lisa A

    2009-12-01

    In inflammatory diseases, circulating neutrophils are recruited to sites of injury. Attractant signals are provided by many different chemotactic molecules, such that blockade of one may not prevent neutrophil recruitment effectively. The Slit family of secreted proteins and their transmembrane receptor, Robo, repel axonal migration during CNS development. Emerging evidence shows that by inhibiting the activation of Rho-family GTPases, Slit2/Robo also inhibit migration of other cell types toward a variety of chemotactic factors in vitro and in vivo. The role of Slit2 in inflammation, however, has been largely unexplored. We isolated primary neutrophils from human peripheral blood and mouse bone marrow and detected Robo-1 expression. Using video-microscopic live cell tracking, we found that Slit2 selectively impaired directional migration but not random movement of neutrophils toward fMLP. Slit2 also inhibited neutrophil migration toward other chemoattractants, namely C5a and IL-8. Slit2 inhibited neutrophil chemotaxis by preventing chemoattractant-induced actin barbed end formation and cell polarization. Slit2 mediated these effects by suppressing inducible activation of Cdc42 and Rac2 but did not impair activation of other major kinase pathways involved in neutrophil migration. We further tested the effects of Slit2 in vivo using mouse models of peritoneal inflammation induced by sodium periodate, C5a, and MIP-2. In all instances, Slit2 reduced neutrophil recruitment effectively (P<0.01). Collectively, these data demonstrate that Slit2 potently inhibits chemotaxis but not random motion of circulating neutrophils and point to Slit2 as a potential new therapeutic for preventing localized inflammation.

  9. Neutrophil swarms require LTB4 and integrins at sites of cell death in vivo

    PubMed Central

    Lämmermann, Tim; Afonso, Philippe V.; Angermann, Bastian R.; Wang, Ji Ming; Kastenmüller, Wolfgang; Parent, Carole A.; Germain, Ronald N.

    2013-01-01

    Neutrophil recruitment from blood to extravascular sites of sterile or infectious tissue damage is a hallmark of early innate immune responses, and the molecular events leading to cell exit from the bloodstream have been well defined1,2. Once outside the vessel, individual neutrophils often show extremely coordinated chemotaxis and cluster formation reminiscent of the swarming behaviour of insects3–11. The molecular players that direct this response at the single-cell and population levels within the complexity of an inflamed tissue are unknown. Using two-photon intravital microscopy in mouse models of sterile injury and infection, we show a critical role for intercellular signal relay among neutrophils mediated by the lipid leukotriene B4, which acutely amplifies local cell death signals to enhance the radius of highly directed interstitial neutrophil recruitment. Integrin receptors are dispensable for long-distance migration12, but have a previously unappreciated role in maintaining dense cellular clusters when congregating neutrophils rearrange the collagenous fibre network of the dermis to form a collagen-free zone at the wound centre. In this newly formed environment, integrins, in concert with neutrophil-derived leukotriene B4 and other chemoattractants, promote local neutrophil interaction while forming a tight wound seal. This wound seal has borders that cease to grow in kinetic concert with late recruitment of monocytes and macrophages at the edge of the displaced collagen fibres. Together, these data provide an initial molecular map of the factors that contribute to neutrophil swarming in the extravascular space of a damaged tissue. They reveal how local events are propagated over large-range distances, and how auto-signalling produces coordinated, self-organized neutrophil-swarming behaviour that isolates the wound or infectious site from surrounding viable tissue. PMID:23708969

  10. Adipocytes and Neutrophils Give a Helping Hand to Pancreatic Cancers.

    PubMed

    Bronte, Vincenzo; Tortora, Giampaolo

    2016-08-01

    Obesity-induced inflammation can build up a confined microenvironment in pancreatic adenocarcinoma that is associated with increased desmoplasia, neutrophil recruitment, reduced delivery of chemotherapeutic drugs, and immune evasion. Targeting molecular pathways empowering this circuit might represent a necessary measure to reach clinical efficacy for combination therapies in patients with excess body weight. Cancer Discov; 6(8); 821-3. ©2016 AACR.See related article by Incio et al., p. 852.

  11. Targeted deletion of tumor suppressor PTEN augments neutrophil function and enhances host defense in neutropenia-associated pneumonia

    PubMed Central

    Li, Yitang; Jia, Yonghui; Pichavant, Muriel; Loison, Fabien; Sarraj, Bara; Kasorn, Anongnard; You, Jian; Robson, Bryanne E.; Umetsu, Dale T.; Mizgerd, Joseph P.; Ye, Keqiang

    2009-01-01

    Neutropenia and related infections are the most important dose-limiting toxicities in anticancer chemotherapy and radiotherapy. In this study, we explored a new strategy for augmenting host defense in neutropenia-related pneumonia. Phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) signaling in neutrophils was elevated by depleting PTEN, a phosphatidylinositol 3′-phosphatase that hydrolyzes PtdIns(3,4,5)P3. In myeloid-specific PTEN knockout mice, significantly more neutrophils were recruited to the inflamed lungs during neutropenia-associated pneumonia. Using an adoptive transfer technique, we demonstrated that this enhancement could be caused directly by PTEN depletion in neutrophils. In addition, disruption of PTEN increased the recruitment of macrophages and elevated proinflammatory cytokines/chemokine levels in the inflamed lungs, which could also be responsible for the enhanced neutrophil recruitment. Depleting PTEN also significantly delayed apoptosis and enhanced the bacteria-killing capability of the recruited neutrophils. Finally, we provide direct evidence that enhancement of neutrophil function by elevating PtdIns(3,4,5)P3 signaling can alleviate pneumonia-associated lung damage and decrease pneumonia-elicited mortality. Collectively, these results not only provide insight into the mechanism of action of PTEN and PtdIns(3,4,5)P3 signaling pathway in modulating neutrophil function during lung infection and inflammation, but they also establish PTEN and related pathways as potential therapeutic targets for treating neutropenia-associated pneumonia. PMID:19286998

  12. Serum and glucocorticoid-regulated kinase 1 regulates neutrophil clearance during inflammation resolution.

    PubMed

    Burgon, Joseph; Robertson, Anne L; Sadiku, Pranvera; Wang, Xingang; Hooper-Greenhill, Edward; Prince, Lynne R; Walker, Paul; Hoggett, Emily E; Ward, Jonathan R; Farrow, Stuart N; Zuercher, William J; Jeffrey, Philip; Savage, Caroline O; Ingham, Philip W; Hurlstone, Adam F; Whyte, Moira K B; Renshaw, Stephen A

    2014-02-15

    The inflammatory response is integral to maintaining health by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralize invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein serum and glucocorticoid-regulated kinase 1 (SGK1). We have characterized the expression patterns and regulation of SGK family members in human neutrophils and shown that inhibition of SGK activity completely abrogates the antiapoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signaling and thus may prove a valuable therapeutic target for the treatment of inflammatory disease.

  13. Serum and Glucocorticoid Regulated Kinase 1 (SGK1) Regulates Neutrophil Clearance During Inflammation Resolution

    PubMed Central

    Burgon, Joseph; Robertson, Anne L.; Sadiku, Pranvera; Wang, Xingang; Hooper-Greenhill, Edward; Prince, Lynne R.; Walker, Paul; Hoggett, Emily E.; Ward, Jonathan R.; Farrow, Stuart N.; Zuercher, William J.; Jeffrey, Philip; Savage, Caroline O.; Ingham, Philip W.; Hurlstone, Adam F.; Whyte, Moira K. B.; Renshaw, Stephen A.

    2013-01-01

    The inflammatory response is integral to maintaining health, by functioning to resist microbial infection and repair tissue damage. Large numbers of neutrophils are recruited to inflammatory sites to neutralise invading bacteria through phagocytosis and the release of proteases and reactive oxygen species into the extracellular environment. Removal of the original inflammatory stimulus must be accompanied by resolution of the inflammatory response, including neutrophil clearance, to prevent inadvertent tissue damage. Neutrophil apoptosis and its temporary inhibition by survival signals provides a target for anti-inflammatory therapeutics, making it essential to better understand this process. GM-CSF, a neutrophil survival factor, causes a significant increase in mRNA levels for the known anti-apoptotic protein Serum and Glucocorticoid Regulated Kinase 1 (SGK1). We have characterised the expression patterns and regulation of SGK family members in human neutrophils, and shown that inhibition of SGK activity completely abrogates the anti-apoptotic effect of GM-CSF. Using a transgenic zebrafish model, we have disrupted sgk1 gene function and shown this specifically delays inflammation resolution, without altering neutrophil recruitment to inflammatory sites in vivo. These data suggest SGK1 plays a key role in regulating neutrophil survival signalling, and thus may prove a valuable therapeutic target for the treatment of inflammatory disease. PMID:24431232

  14. Neutrophil swarming toward Cryptococcus neoformans is mediated by complement and leukotriene B4.

    PubMed

    Sun, Donglei; Shi, Meiqing

    2016-09-01

    Swarming behavior of neutrophils has been noticed in both sterile injury and infection models and the mechanisms are being unveiled. So far, no in vitro model has been established to study neutrophil swarming to microbes. In the current study, using live-cell imaging, we observed in vitro neutrophil swarming toward Cryptococcus neoformans, a fungal pathogen causing human meningoencephalitis. Complement C3 and CD11b expression are essential for neutrophils to form cell swarms surrounding C. neoformans. Leukotriene B4 (LTB4) was quickly released by neutrophils during their interactions with C. neoformans. Blockade of LTB4 synthesis inhibited the swarming response to C. neoformans. Importantly, blockade of LTB4 synthesis also significantly reduced neutrophil recruitment in the lung vasculature of mice infected intravenously with C. neoformans, demonstrating a critical role of LTB4 in intravascular neutrophil swarming during infection. Together, this is the first report of neutrophil dynamics of swarming toward a microorganism in vitro, mediated by complement and LTB4. PMID:27402276

  15. Multifaceted effects of Francisella tularensis on human neutrophil function and lifespan.

    PubMed

    Kinkead, Lauren C; Allen, Lee-Ann H

    2016-09-01

    Francisella tularensis in an intracellular bacterial pathogen that causes a potentially lethal disease called tularemia. Studies performed nearly 100 years ago revealed that neutrophil accumulation in infected tissues correlates directly with the extent of necrotic damage during F. tularensis infection. However, the dynamics and details of bacteria-neutrophil interactions have only recently been studied in detail. Herein, we review current understanding regarding the mechanisms that recruit neutrophils to F. tularensis-infected lungs, opsonization and phagocytosis, evasion and inhibition of neutrophil defense mechanisms, as well as the ability of F. tularensis to prolong neutrophil lifespan. In addition, we discuss distinctive features of the bacterium, including its ability to act at a distance to alter overall neutrophil responsiveness to exogenous stimuli, and the evidence which suggests that macrophages and neutrophils play distinct roles in tularemia pathogenesis, such that macrophages are major vehicles for intracellular growth and dissemination, whereas neutrophils drive tissue destruction by dysregulation of the inflammatory response. PMID:27558340

  16. Neutrophil-derived IL-1β is sufficient for abscess formation in immunity against Staphylococcus aureus in mice.

    PubMed

    Cho, John S; Guo, Yi; Ramos, Romela Irene; Hebroni, Frank; Plaisier, Seema B; Xuan, Caiyun; Granick, Jennifer L; Matsushima, Hironori; Takashima, Akira; Iwakura, Yoichiro; Cheung, Ambrose L; Cheng, Genhong; Lee, Delphine J; Simon, Scott I; Miller, Lloyd S

    2012-01-01

    Neutrophil abscess formation is critical in innate immunity against many pathogens. Here, the mechanism of neutrophil abscess formation was investigated using a mouse model of Staphylococcus aureus cutaneous infection. Gene expression analysis and in vivo multispectral noninvasive imaging during the S. aureus infection revealed a strong functional and temporal association between neutrophil recruitment and IL-1β/IL-1R activation. Unexpectedly, neutrophils but not monocytes/macrophages or other MHCII-expressing antigen presenting cells were the predominant source of IL-1β at the site of infection. Furthermore, neutrophil-derived IL-1β was essential for host defense since adoptive transfer of IL-1β-expressing neutrophils was sufficient to restore the impaired neutrophil abscess formation in S. aureus-infected IL-1β-deficient mice. S. aureus-induced IL-1β production by neutrophils required TLR2, NOD2, FPR1 and the ASC/NLRP3 inflammasome in an α-toxin-dependent mechanism. Taken together, IL-1β and neutrophil abscess formation during an infection are functionally, temporally and spatially linked as a consequence of direct IL-1β production by neutrophils.

  17. A Critical Role for P2X7 Receptor-Induced VCAM-1 Shedding and Neutrophil Infiltration during Acute Lung Injury.

    PubMed

    Mishra, Amarjit; Guo, Yujie; Zhang, Li; More, Sunil; Weng, Tingting; Chintagari, Narendranath Reddy; Huang, Chaoqun; Liang, Yurong; Pushparaj, Samuel; Gou, Deming; Breshears, Melanie; Liu, Lin

    2016-10-01

    Pulmonary neutrophils are the initial inflammatory cells that are recruited during lung injury and are crucial for innate immunity. However, pathological recruitment of neutrophils results in lung injury. The objective of this study is to determine whether the novel neutrophil chemoattractant, soluble VCAM-1 (sVCAM-1), recruits pathological levels of neutrophils to injury sites and amplifies lung inflammation during acute lung injury. The mice with P2X7 receptor deficiency, or treated with a P2X7 receptor inhibitor or anti-VCAM-1 Abs, were subjected to a clinically relevant two-hit LPS and mechanical ventilation-induced acute lung injury. Neutrophil infiltration and lung inflammation were measured. Neutrophil chemotactic activities were determined by a chemotaxis assay. VCAM-1 shedding and signaling pathways were assessed in isolated lung epithelial cells. Ab neutralization of sVCAM-1 or deficiency or antagonism of P2X7R reduced neutrophil infiltration and proinflammatory cytokine levels. The ligands for sVCAM-1 were increased during acute lung injury. sVCAM-1 had neutrophil chemotactic activities and activated alveolar macrophages. VCAM-1 is released into the alveolar airspace from alveolar epithelial type I cells through P2X7 receptor-mediated activation of the metalloproteinase ADAM-17. In conclusion, sVCAM-1 is a novel chemoattractant for neutrophils and an activator for alveolar macrophages. Targeting sVCAM-1 provides a therapeutic intervention that could block pathological neutrophil recruitment, without interfering with the physiological recruitment of neutrophils, thus avoiding the impairment of host defenses. PMID:27559050

  18. Neutrophil Elastase Inhibitors

    PubMed Central

    Groutas, William C.; Dou, Dengfeng; Alliston, Kevin R.

    2011-01-01

    Introduction Chronic obstructive pulmonary disease (COPD) constitutes a worldwide health problem. There is currently an urgent and unmet need for the development of small molecule therapeutics capable of blocking and/or reversing the progression of the disorder. Recent studies have greatly illuminated our understanding of the multiple pathogenic processes associated with COPD. Of paramount importance is the key role played by proteases, oxidative stress, apoptosis, and inflammation. Insights gained from these studies have made possible the exploration of new therapeutic approaches. Areas covered An overview of major developments in COPD research with emphasis on low molecular weight neutrophil elastase inhibitors is described in this review. Expert opinion Great strides have been made toward our understanding of the biochemical and cellular events associated with COPD. However, our knowledge regarding the inter-relationships among the multiple pathogenic mechanisms and their mediators involved is till limited. The problem is further compounded by the unavailability of suitable validated biomarkers for assessing the efficacy of potential therapeutic interventions. The complexity of COPD suggests that effective therapeutic interventions may require the administration of more than one agent such as, for instance, an HNE or MMP-12 inhibitor with an anti-inflammatory agent such as a phosphodiesterase-4 inhibitor, or a dual function agent capable of disrupting the cycle of proteolysis, apoptosis, inflammation and oxidative stress PMID:21235378

  19. AUTOINFLAMMATORY PUSTULAR NEUTROPHILIC DISEASES

    PubMed Central

    Naik, Haley B.; Cowen, Edward W.

    2013-01-01

    SYNOPSIS The inflammatory pustular dermatoses constitute a spectrum of non-infectious conditions ranging from localized involvement to generalized disease with associated acute systemic inflammation and multi-organ involvement. Despite the variability in extent and severity of cutaneous presentation, each of these diseases is characterized by non-infectious neutrophilic intra-epidermal microabscesses. Many share systemic findings including fever, elevated inflammatory markers, inflammatory bowel disease and/or osteoarticular involvement, suggesting potential common pathogenic links (Figure 1). The recent discoveries of several genes responsible for heritable pustular diseases have revealed a distinct link between pustular skin disease and regulation of innate immunity. These genetic advances have led to a deeper exploration of common pathways in pustular skin disease and offer the potential for a new era of biologic therapy which targets these shared pathways. This chapter provides a new categorization of inflammatory pustular dermatoses in the context of recent genetic and biologic insights. We will discuss recently-described monogenic diseases with pustular phenotypes, including deficiency of IL-1 receptor antagonist (DIRA), deficiency of the IL-36 receptor antagonist (DITRA), CARD14-associated pustular psoriasis (CAMPS), and pyogenic arthritis, pyoderma gangrenosum, acne (PAPA). We will then discuss how these new genetic advancements may inform how we view previously described pustular diseases, including pustular psoriasis and its clinical variants, with a focus on historical classification by clinical phenotype. PMID:23827244

  20. Neutrophils rapidly transit inflamed lymphatic vessel endothelium via integrin-dependent proteolysis and lipoxin-induced junctional retraction.

    PubMed

    Rigby, David A; Ferguson, David J P; Johnson, Louise A; Jackson, David G

    2015-12-01

    Neutrophils are the first leukocyte population to be recruited from the circulation following tissue injury or infection, where they play key roles in host defense. However, recent evidence indicates recruited neutrophils can also enter lymph and shape adaptive immune responses downstream in draining lymph nodes. At present, the cellular mechanisms regulating neutrophil entry to lymphatic vessels and migration to lymph nodes are largely unknown. Here, we have investigated these events in an in vivo mouse Mycobacterium bovis bacillus Calmette-Guérin vaccination model, ex vivo mouse dermal explants, and in vitro Transwell system comprising monolayers of primary human dermal lymphatic endothelial cells. We demonstrate that neutrophils are reliant on endothelial activation for adhesion, initially via E-selectin and subsequently, by integrin-mediated binding to ICAM-1 and VCAM-1, combined with CXCL8-dependent chemotaxis. Moreover, we reveal that integrin-mediated neutrophil adhesion plays a pivotal role in subsequent transmigration by focusing the action of matrix metalloproteinases and the 15-lipoxygenase-1-derived chemorepellent 12(S)-hydroxyeicosatetraenoic acid at neutrophil:endothelial contact sites to induce transient endothelial junctional retraction and rapid, selective neutrophil trafficking. These findings reveal an unexpectedly intimate collaboration between neutrophils and the lymphatic vessel endothelium, in which these phagocytic leukocytes act as pathfinders for their own transit during inflammation.

  1. TRPC6 regulates CXCR2-mediated chemotaxis of murine neutrophils.

    PubMed

    Lindemann, Otto; Umlauf, Daniel; Frank, Svetlana; Schimmelpfennig, Sandra; Bertrand, Jessica; Pap, Thomas; Hanley, Peter J; Fabian, Anke; Dietrich, Alexander; Schwab, Albrecht

    2013-06-01

    Unraveling the mechanisms involved in chemotactic navigation of immune cells is of particular interest for the development of new immunoregulatory therapies. It is generally agreed upon that members of the classical transient receptor potential channel family (TRPC) are involved in chemotaxis. However, the regulatory role of TRPC channels in chemoattractant receptor-mediated signaling has not yet been clarified in detail. In this study, we demonstrate that the TRPC6 channels play a pronounced role in CXCR2-mediated intermediary chemotaxis, whereas N-formyl-methionine-leucine-phenylalanine receptor-mediated end-target chemotaxis is TRPC6 independent. The knockout of TRPC6 channels in murine neutrophils led to a strongly impaired intermediary chemotaxis after CXCR2 activation which is not further reinforced by CXCR2, PI3K, or p38 MAPK inhibition. Furthermore, CXCR2-mediated Ca(2+) influx but not Ca(2+) store release was attenuated in TRPC6(-/-) neutrophils. We demonstrate that the TRPC6 deficiency affected phosphorylation of AKT and MAPK downstream of CXCR2 receptor activation and led to altered remodeling of actin. The relevance of this TRPC6-depending defect in neutrophil chemotaxis is underscored by our in vivo findings. A nonseptic peritoneal inflammation revealed an attenuated recruitment of neutrophils in the peritoneal cavity of TRPC6(-/-) mice. In summary, this paper defines a specific role of TRPC6 channels in CXCR2-induced intermediary chemotaxis. In particular, TRPC6-mediated supply of calcium appears to be critical for activation of downstream signaling components.

  2. Neutrophil attractant/activating peptide-1/interleukin-8 in term and preterm parturition.

    PubMed

    Romero, R; Ceska, M; Avila, C; Mazor, M; Behnke, E; Lindley, I

    1991-10-01

    The neutrophil is the leukocyte most frequently recruited into the amniotic fluid in cases of microbial invasion of the amniotic cavity. Neutrophil attractant/activating peptide-1/interleukin-8 is a newly identified cytokine that is capable of inducing selective neutrophil chemotaxis and activation. The purpose of this study was to examine the relationship between amniotic fluid concentrations of neutrophil attractant/activating peptide-1/interleukin-8, microbial invasion of the amniotic cavity, and parturition (term and preterm). Amniotic fluid neutrophil attractant/activating peptide-1/interleukin-8 was measured with an immunoassay validated for human amniotic fluid (sensitivity 0.3 ng/ml). Fluid was obtained from women in the following groups: midtrimester (n = 38), term not in labor (n = 38), term in active labor (n = 67), and preterm labor with intact membranes (n = 62). Fluid was cultured for aerobic and anaerobic bacterial and Mycoplasma. Sterile amniotic fluid from most women in the midtrimester of pregnancy and women at term not in labor did not contain immunoreactive neutrophil attractant/activating peptide-1/interleukin-8. Microbial invasion of the amniotic cavity was associated with increased concentrations of neutrophil attractant/activating peptide-1/interleukin-8. The amniotic fluid of women with preterm labor and sterile amniotic fluid who had preterm delivery contained higher neutrophil attractant/activating peptide-1/interleukin-8 levels than did the amniotic fluid of women who responded to tocolysis and had delivery at term. Term parturition is associated with increased concentrations of neutrophil attractant/activating peptide-1/interleukin-8 in the amniotic fluid. We conclude that neutrophil attractant/activating peptide-1/interleukin-8 is part of the host response to microbial invasion of the amniotic cavity and that increased amniotic fluid availability of this cytokine occurs in term and preterm parturition. PMID:1951537

  3. Role of osteopontin in hepatic neutrophil infiltration during alcoholic steatohepatitis

    SciTech Connect

    Apte, Udayan M.; Banerjee, Atrayee; McRee, Rachel; Wellberg, Elizabeth; Ramaiah, Shashi K. . E-mail: sramaiah@cvm.tamu.edu

    2005-08-22

    Alcoholic liver disease (ALD) is a major complication of heavy alcohol (EtOH) drinking and is characterized by three progressive stages of pathology: steatosis, steatohepatitis, and fibrosis/cirrhosis. Alcoholic steatosis (AS) is the initial stage of ALD and consists of fat accumulation in the liver accompanied by minimal liver injury. AS is known to render the hepatocytes increasingly sensitive to toxicants such as bacterial endotoxin (LPS). Alcoholic steatohepatitis (ASH), the second and rate-limiting step in the progression of ALD, is characterized by hepatic fat accumulation, neutrophil infiltration, and neutrophil-mediated parenchymal injury. However, the pathogenesis of ASH is poorly defined. It has been theorized that the pathogenesis of ASH involves interaction of increased circulating levels of LPS with hepatocytes being rendered highly sensitive to LPS due to heavy EtOH consumption. We hypothesize that osteopontin (OPN), a matricellular protein (MCP), plays an important role in the hepatic neutrophil recruitment due to its enhanced expression during the early phase of ALD (AS and ASH). To study the role of OPN in the pathogenesis of ASH, we induced AS in male Sprague-Dawley rats by feeding EtOH-containing Lieber-DeCarli liquid diet for 6 weeks. AS rats experienced extensive fat accumulation and minimal liver injury. Moderate induction in OPN was observed in AS group. ASH was induced by feeding male Sprague-Dawley rats EtOH-containing Lieber-DeCarli liquid diet for 6 weeks followed by LPS injection. The ASH rats had substantial neutrophil infiltration, coagulative oncotic necrosis, and developed higher liver injury. Significant increases in the hepatic and circulating levels of OPN was observed in the ASH rats. Higher levels of the active, thrombin-cleaved form of OPN in the liver in ASH group correlated remarkably with hepatic neutrophil infiltration. Finally, correlative studies between OPN and hepatic neutrophil infiltration was corroborated in a simple

  4. Do neutrophil extracellular traps contribute to the heightened risk of thrombosis in inflammatory diseases?

    PubMed Central

    Rao, Ashish N; Kazzaz, Nayef M; Knight, Jason S

    2015-01-01

    Thrombotic events, both arterial and venous, are a major health concern worldwide. Further, autoimmune diseases, such as systemic lupus erythematosus, anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis, and antiphospholipid syndrome, predispose to thrombosis, and thereby push the risk for these morbid events even higher. In recent years, neutrophils have been identified as important players in both arterial and venous thrombosis. Specifically, chromatin-based structures called neutrophil extracellular traps (NETs) play a key role in activating the coagulation cascade, recruiting platelets, and serving as scaffolding upon which the thrombus can be assembled. At the same time, neutrophils and NETs are emerging as important mediators of pathogenic inflammation in the aforementioned autoimmune diseases. Here, we first review the general role of NETs in thrombosis. We then posit that exaggerated NET release contributes to the prothrombotic diatheses of systemic lupus erythematosus, ANCA-associated vasculitis, and antiphospholipid syndrome. PMID:26730289

  5. Neutrophil Crawling in Capillaries; A Novel Immune Response to Staphylococcus aureus

    PubMed Central

    Harding, Mark Geoffrey; Zhang, Kunyan; Conly, John; Kubes, Paul

    2014-01-01

    Methicillin-resistant Staphylococcus aureus (MRSA), particularly the USA300 strain, is a highly virulent pathogen responsible for an increasing number of skin and soft tissue infections globally. Furthermore, MRSA-induced soft tissue infections can rapidly progress into life-threatening conditions, such as sepsis and necrotizing fasciitis. The importance of neutrophils in these devastating soft tissue infections remains ambiguous, partly because of our incomplete understanding of their behaviour. Spinning disk confocal microscopy was used to visualize the behaviour of GR1-labelled neutrophils in subcutaneous tissue in response to GFP-expressing MRSA attached to a foreign particle (agarose bead). We observed significant directional neutrophil recruitment towards the S. aureus agarose bead but not a control agarose bead. A significant increase in neutrophil crawling within the capillaries surrounding the infectious nidus was noted, with impaired capillary perfusion in these vessels and increased parenchymal cell death. No neutrophils were able to emigrate from capillaries. The crawling within these capillaries was mediated by the β2 and α4 integrins and blocking these integrins 2 hours post infection eliminated neutrophil crawling, improved capillary perfusion, reduced cell death and reduced lesion size. Blocking prior to infection increased pathology. Neutrophil crawling within capillaries during MRSA soft tissue infections, while potentially contributing to walling off or preventing early dissemination of the pathogen, resulted in impaired perfusion and increased tissue injury with time. PMID:25299673

  6. BLT1 signalling protects the liver against acetaminophen hepatotoxicity by preventing excessive accumulation of hepatic neutrophils.

    PubMed

    Kojo, Ken; Ito, Yoshiya; Eshima, Koji; Nishizawa, Nobuyuki; Ohkubo, Hirotoki; Yokomizo, Takehiko; Shimizu, Takao; Watanabe, Masahiko; Majima, Masataka

    2016-01-01

    Leukotriene B4 (LTB4) is a potent chemoattractant for neutrophils. Signalling of LTB4 receptor type 1 (BLT1) has pro-inflammatory functions through neutrophil recruitment. In this study, we investigated whether BLT1 signalling plays a role in acetaminophen (APAP)-induced liver injury by affecting inflammatory responses including the accumulation of hepatic neutrophils. BLT1-knockout (BLT1(-/-)) mice and their wild-type (WT) counterparts were subjected to a single APAP overdose (300 mg/kg), and various parameters compared within 24 h after treatment. Compared with WT mice, BLT1(-/-) mice exhibited exacerbation of APAP-induced liver injury as evidenced by enhancement of alanine aminotransferase level, necrotic area, hepatic neutrophil accumulation, and expression of cytokines and chemokines. WT mice co-treated with APAP and ONO-0457, a specific antagonist for BLT1, displayed amplification of the injury, and similar results to those observed in BLT1(-/-) mice. Hepatic neutrophils in BLT1(-/-) mice during APAP hepatotoxicity showed increases in the production of reactive oxygen species and matrix metalloproteinase-9. Administration of isolated BLT1-deficient neutrophils into WT mice aggravated the liver injury elicited by APAP. These results demonstrate that BLT1 signalling dampens the progression of APAP hepatotoxicity through inhibiting an excessive accumulation of activated neutrophils. The development of a specific agonist for BLT1 could be useful for the prevention of APAP hepatotoxicity. PMID:27404729

  7. TRPC1 regulates fMLP-stimulated migration and chemotaxis of neutrophil granulocytes.

    PubMed

    Lindemann, O; Strodthoff, C; Horstmann, M; Nielsen, N; Jung, F; Schimmelpfennig, S; Heitzmann, M; Schwab, A

    2015-09-01

    Neutrophils form the first line of defense of the innate immune system and are rapidly recruited by chemotactic signals to sites of inflammation. Understanding the mechanisms of neutrophil chemotaxis is therefore of great interest for the potential development of new immunoregulatory therapies. It has been shown that members of the transient receptor potential (TRP) family of cation channels are involved in both cell migration and chemotaxis. In this study, we demonstrate that TRPC1 channels play an important role in fMLP mediated chemotaxis and migration of murine neutrophils. The knock-out of TRPC1 channels leads to an impaired migration, transmigration and chemotaxis of the neutrophils. In contrast, Ca²⁺ influx but not store release after activation of the TRPC1(-/-) neutrophils with fMLP is strongly enhanced. We show that the enhanced Ca²⁺ influx in the TRPC1(-/-) neutrophils is associated with a steepened front to rear gradient of the intracellular Ca²⁺ concentration with higher levels at the cell rear. Taken together, this paper highlights a distinct role of TRPC1 in neutrophil migration and chemotaxis. We propose that TRPC1 controls the activity of further Ca²⁺ influx channels and thus regulates the maintenance of intracellular Ca²⁺ gradients which are critical for cell migration. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

  8. Rapid and label-free microfluidic neutrophil purification and phenotyping in diabetes mellitus

    NASA Astrophysics Data System (ADS)

    Hou, Han Wei; Petchakup, Chayakorn; Tay, Hui Min; Tam, Zhi Yang; Dalan, Rinkoo; Chew, Daniel Ek Kwang; Li, King Ho Holden; Boehm, Bernhard O.

    2016-07-01

    Advanced management of dysmetabolic syndromes such as diabetes will benefit from a timely mechanistic insight enabling personalized medicine approaches. Herein, we present a rapid microfluidic neutrophil sorting and functional phenotyping strategy for type 2 diabetes mellitus (T2DM) patients using small blood volumes (fingerprick ~100 μL). The developed inertial microfluidics technology enables single-step neutrophil isolation (>90% purity) without immuno-labeling and sorted neutrophils are used to characterize their rolling behavior on E-selectin, a critical step in leukocyte recruitment during inflammation. The integrated microfluidics testing methodology facilitates high throughput single-cell quantification of neutrophil rolling to detect subtle differences in speed distribution. Higher rolling speed was observed in T2DM patients (P < 0.01) which strongly correlated with neutrophil activation, rolling ligand P-selectin glycoprotein ligand 1 (PSGL-1) expression, as well as established cardiovascular risk factors (cholesterol, high-sensitive C-reactive protein (CRP) and HbA1c). Rolling phenotype can be modulated by common disease risk modifiers (metformin and pravastatin). Receiver operating characteristics (ROC) and principal component analysis (PCA) revealed neutrophil rolling as an important functional phenotype in T2DM diagnostics. These results suggest a new point-of-care testing methodology, and neutrophil rolling speed as a functional biomarker for rapid profiling of dysmetabolic subjects in clinical and patient-oriented settings.

  9. In vivo two-photon imaging reveals monocyte-dependent neutrophil extravasation during pulmonary inflammation

    PubMed Central

    Kreisel, Daniel; Nava, Ruben G.; Li, Wenjun; Zinselmeyer, Bernd H.; Wang, Baomei; Lai, Jiaming; Pless, Robert; Gelman, Andrew E.; Krupnick, Alexander S.; Miller, Mark J.

    2010-01-01

    Immune-mediated pulmonary diseases are a significant public health concern. Analysis of leukocyte behavior in the lung is essential for understanding cellular mechanisms that contribute to normal and diseased states. Here, we used two-photon imaging to study neutrophil extravasation from pulmonary vessels and subsequent interstitial migration. We found that the lungs contained a significant pool of tissue-resident neutrophils in the steady state. In response to inflammation produced by bacterial challenge or transplant-mediated, ischemia-reperfusion injury, neutrophils were rapidly recruited from the circulation and patrolled the interstitium and airspaces of the lung. Motile neutrophils often aggregated in dynamic clusters that formed and dispersed over tens of minutes. These clusters were associated with CD115+ F4/80+ Ly6C+ cells that had recently entered the lung. The depletion of blood monocytes with clodronate liposomes reduced neutrophil clustering in the lung, but acted by inhibiting neutrophil transendothelial migration upstream of interstitial migration. Our results suggest that a subset of monocytes serve as key regulators of neutrophil extravasation in the lung and may be an attractive target for the treatment of inflammatory pulmonary diseases. PMID:20923880

  10. Rapid and label-free microfluidic neutrophil purification and phenotyping in diabetes mellitus

    PubMed Central

    Hou, Han Wei; Petchakup, Chayakorn; Tay, Hui Min; Tam, Zhi Yang; Dalan, Rinkoo; Chew, Daniel Ek Kwang; Li, King Ho Holden; Boehm, Bernhard O.

    2016-01-01

    Advanced management of dysmetabolic syndromes such as diabetes will benefit from a timely mechanistic insight enabling personalized medicine approaches. Herein, we present a rapid microfluidic neutrophil sorting and functional phenotyping strategy for type 2 diabetes mellitus (T2DM) patients using small blood volumes (fingerprick ~100 μL). The developed inertial microfluidics technology enables single-step neutrophil isolation (>90% purity) without immuno-labeling and sorted neutrophils are used to characterize their rolling behavior on E-selectin, a critical step in leukocyte recruitment during inflammation. The integrated microfluidics testing methodology facilitates high throughput single-cell quantification of neutrophil rolling to detect subtle differences in speed distribution. Higher rolling speed was observed in T2DM patients (P < 0.01) which strongly correlated with neutrophil activation, rolling ligand P-selectin glycoprotein ligand 1 (PSGL-1) expression, as well as established cardiovascular risk factors (cholesterol, high-sensitive C-reactive protein (CRP) and HbA1c). Rolling phenotype can be modulated by common disease risk modifiers (metformin and pravastatin). Receiver operating characteristics (ROC) and principal component analysis (PCA) revealed neutrophil rolling as an important functional phenotype in T2DM diagnostics. These results suggest a new point-of-care testing methodology, and neutrophil rolling speed as a functional biomarker for rapid profiling of dysmetabolic subjects in clinical and patient-oriented settings. PMID:27381673

  11. BLT1 signalling protects the liver against acetaminophen hepatotoxicity by preventing excessive accumulation of hepatic neutrophils

    PubMed Central

    Kojo, Ken; Ito, Yoshiya; Eshima, Koji; Nishizawa, Nobuyuki; Ohkubo, Hirotoki; Yokomizo, Takehiko; Shimizu, Takao; Watanabe, Masahiko; Majima, Masataka

    2016-01-01

    Leukotriene B4 (LTB4) is a potent chemoattractant for neutrophils. Signalling of LTB4 receptor type 1 (BLT1) has pro-inflammatory functions through neutrophil recruitment. In this study, we investigated whether BLT1 signalling plays a role in acetaminophen (APAP)-induced liver injury by affecting inflammatory responses including the accumulation of hepatic neutrophils. BLT1-knockout (BLT1−/−) mice and their wild-type (WT) counterparts were subjected to a single APAP overdose (300 mg/kg), and various parameters compared within 24 h after treatment. Compared with WT mice, BLT1−/− mice exhibited exacerbation of APAP-induced liver injury as evidenced by enhancement of alanine aminotransferase level, necrotic area, hepatic neutrophil accumulation, and expression of cytokines and chemokines. WT mice co-treated with APAP and ONO-0457, a specific antagonist for BLT1, displayed amplification of the injury, and similar results to those observed in BLT1−/− mice. Hepatic neutrophils in BLT1−/− mice during APAP hepatotoxicity showed increases in the production of reactive oxygen species and matrix metalloproteinase-9. Administration of isolated BLT1-deficient neutrophils into WT mice aggravated the liver injury elicited by APAP. These results demonstrate that BLT1 signalling dampens the progression of APAP hepatotoxicity through inhibiting an excessive accumulation of activated neutrophils. The development of a specific agonist for BLT1 could be useful for the prevention of APAP hepatotoxicity. PMID:27404729

  12. The effects of extracellular matrix proteins on neutrophil-endothelial interaction--a roadway to multiple therapeutic opportunities.

    PubMed

    Padmanabhan, Jagannath; Gonzalez, Anjelica L

    2012-06-01

    Polymorphoneuclear leukocytes or neutrophils, a major component of white blood cells, contribute to the innate immune response in humans. Upon sensing changes in the microenvironment, neutrophils adhere to the vascular wall, migrate through the endothelial cell (EC)-pericyte bilayer, and subsequently through the extracellular matrix to reach the site of inflammation. These cells are capable of destroying microbes, cell debris, and foreign proteins by oxidative and non-oxidative processes. While primarily mediators of tissue homeostasis, there are an increasing number of studies indicating that neutrophil recruitment and transmigration can also lead to host-tissue injury and subsequently inflammation-related diseases. Neutrophil-induced tissue injury is highly regulated by the microenvironment of the infiltrated tissue, which includes cytokines, chemokines, and the provisional extracellular matrix, remodeled through increased vascular permeability and other cellular infiltrates. Thus, investigation of the effects of matrix proteins on neutrophil-EC interaction and neutrophil transmigration may help identify the proteins that induce pro- or anti-inflammatory responses. This area of research presents an opportunity to identify therapeutic targets in inflammation-related diseases. This review will summarize recent literature on the role of neutrophils and the effects of matrix proteins on neutrophil-EC interactions, with focus on three different disease models: 1) atherosclerosis, 2) COPD, and 3) tumor growth and progression. For each disease model, inflammatory molecules released by neutrophils, important regulatory matrix proteins, current anti-inflammatory treatments, and the scope for further research will be summarized.

  13. Real-time detection of implant-associated neutrophil responses using a formyl peptide receptor-targeting NIR nanoprobe

    PubMed Central

    Zhou, Jun; Tsai, Yi-Ting; Weng, Hong; Tang, Ewin N; Nair, Ashwin; Davé, Digant P; Tang, Liping

    2012-01-01

    Neutrophils play an important role in implant-mediated inflammation and infection. Unfortunately, current methods which monitor neutrophil activity, including enzyme measurements and histological evaluation, require many animals and cannot be used to accurately depict the dynamic cellular responses. To understand the neutrophil interactions around implant-mediated inflammation and infection it is critical to develop methods which can monitor in vivo cellular activity in real time. In this study, formyl peptide receptor (FPR)-targeting near-infrared nanoprobes were fabricated. This was accomplished by conjugating near-infrared dye with specific peptides having a high affinity to the FPRs present on activated neutrophils. The ability of FPR-targeting nanoprobes to detect and quantify activated neutrophils was assessed both in vitro and in vivo. As expected, FPR-targeting nanoprobes preferentially accumulated on activated neutrophils in vitro. Following transplantation, FPR-targeting nanoprobes preferentially accumulated at the biomaterial implantation site. Equally important, a strong relationship was observed between the extent of fluorescence intensity in vivo and the number of recruited neutrophils at the implantation site. Furthermore, FPR-targeting nanoprobes may be used to detect and quantify the number of neutrophils responding to a catheter-associated infection. The results show that FPR-targeting nanoprobes may serve as a powerful tool to monitor and measure the extent of neutrophil responses to biomaterial implants in vivo. PMID:22619542

  14. Neutrophil functional disorder in childhood.

    PubMed

    Mironska, K

    2015-01-01

    Neutrophil functional disorders thought to be uncommon, yet important as a cause of morbidity and mortality in infants and children. During the first years of life, when the immune system is still not completely mature, when the viral infections are frequent and antibiotic overuse can damage and alter the immune response, the inadequate nutrition followed with iron deficient anemia and malnutrition can lead the child`s organism in state of immunodeficiency. Sometimes is difficult to distinguish at the beginning weather the cause of patient suffering from frequent infections is existing of primary immunodeficiency disorder or the cause of the immunodeficiency state is just from exogenous factors. Fortunately, primary immune deficiencies are rare diseases and only 6-7% of all of them, due to the neutrophilic functional disorders. Unfortunately, many exogenous and environmental factors have influence to the immune system, and the percentage of secondary caused neutrophilic functional disorders is much higher and should be considered when children are investigated for immunodeficiency. So, when to suspect neutrophil functional disorder? The hallmarks for diseases related to the neutrophilic functional disorders are discussed in this article.

  15. Neutrophil Extracellular Traps Go Viral

    PubMed Central

    Schönrich, Günther; Raftery, Martin J.

    2016-01-01

    Neutrophils are the most numerous immune cells. Their importance as the first line of defense against bacterial and fungal pathogens is well described. In contrast, the role of neutrophils in controlling viral infections is less clear. Bacterial and fungal pathogens can stimulate neutrophils extracellular traps (NETs) in a process called NETosis. Although NETosis has previously been described as a special form of programmed cell death, there are forms of NET production that do not end with the demise of neutrophils. As an end result of NETosis, genomic DNA complexed with microbicidal proteins is expelled from neutrophils. These structures can kill pathogens or at least prevent their local spread within host tissue. On the other hand, disproportionate NET formation can cause local or systemic damage. Only recently, it was recognized that viruses can also induce NETosis. In this review, we discuss the mechanisms by which NETs are produced in the context of viral infection and how this may contribute to both antiviral immunity and immunopathology. Finally, we shed light on viral immune evasion mechanisms targeting NETs. PMID:27698656

  16. Neutrophils in type 1 diabetes.

    PubMed

    Huang, Juan; Xiao, Yang; Xu, Aimin; Zhou, Zhiguang

    2016-09-01

    Type 1 diabetes is an autoimmune disease that afflicts millions of people worldwide. It occurs as the consequence of destruction of insulin-producing pancreatic β-cells triggered by genetic and environmental factors. The initiation and progression of the disease involves a complicated interaction between β-cells and immune cells of both innate and adaptive systems. Immune cells, such as T cells, macrophages and dendritic cells, have been well documented to play crucial roles in type 1 diabetes pathogenesis. However, the particular actions of neutrophils, which are the most plentiful immune cell type and the first immune cells responding to inflammation, in the etiology of this disease might indeed be unfairly ignored. Progress over the past decades shows that neutrophils might have essential effects on the onset and perpetuation of type 1 diabetes. Neutrophil-derived cytotoxic substances, including degranulation products, cytokines, reactive oxygen species and extracellular traps that are released during the process of neutrophil maturation or activation, could cause destruction to islet cells. In addition, these cells can initiate diabetogenic T cell response and promote type 1 diabetes development through cell-cell interactions with other immune and non-immune cells. Furthermore, relevant antineutrophil therapies have been shown to delay and dampen the progression of insulitis and autoimmune diabetes. Here, we discuss the relationship between neutrophils and autoimmune type 1 diabetes from the aforementioned aspects to better understand the roles of these cells in the initiation and development of type 1 diabetes. PMID:27181374

  17. Neutrophil Extracellular Traps Go Viral

    PubMed Central

    Schönrich, Günther; Raftery, Martin J.

    2016-01-01

    Neutrophils are the most numerous immune cells. Their importance as the first line of defense against bacterial and fungal pathogens is well described. In contrast, the role of neutrophils in controlling viral infections is less clear. Bacterial and fungal pathogens can stimulate neutrophils extracellular traps (NETs) in a process called NETosis. Although NETosis has previously been described as a special form of programmed cell death, there are forms of NET production that do not end with the demise of neutrophils. As an end result of NETosis, genomic DNA complexed with microbicidal proteins is expelled from neutrophils. These structures can kill pathogens or at least prevent their local spread within host tissue. On the other hand, disproportionate NET formation can cause local or systemic damage. Only recently, it was recognized that viruses can also induce NETosis. In this review, we discuss the mechanisms by which NETs are produced in the context of viral infection and how this may contribute to both antiviral immunity and immunopathology. Finally, we shed light on viral immune evasion mechanisms targeting NETs.

  18. Neutrophils and Granulocytic MDSC: The Janus God of Cancer Immunotherapy.

    PubMed

    Zilio, Serena; Serafini, Paolo

    2016-01-01

    Neutrophils are the most abundant circulating blood cell type in humans, and are the first white blood cells recruited at the inflammation site where they orchestrate the initial immune response. Although their presence at the tumor site was recognized in the 1970s, until recently these cells have been neglected and considered to play just a neutral role in tumor progression. Indeed, in recent years neutrophils have been recognized to play a dual role in tumor development by either assisting the growth, angiogenesis, invasion, and metastasis or by exerting tumoricidal action directly via the secretion of antitumoral compounds, or indirectly via the orchestration of antitumor immunity. Understanding the biology of these cells and influencing their polarization in the tumor micro- and macro-environment may be the key for the development of new therapeutic strategies, which may finally hold the promise of an effective immunotherapy for cancer.

  19. Neutrophils and Granulocytic MDSC: The Janus God of Cancer Immunotherapy

    PubMed Central

    Zilio, Serena; Serafini, Paolo

    2016-01-01

    Neutrophils are the most abundant circulating blood cell type in humans, and are the first white blood cells recruited at the inflammation site where they orchestrate the initial immune response. Although their presence at the tumor site was recognized in the 1970s, until recently these cells have been neglected and considered to play just a neutral role in tumor progression. Indeed, in recent years neutrophils have been recognized to play a dual role in tumor development by either assisting the growth, angiogenesis, invasion, and metastasis or by exerting tumoricidal action directly via the secretion of antitumoral compounds, or indirectly via the orchestration of antitumor immunity. Understanding the biology of these cells and influencing their polarization in the tumor micro- and macro-environment may be the key for the development of new therapeutic strategies, which may finally hold the promise of an effective immunotherapy for cancer. PMID:27618112

  20. Neutrophils and Granulocytic MDSC: The Janus God of Cancer Immunotherapy.

    PubMed

    Zilio, Serena; Serafini, Paolo

    2016-01-01

    Neutrophils are the most abundant circulating blood cell type in humans, and are the first white blood cells recruited at the inflammation site where they orchestrate the initial immune response. Although their presence at the tumor site was recognized in the 1970s, until recently these cells have been neglected and considered to play just a neutral role in tumor progression. Indeed, in recent years neutrophils have been recognized to play a dual role in tumor development by either assisting the growth, angiogenesis, invasion, and metastasis or by exerting tumoricidal action directly via the secretion of antitumoral compounds, or indirectly via the orchestration of antitumor immunity. Understanding the biology of these cells and influencing their polarization in the tumor micro- and macro-environment may be the key for the development of new therapeutic strategies, which may finally hold the promise of an effective immunotherapy for cancer. PMID:27618112

  1. Infiltrating neutrophils increase bladder cancer cell invasion via modulation of androgen receptor (AR)/MMP13 signals

    PubMed Central

    Yeh, Shuyuan; Li, Lei; Chang, Chawnshang

    2015-01-01

    Early studies indicated that several inflammatory immune cells, including macrophages, mast cells, B and T cells in the tumor microenvironment, might influence cancer progression. Here we found that bladder cancer (BCa) cells could recruit more neutrophils than normal bladder cells. The consequences of recruiting more neutrophils might then increase BCa cell invasion via up-regulating androgen receptor (AR) signals. Mechanism dissection revealed infiltrating neutrophils could up-regulate AR signals via either increased AR mRNA/protein expression or increased AR transactivation. The increased AR signals might then enhance BCa cell invasion via increasing MMP13 expression. Together, these results might provide us a new potential therapeutic approach to better battle BCa metastasis via targeting the newly identified signaling from infiltrating neutrophils to BCa through AR to MMP13 signals. PMID:26517808

  2. Neutrophil activation during acetaminophen hepatotoxicity and repair in mice and humans

    SciTech Connect

    Williams, C. David; Bajt, Mary Lynn; Sharpe, Matthew R.; McGill, Mitchell R.; Farhood, Anwar; Jaeschke, Hartmut

    2014-03-01

    Following acetaminophen (APAP) overdose there is an inflammatory response triggered by the release of cellular contents from necrotic hepatocytes into the systemic circulation which initiates the recruitment of neutrophils into the liver. It has been demonstrated that neutrophils do not contribute to APAP-induced liver injury, but their role and the role of NADPH oxidase in injury resolution are controversial. C57BL/6 mice were subjected to APAP overdose and neutrophil activation status was determined during liver injury and liver regeneration. Additionally, human APAP overdose patients (ALT: > 800 U/L) had serial blood draws during the injury and recovery phases for the determination of neutrophil activation. Neutrophils in the peripheral blood of mice showed an increasing activation status (CD11b expression and ROS priming) during and after the peak of injury but returned to baseline levels prior to complete injury resolution. Hepatic sequestered neutrophils showed an increased and sustained CD11b expression, but no ROS priming was observed. Confirming that NADPH oxidase is not critical to injury resolution, gp91{sup phox}−/− mice following APAP overdose displayed no alteration in injury resolution. Peripheral blood from APAP overdose patients also showed increased neutrophil activation status after the peak of liver injury and remained elevated until discharge from the hospital. In mice and humans, markers of activation, like ROS priming, were increased and sustained well after active liver injury had subsided. The similar findings between surviving patients and mice indicate that neutrophil activation may be a critical event for host defense or injury resolution following APAP overdose, but not a contributing factor to APAP-induced injury. - Highlights: • Neutrophil (PMN) function increases during liver repair after acetaminophen overdose. • Liver repair after acetaminophen (APAP)-overdose is not dependent on NADPH oxidase. • Human PMNs do not appear

  3. Neutrophil dynamics in the blood and milk of crossbred cows naturally infected with Staphylococcus aureus

    PubMed Central

    Swain, Dilip K.; Kushwah, Mohar Singh; Kaur, Mandheer; Dang, Ajay K.

    2015-01-01

    Aim: The present study was designed to evaluate the neutrophil dynamics in terms of the functional competence during subclinical mastitis (SCM) and clinical mastitis (CM). Materials and Methods: A total of 146 Karan fries cows were screened and were divided into three groups as control (n=12), SCM, n=12 and CM, n=12 groups on the basis of California mastitis test scoring, bacteriological evaluation, gross and morphological changes in milk and by counting milk somatic cell count (SCC). Both blood and milk polymorphonuclear neutrophils (PMNs) were isolated in the study. Phagocytic activity (PA) was studied by spectrophotometrically; neutrophil extracelluar traps (NETs) were studied by scanning electron microscopy (SEM); CD44 was quantified by flow cytometry and apoptosis was studied by fluorescent microscopy. Results: Significantly (p<0.05) higher SCC, PA was found in milk of CM cows as compared to SCM and control cows. Significantly lower (p<0.05) apoptosis was observed in PMNs isolated from both blood and milk of CM group of cows when compared to control and SCM group. The milk neutrophils of CM group of cows formed NETs as evidenced from the SEM images. Surface expression of CD44 revealed a significantly (p<0.05) lower expression in milk neutrophils of CM group of cows when compared to SCM and control group of cows. Conclusion: The study indicated a positive correlation between delayed neutrophil apoptosis, persistent staying of neutrophils at the site of infection along with formation of NETs as the strategies to fight against the pathogens in the udder during Staphylococcal mastitis. The study forms a strong base for future molecular research in terms of neutrophil recruitment and neutrophil removal from the site of infection. PMID:27047094

  4. Interaction of CD44 and hyaluronan is the dominant mechanism for neutrophil sequestration in inflamed liver sinusoids

    PubMed Central

    McDonald, Braedon; McAvoy, Erin F.; Lam, Florence; Gill, Varinder; de la Motte, Carol; Savani, Rashmin C.; Kubes, Paul

    2008-01-01

    Adhesion molecules known to be important for neutrophil recruitment in many other organs are not involved in recruitment of neutrophils into the sinusoids of the liver. The prevailing view is that neutrophils become physically trapped in inflamed liver sinusoids. In this study, we used a biopanning approach to identify hyaluronan (HA) as disproportionately expressed in the liver versus other organs under both basal and inflammatory conditions. Spinning disk intravital microscopy revealed that constitutive HA expression was restricted to liver sinusoids. Blocking CD44–HA interactions reduced neutrophil adhesion in the sinusoids of endotoxemic mice, with no effect on rolling or adhesion in postsinusoidal venules. Neutrophil but not endothelial CD44 was required for adhesion in sinusoids, yet neutrophil CD44 avidity for HA did not increase significantly in endotoxemia. Instead, activation of CD44–HA engagement via qualitative modification of HA was demonstrated by a dramatic induction of serum-derived HA-associated protein in sinusoids in response to lipopolysaccharide (LPS). LPS-induced hepatic injury was significantly reduced by blocking CD44–HA interactions. Administration of anti-CD44 antibody 4 hours after LPS rapidly detached adherent neutrophils in sinusoids and improved sinusoidal perfusion in endotoxemic mice, revealing CD44 as a potential therapeutic target in systemic inflammatory responses involving the liver. PMID:18362172

  5. Neutral serine proteases of neutrophils.

    PubMed

    Kettritz, Ralph

    2016-09-01

    Neutrophil serine proteases (NSPs) exercise tissue-degrading and microbial-killing effects. The spectrum of NSP-mediated functions grows continuously, not least because of methodological progress. Sensitive and specific FRET substrates were developed to study the proteolytic activity of each NSP member. Advanced biochemical methods are beginning to characterize common and specific NSP substrates. The resulting novel information indicates that NSPs contribute not only to genuine inflammatory neutrophil functions but also to autoimmunity, metabolic conditions, and cancer. Tight regulatory mechanisms control the proteolytic potential of NSPs. However, not all NSP functions depend on their enzymatic activity. Proteinase-3 (PR3) is somewhat unique among the NSPs for PR3 functions as an autoantigen. Patients with small-vessel vasculitis develop autoantibodies to PR3 that bind their target antigens on the neutrophil surface and trigger neutrophil activation. These activated cells subsequently contribute to vascular necrosis with life-threatening multiorgan failure. This article discusses various aspects of NSP biology and highlights translational aspects with strong clinical implications. PMID:27558338

  6. Type I interferon transcriptional signature in neutrophils and high frequency of low-density granulocytes are associated with tissue damage in malaria

    PubMed Central

    Rocha, Bruno Coelho; Marques, Pedro Elias; Leoratti, Fabiana Maria de Souza; Junqueira, Caroline; Pereira, Dhelio Batista; Antonelli, Lis Ribeiro do Valle; Menezes, Gustavo Batista

    2015-01-01

    SUMMARY Neutrophils are the most abundant leukocyte population in the bloodstream, the primary compartment of Plasmodium sp. infection. Yet, the role of these polymorphonuclear cells in mediating either resistance or pathogenesis of malaria is poorly understood. We report that circulating neutrophils from malaria patients are highly activated, as indicated by a strong type I interferon transcriptional signature, increased expression of surface activation markers, the enhanced release of reactive oxygen species and myeloperoxidase, as well as the high frequency of low-density granulocytes. The activation of neutrophils was associated with increased levels of serum alanine and aspartate aminotransferases, indicating liver damage. In a rodent malaria model, we observed an intense recruitment of neutrophils to liver sinusoids. Neutrophil migration, IL-1β and chemokine expression as well as liver damage were all dependent on type I interferon signaling. The data suggests that type I interferon signaling have a central role in neutrophil activation and malaria pathogenesis. PMID:26711347

  7. Microbe-specific unconventional T-cells induce human neutrophil differentiation into antigen cross-presenting cells

    PubMed Central

    Liuzzi, Anna Rita; Tyler, Christopher J.; Khan, Mohd Wajid A.; Szakmany, Tamas; Hall, Judith E.; Moser, Bernhard; Eberl, Matthias

    2014-01-01

    The early immune response to microbes is dominated by the recruitment of neutrophils whose primary function is to clear invading pathogens. However, there is emerging evidence that neutrophils play additional effector and regulatory roles. The present study demonstrates that human neutrophils assume antigen cross-presenting functions, and suggests a plausible scenario for the local generation of APC-like neutrophils through the mobilization of unconventional T-cells in response to microbial metabolites. Vγ9/Vδ2 T-cells and MAIT cells are abundant in blood, inflamed tissues and mucosal barriers. Here, both human cell types responded rapidly to neutrophils after phagocytosis of Gram-positive and Gram-negative bacteria producing the corresponding ligands, and in turn mediated the differentiation of neutrophils into APCs for both CD4+ and CD8+ T-cells through secretion of GM-CSF, IFN-γ and TNF-α. In patients with acute sepsis, circulating neutrophils displayed a similar APC-like phenotype and readily processed soluble proteins for cross-presentation of antigenic peptides to CD8+ T-cells, at a time when peripheral Vγ9/Vδ2 T-cells were highly activated. Our findings indicate that unconventional T-cells represent key controllers of neutrophil-driven innate and adaptive responses to a broad range of pathogens. PMID:25165152

  8. Reverse-migrated neutrophils regulated by JAM-C are involved in acute pancreatitis-associated lung injury

    PubMed Central

    Wu, Deqing; Zeng, Yue; Fan, Yuting; Wu, Jianghong; Mulatibieke, Tunike; Ni, Jianbo; Yu, Ge; Wan, Rong; Wang, Xingpeng; Hu, Guoyong

    2016-01-01

    Junctional adhesion molecule-C (JAM-C) plays a key role in the promotion of the reverse transendothelial migration (rTEM) of neutrophils, which contributes to the dissemination of systemic inflammation and to secondary organ damage. During acute pancreatitis (AP), systemic inflammatory responses lead to distant organ damage and typically result in acute lung injury (ALI). Here, we investigated the role of rTEM neutrophils in AP-associated ALI and the molecular mechanisms by which JAM-C regulates neutrophil rTEM in this disorder. In this study, rTEM neutrophils were identified in the peripheral blood both in murine model of AP and human patients with AP, which elevated with increased severity of lung injury. Pancreatic JAM-C was downregulated during murine experimental pancreatitis, whose expression levels were inversely correlated with both increased neutrophil rTEM and severity of lung injury. Knockout of JAM-C resulted in more severe lung injury and systemic inflammation. Significantly greater numbers of rTEM neutrophils were present both in the circulation and pulmonary vascular washout in JAM-C knockout mice with AP. This study demonstrates that during AP, neutrophils that are recruited to the pancreas may migrate back into the circulation and then contribute to ALI. JAM-C downregulation may contribute to AP-associated ALI via promoting neutrophil rTEM. PMID:26841848

  9. S. aureus blocks efferocytosis of neutrophils by macrophages through the activity of its virulence factor alpha toxin

    PubMed Central

    Cohen, Taylor S.; Jones-Nelson, Omari; Hotz, Meghan; Cheng, Lily; Miller, Lloyd S.; Suzich, JoAnn; Stover, C. Kendall; Sellman, Bret R.

    2016-01-01

    Bacterial pneumonia, such as those caused by Staphylococcus aureus, is associated with an influx of inflammatory neutrophils into the lung tissue and airways. Regulation and clearance of recruited neutrophils is essential for preventing tissue damage by “friendly fire”, a responsibility of macrophages in a process called efferocytosis. We hypothesized that S. aureus impairs efferocytosis by alveolar macrophages (AMs) through the activity of the secreted virulence factor alpha toxin (AT), which has been implicated in altering the antimicrobial function of AMs. Infection of mice lacking AMs resulted in significantly increased numbers of neutrophils in the lung, while clearance of neutrophils delivered intranasally into uninfected mice was reduced in AM depleted animals. In vitro, sublytic levels of AT impaired uptake of apoptotic neutrophils by purified AMs. In vivo, the presence of AT reduced uptake of neutrophils by AMs. Differential uptake of neutrophils was not due to changes in either the CD47/CD172 axis or CD36 levels. AT significantly reduced lung expression of CCN1 and altered AM surface localization of DD1α, two proteins known to influence efferocytosis. We conclude that AT may contribute to tissue damage during S. aureus pneumonia by inhibiting the ability of AM to clear neutrophils at the site of infection. PMID:27739519

  10. Type 2 Interleukin-4 Receptor Signaling in Neutrophils Antagonizes Their Expansion and Migration during Infection and Inflammation.

    PubMed

    Woytschak, Janine; Keller, Nadia; Krieg, Carsten; Impellizzieri, Daniela; Thompson, Robert W; Wynn, Thomas A; Zinkernagel, Annelies S; Boyman, Onur

    2016-07-19

    Neutrophils are the first immune cells recruited to sites of inflammation and infection. However, patients with allergic disorders such as atopic dermatitis show a paucity of skin neutrophils and are prone to bacterial skin infections, suggesting that allergic inflammation curtails neutrophil responses. Here we have shown that the type 2 cell signature cytokine interleukin-4 (IL-4) hampers neutrophil expansion and migration by antagonizing granulocyte colony-stimulating factor (G-CSF) and chemokine receptor-mediated signals. Cutaneous bacterial infection in mice was exacerbated by IL-4 signaling and improved with IL-4 inhibition, each outcome inversely correlating with neutrophil migration to skin. Likewise, systemic bacterial infection was worsened by heightened IL-4 activity, with IL-4 restricting G-CSF-induced neutrophil expansion and migration to tissues by affecting CXCR2-CXCR4 chemokine signaling in neutrophils. These effects were dependent on IL-4 acting through type 2 IL-4 receptors on neutrophils. Thus, targeting IL-4 might be beneficial in neutropenic conditions with increased susceptibility to bacterial infections. PMID:27438770

  11. Role of neutrophils in IL-17-dependent immunity to mucosal candidiasis.

    PubMed

    Huppler, Anna R; Conti, Heather R; Hernández-Santos, Nydiaris; Darville, Toni; Biswas, Partha S; Gaffen, Sarah L

    2014-02-15

    Oropharyngeal candidiasis (OPC), caused by the commensal fungus Candida albicans, is an opportunistic infection associated with infancy, AIDS, and IL-17-related primary immunodeficiencies. The Th17-associated cytokines IL-23 and IL-17 are crucial for immunity to OPC, but the mechanisms by which they mediate immunity are poorly defined. IL-17RA-deficient humans and mice are strongly susceptible to OPC, with reduced levels of CXC chemokines and concomitantly impaired neutrophil recruitment to the oral mucosa. Paradoxically, humans with isolated neutropenia are typically not susceptible to candidiasis. To determine whether immunity to OPC is mediated via neutrophil recruitment, mice lacking CXCR2 were subjected to OPC and were found to be highly susceptible, although there was no dissemination of fungi to peripheral organs. To assess whether the entire neutrophil response is IL-17 dependent, IL-17RA(-/-) and IL-23(-/-) mice were administered neutrophil-depleting Abs and subjected to OPC. These mice displayed increased oral fungal burdens compared with IL-17RA(-/-) or IL-23(-/-) mice alone, indicating that additional IL-17-independent signals contribute to the neutrophil response. WT mice treated with anti-Gr-1 Abs exhibited a robust infiltrate of CD11b(+)Ly-6G(low)F4/80(-) cells to the oral mucosa but were nonetheless highly susceptible to OPC, indicating that this monocytic influx is insufficient for host defense. Surprisingly, Ly-6G Ab treatment did not induce the same strong susceptibility to OPC in WT mice. Thus, CXCR2(+) and Gr-1(+) neutrophils play a vital role in host defense against OPC. Moreover, defects in the IL-23/17 axis cause a potent but incomplete deficiency in the neutrophil response to oral candidiasis.

  12. Endothelial LSP1 Modulates Extravascular Neutrophil Chemotaxis by Regulating Nonhematopoietic Vascular PECAM-1 Expression.

    PubMed

    Hossain, Mokarram; Qadri, Syed M; Xu, Najia; Su, Yang; Cayabyab, Francisco S; Heit, Bryan; Liu, Lixin

    2015-09-01

    During inflammation, leukocyte-endothelial cell interactions generate molecular signals that regulate cell functions. The Ca(2+)- and F-actin-binding leukocyte-specific protein 1 (LSP1) expressed in leukocytes and nonhematopoietic endothelial cells is pivotal in regulating microvascular permeability and leukocyte recruitment. However, cell-specific function of LSP1 during leukocyte recruitment remains elusive. Using intravital microscopy of cremasteric microvasculature of chimeric LSP1-deficient mice, we show that not neutrophil but endothelial LSP1 regulates neutrophil transendothelial migration and extravascular directionality without affecting the speed of neutrophil migration in tissue in response to CXCL2 chemokine gradient. The expression of PECAM-1-sensitive α6β1 integrins on the surface of transmigrated neutrophils was blunted in mice deficient in endothelial LSP1. Functional blocking studies in vivo and in vitro elucidated that α6β1 integrins orchestrated extravascular directionality but not the speed of neutrophil migration. In LSP1-deficient mice, PECAM-1 expression was reduced in endothelial cells, but not in neutrophils. Similarly, LSP1-targeted small interfering RNA silencing in murine endothelial cells mitigated mRNA and protein expression of PECAM-1, but not ICAM-1 or VCAM-1. Overexpression of LSP1 in endothelial cells upregulated PECAM-1 expression. Furthermore, the expression of transcription factor GATA-2 that regulates endothelial PECAM-1 expression was blunted in LSP1-deficient or LSP1-silenced endothelial cells. The present study unravels endothelial LSP1 as a novel cell-specific regulator of integrin α6β1-dependent neutrophil extravascular chemotactic function in vivo, effective through GATA-2-dependent transcriptional regulation of endothelial PECAM-1 expression. PMID:26238489

  13. Peeking into the secret life of neutrophils.

    PubMed

    Li, Jackson LiangYao; Ng, Lai Guan

    2012-09-01

    The migration of neutrophils between tissue compartments is an important aspect of innate immune surveillance. This process is regulated by a cascade of cellular and molecular signals to avoid unnecessary crowding of neutrophils at the periphery, to allow rapid mobilization of neutrophils in response to inflammatory stimuli, and to return to a state of homeostasis after the response. Intravital microscopy approaches have been fundamental in unraveling many aspects of neutrophil behavior, providing important mechanistic information on the processes involved in basal and disease states. Here, we provide a broad overview of the current state of research on neutrophil biology, describing the processes in the typical life cycle of neutrophils, from their first appearance in the bone marrow until their eventual destruction. We will focus on novel aspects of neutrophil behavior, which had previously been elusive until their recent elucidation by advanced intravital microscopy techniques. PMID:22407577

  14. Neutrophil extracellular traps - the dark side of neutrophils.

    PubMed

    Sørensen, Ole E; Borregaard, Niels

    2016-05-01

    Neutrophil extracellular traps (NETs) were discovered as extracellular strands of decondensed DNA in complex with histones and granule proteins, which were expelled from dying neutrophils to ensnare and kill microbes. NETs are formed during infection in vivo by mechanisms different from those originally described in vitro. Citrullination of histones by peptidyl arginine deiminase 4 (PAD4) is central for NET formation in vivo. NETs may spur formation of autoantibodies and may also serve as scaffolds for thrombosis, thereby providing a link among infection, autoimmunity, and thrombosis. In this review, we present the mechanisms by which NETs are formed and discuss the physiological and pathophysiological consequences of NET formation. We conclude that NETs may be of more importance in autoimmunity and thrombosis than in innate immune defense.

  15. Characterization of a polysaccharide from Rosa davurica and inhibitory activity against neutrophil migration.

    PubMed

    Tong, Haibin; Jiang, Guiquan; Guan, Xingang; Wu, Hong; Song, Kangxing; Cheng, Kuan; Sun, Xin

    2016-08-01

    The rapid recruitment of neutrophils from peripheral blood into infected sites is critical step for inflammatory responses; however, the excessive and improper recruitment can lead to serious tissue damages. Thus, it is a promising strategy to inhibit their excessive recruitment for treating inflammation-related disease. Here, we isolated a polysaccharide (RDPA1) from Rosa davurica, to evaluate its physicochemical property and inhibitory effects on neutrophil migration. RDPA1 was obtained by hot-water extraction, ethanol precipitation, and fractionated by DEAE-cellulose and Sepharose CL-6B columns. RDPA1 significantly inhibited in vitro migration of human neutrophils evaluated by transwell chamber and impacted the migratory behavior observed by time-lapsed microscopy, we found the migrated distance and average velocity of RDPA1-treated cells were greatly reduced. In addition, RDPA1 treatment impaired in vivo neutrophil infiltration in the peritonitis mice. RDPA1 exhibited significant blocking capacity of the interaction between β2 integrins and ICAM-1 evaluated by flow cytometry and in vitro protein binding assay. Together, these results suggest RDPA1 could be considered as a potential candidate for developing a novel anti-inflammatory agent. PMID:27112979

  16. Activation of Neutrophils via IP3 Pathway Following Exposure to Demodex-Associated Bacterial Proteins.

    PubMed

    McMahon, Fred; Banville, Nessa; Bergin, David A; Smedman, Christian; Paulie, Staffan; Reeves, Emer; Kavanagh, Kevin

    2016-02-01

    Rosacea is a chronic inflammatory condition that predominantly affects the skin of the face. Sera from rosacea patients display elevated reactivity to proteins from a bacterium (Bacillus oleronius) originally isolated from a Demodex mite from a rosacea patient suggesting a possible role for bacteria in the induction and persistence of this condition. This work investigated the ability of B. oleronius proteins to activate neutrophils and demonstrated activation via the IP3 pathway. Activated neutrophils displayed increased levels of IP1 production, F-actin formation, chemotaxis, and production of the pro-inflammatory cytokines IL-1β and IL-6 following stimulation by pure and crude B. oleronius protein preparations (2 μg/ml), respectively. In addition, neutrophils exposed to pure and crude B. oleronius proteins (2 μg/ml) demonstrated increased release of internally stored calcium (Ca(2+)), a hallmark of the IP3 pathway of neutrophil activation. Neutrophils play a significant role in the inflammation associated with rosacea, and this work demonstrates how B. oleronius proteins can induce neutrophil recruitment and activation. PMID:26433579

  17. Neutrophil-dependent, oxygen-radical mediated lung injury associated with acute pancreatitis.

    PubMed Central

    Guice, K S; Oldham, K T; Caty, M G; Johnson, K J; Ward, P A

    1989-01-01

    Cerulein-induced acute pancreatitis in rats is associated with a reversible lung injury that is characterized by alveolar capillary endothelial-cell injury, increased microvascular permeability, interstitial edema formation, and intraalveolar hemorrhage and fibrin deposition. The role of mediators in this injury was analyzed using gravimetric data, microvascular permeability indices, electron microscopy, and a quantitative morphometric analysis. Neutrophil depletion induced by a specific antibody was highly protective against lung injury. Interruption of the complement pathway (using low dose Naja naja cobra venom factor) also protected against lung injury. Catalase and superoxide dismutase were also protective. The iron chelator deferoxamine and the hydroxyl radical scavenger, dimethylsulfoxide, were not protective against acute lung injury. These data suggest that complement, neutrophils, and neutrophil-derived (H2O2-dependent) oxygen products mediate lung injury that occurs secondary to cerulein-induced pancreatitis. In contrast to other models of neutrophil-dependent, oxygen-radical-mediated lung injury, this lung injury does not appear to be an iron-dependent and hydroxyl-radical mediated injury. We postulate that the process of acute pancreatitis leads to complement activation followed by neutrophil recruitment, sequestration, and adherence to alveolar capillary endothelial cells. Ultimately lung injury appears to result from local endothelial-cell injury secondary to neutrophil-generated oxygen products that may be myeloperoxidase dependent. Images Figs. 3A-D. PMID:2589887

  18. IL-17 attenuates the anti-apoptotic effects of GM-CSF in human neutrophils.

    PubMed

    Dragon, Stéphane; Saffar, Arash Shoja; Shan, Lianyu; Gounni, Abdelilah Soussi

    2008-01-01

    Interleukin (IL)-17A is a pleiotropic, pro-inflammatory cytokine that is implicated in chronic inflammatory and degenerative disorders. IL-17 has been demonstrated to link activated T-lymphocyte with the recruitment of neutrophils at sites of inflammation, however whether IL-17 can mediate neutrophil survival and subsequently affect inflammatory responses has not fully been elucidated. In our study, we demonstrate that human peripheral blood and HL-60 differentiated neutrophils express mRNA and cell surface IL-17A receptor. IL-17A does not affect the rate of spontaneous neutrophil apoptosis, however significantly decreased granulocyte macrophage-colony stimulating factor (GM-CSF)-mediated survival by antagonizing the signal transduction pathways of p38, Erk1/2 and signal transducer and activator of transcription (STAT) 5B. These events were associated with reduced myeloid cell lymphoma-1 (Mcl-1) protein levels, increased translocation and aggregation of Bax to mitochondria, decreased mitochondrial transmembrane potential and in an increase in caspase-3/7 activity. These events were independent of increased Fas or soluble Fas ligand expression levels. Taken together, our findings suggest that IL-17 may regulate neutrophil homeostasis and favor the resolution of inflamed tissues by attenuating the delay in neutrophil apoptosis induced by inflammatory cytokines.

  19. Effect of glutamine supplementation on neutrophil function in male judoists.

    PubMed

    Sasaki, Eiji; Umeda, Takashi; Takahashi, Ippei; Arata, Kojima; Yamamoto, Yousuke; Tanabe, Masaru; Oyamada, Kazuyuki; Hashizume, Erika; Nakaji, Shigeyuki

    2013-01-01

    Glutamine is an important amino acid for immune function. Though high intensity and prolonged exercise decreases plasma glutamine concentration and causes immune suppression, the relationship between neutrophil functions and glutamine has not yet been found. The purpose of this study was to investigate the impacts of glutamine supplementation on neutrophil function. Twenty-six male university judoists were recruited. Subjects were classified into glutamine and control groups. The glutamine group ingested 3000 mg of glutamine per day and the control group ingested placebo for 2 weeks. Examinations were performed at the start of preunified loading exercise (pre-ULE), then 1 and 2 weeks after ULE (post-ULE). Reactive oxygen species (ROS) production, phagocytic activity, serum opsonic activity and serum myogenic enzymes were measured. Differences between the levels obtained in pre-ULE and post-ULE for the two groups were compared. In the glutamine group, ROS production activity increased 1 week after ULE, whereas it was not observed in the control group (P < 0.001). Though myogenic enzymes increased significantly after ULE (P < 0.001), the glutamine group remained unchanged by supplementation during ULE. Glutamine supplementation has prevented excessive muscle damage and suppression of neutrophil function, especially in ROS production activity, even during an intensive training period. PMID:23348981

  20. Effect of glutamine supplementation on neutrophil function in male judoists.

    PubMed

    Sasaki, Eiji; Umeda, Takashi; Takahashi, Ippei; Arata, Kojima; Yamamoto, Yousuke; Tanabe, Masaru; Oyamada, Kazuyuki; Hashizume, Erika; Nakaji, Shigeyuki

    2013-01-01

    Glutamine is an important amino acid for immune function. Though high intensity and prolonged exercise decreases plasma glutamine concentration and causes immune suppression, the relationship between neutrophil functions and glutamine has not yet been found. The purpose of this study was to investigate the impacts of glutamine supplementation on neutrophil function. Twenty-six male university judoists were recruited. Subjects were classified into glutamine and control groups. The glutamine group ingested 3000 mg of glutamine per day and the control group ingested placebo for 2 weeks. Examinations were performed at the start of preunified loading exercise (pre-ULE), then 1 and 2 weeks after ULE (post-ULE). Reactive oxygen species (ROS) production, phagocytic activity, serum opsonic activity and serum myogenic enzymes were measured. Differences between the levels obtained in pre-ULE and post-ULE for the two groups were compared. In the glutamine group, ROS production activity increased 1 week after ULE, whereas it was not observed in the control group (P < 0.001). Though myogenic enzymes increased significantly after ULE (P < 0.001), the glutamine group remained unchanged by supplementation during ULE. Glutamine supplementation has prevented excessive muscle damage and suppression of neutrophil function, especially in ROS production activity, even during an intensive training period.

  1. 11β-Hydroxysteroid Dehydrogenase Type 1 Is Expressed in Neutrophils and Restrains an Inflammatory Response in Male Mice

    PubMed Central

    Coutinho, Agnes E.; Kipari, Tiina M. J.; Zhang, Zhenguang; Esteves, Cristina L.; Lucas, Christopher D.; Gilmour, James S.; Webster, Scott P.; Walker, Brian R.; Hughes, Jeremy; Savill, John S.; Seckl, Jonathan R.; Rossi, Adriano G.

    2016-01-01

    Endogenous glucocorticoid action within cells is enhanced by prereceptor metabolism by 11β-hydroxysteroid dehydrogenase type 1 (11β-HSD1), which converts intrinsically inert cortisone and 11-dehydrocorticosterone into active cortisol and corticosterone, respectively. 11β-HSD1 is highly expressed in immune cells elicited to the mouse peritoneum during thioglycollate-induced peritonitis and is down-regulated as the inflammation resolves. During inflammation, 11β-HSD1-deficient mice show enhanced recruitment of inflammatory cells and delayed acquisition of macrophage phagocytic capacity. However, the key cells in which 11β-HSD1 exerts these effects remain unknown. Here we have identified neutrophils (CD11b+,Ly6G+,7/4+ cells) as the thioglycollate-recruited cells that most highly express 11β-HSD1 and show dynamic regulation of 11β-HSD1 in these cells during an inflammatory response. Flow cytometry showed high expression of 11β-HSD1 in peritoneal neutrophils early during inflammation, declining at later states. In contrast, expression in blood neutrophils continued to increase during inflammation. Ablation of monocytes/macrophages by treatment of CD11b-diphtheria-toxin receptor transgenic mice with diphtheria toxin prior to thioglycollate injection had no significant effect on 11β-HSD1 activity in peritoneal cells, consistent with neutrophils being the predominant 11β-HSD1 expressing cell type at this time. Similar to genetic deficiency in 11β-HSD1, acute inhibition of 11β-HSD1 activity during thioglycollate-induced peritonitis augmented inflammatory cell recruitment to the peritoneum. These data suggest that neutrophil 11β-HSD1 increases during inflammation to contribute to the restraining effect of glucocorticoids upon neutrophil-mediated inflammation. In human neutrophils, lipopolysaccharide activation increased 11β-HSD1 expression, suggesting the antiinflammatory effects of 11β-HSD1 in neutrophils may be conserved in humans. PMID:27145012

  2. Recruitment by Brainstorming.

    ERIC Educational Resources Information Center

    Hankin, Joseph N.

    1982-01-01

    Reports on Westchester Community College's (WCC's) campuswide brainstorming meetings which prepared strategies, programs, and recommendations for realizing recruitment goals. Includes a draft of WCC's 1981 Marketing Recruitment Plan, which covers programs and instruction, services, admissions office and processes, publications, and…

  3. Coccidioides Endospores and Spherules Draw Strong Chemotactic, Adhesive, and Phagocytic Responses by Individual Human Neutrophils

    PubMed Central

    Lee, Cheng-Yuk; Thompson III, George R.; Hastey, Christine J.; Hodge, Gregory C.; Lunetta, Jennine M.; Pappagianis, Demosthenes; Heinrich, Volkmar

    2015-01-01

    Coccidioides spp. are dimorphic pathogenic fungi whose parasitic forms cause coccidioidomycosis (Valley fever) in mammalian hosts. We use an innovative interdisciplinary approach to analyze one-on-one encounters between human neutrophils and two forms of Coccidioides posadasii. To examine the mechanisms by which the innate immune system coordinates different stages of the host response to fungal pathogens, we dissect the immune-cell response into chemotaxis, adhesion, and phagocytosis. Our single-cell technique reveals a surprisingly strong response by initially quiescent neutrophils to close encounters with C. posadasii, both from a distance (by complement-mediated chemotaxis) as well as upon contact (by serum-dependent adhesion and phagocytosis). This response closely resembles neutrophil interactions with Candida albicans and zymosan particles, and is significantly stronger than the neutrophil responses to Cryptococcus neoformans, Aspergillus fumigatus, and Rhizopus oryzae under identical conditions. The vigorous in vitro neutrophil response suggests that C. posadasii evades in vivo recognition by neutrophils through suppression of long-range mobilization and recruitment of the immune cells. This observation elucidates an important paradigm of the recognition of microbes, i.e., that intact immunotaxis comprises an intricate spatiotemporal hierarchy of distinct chemotactic processes. Moreover, in contrast to earlier reports, human neutrophils exhibit vigorous chemotaxis toward, and frustrated phagocytosis of, the large spherules of C. posadasii under physiological-like conditions. Finally, neutrophils from healthy donors and patients with chronic coccidioidomycosis display subtle differences in their responses to antibody-coated beads, even though the patient cells appear to interact normally with C. posadasii endospores. PMID:26070210

  4. Gcsf-Chr19 promotes neutrophil migration to damaged tissue through blood vessels in zebrafish.

    PubMed

    Galdames, Jorge A; Zuñiga-Traslaviña, Constanza; Reyes, Ariel E; Feijóo, Carmen G

    2014-07-01

    G-CSF is an essential cytokine that regulates proliferation and differentiation of granulocytes from hematopoietic stem and progenitor cells. In mammals G-CSF has been identified as a key factor that promotes the release of neutrophils from the bone marrow into the blood circulation. In silico analysis indicates that zebrafish has two gcsf genes, gcsf-chr12 in chromosome 12 and gcsf-chr19 in chromosome 19. Gcsf-Chr12 participates in emergency myelopoiesis, but, in contrast to its mammalian orthologue, is not involved in neutrophil migration toward damaged tissue. In turn, the function of Gcsf-Chr19 has not been examined yet. In this study, we analyzed the role of Gcsf-Chr19 in regulating neutrophil migration toward the wound. Our results indicated that during the first h after caudal fin transection, neutrophils migrate from the hematopoietic tissue toward the injury, using the extracellular matrix as a substrate. Later, between 3 and 4 h postdamage, the recruitment mainly occurs through the bloodstream, and only a few neutrophils still use the extracellular matrix to migrate. During this process, the transcriptional levels of gcsf-chr19 are considerably increased, reaching a peak 1 h postdamage. The knockdown of Gcsf-chr19 indicated that the percentage of neutrophils that reach the wound decreased after the first h postinjury, suggesting that the knockdown specifically affects neutrophils that travel to the wound through blood vessels. Together, our data provide novel information about the regulation of neutrophil migration in zebrafish, positioning Gcsf-Chr19 as a key signal during the course of an inflammatory process triggered by severe damage.

  5. Adult Recruitment Practices.

    ERIC Educational Resources Information Center

    Kaufman, Juliet, Ed.; And Others

    Findings of an American College Testing Program 1981 survey on college recruitment of adult students are summarized, and 12 articles on adult recruitment are presented. Titles and authors are as follows: "Adult Recruitment Practices: A Report of a National Survey" (Patricia Spratt, Juliet Kaufmann, Lee Noel); "Three Programs for Adults in Shopping…

  6. Recruitment: Some Unanswered Questions

    ERIC Educational Resources Information Center

    Ericson, Robert W.

    1974-01-01

    The author summarizes various past studies on job recruitment methods and raises further unanswered questions, especially important to policy-makers, pertaining to the: (1) impact on employers, workers, and society; (2) level of sophistication of employer recruitment method selection; (3) major factors influencing a recruitment selection method;…

  7. Recruitment and Training. Symposium.

    ERIC Educational Resources Information Center

    2002

    This document contains three papers from a symposium on recruitment and training. "College Choice: The State of Marketing and Effective Student Recruitment Strategies" (Fredrick Muyia Nafukho, Michael F. Burnett) reports on a study of the recruitment strategies used by Louisiana State University's admissions office and College of Agriculture that…

  8. Neutrophil ageing is regulated by the microbiome

    PubMed Central

    Zhang, Dachuan; Chen, Grace; Manwani, Deepa; Mortha, Arthur; Xu, Chunliang; Faith, Jeremiah J.; Burk, Robert D.; Kunisaki, Yuya; Jang, Jung-Eun; Scheiermann, Christoph; Merad, Miriam; Frenette, Paul S.

    2015-01-01

    Blood polymorphonuclear neutrophils provide immune protection against pathogens but also may promote tissue injury in inflammatory diseases1,2. Although neutrophils are generally considered as a relatively homogeneous population, evidence for heterogeneity is emerging3,4. Under steady-state conditions, neutrophil heterogeneity may arise from ageing and the replenishment by newly released neutrophils from the bone marrow5. Aged neutrophils up-regulate CXCR4, a receptor allowing their clearance in the bone marrow6,7, with feedback inhibition of neutrophil production via the IL17/G-CSF axis8, and rhythmic modulation of the haematopoietic stem cell niche5. The aged subset also expresses low levels of L-selectin (CD62L)5,9. Previous studies have suggested that in vitro-aged neutrophils exhibit impaired migration and reduced pro-inflammatory properties6,10. Here, we show using in vivo ageing analyses that the neutrophil pro-inflammatory activity correlates positively with their ageing in the circulation. Aged neutrophils represent an overly active subset exhibiting enhanced αMβ2 integrin (Mac-1) activation and neutrophil extracellular trap (NET) formation under inflammatory conditions. Neutrophil ageing is driven by the microbiota via Toll-like receptors (TLRs)- and myeloid differentiation factor 88 (Myd88)-mediated signalling pathways. Depletion of the microbiota significantly reduces the number of circulating aged neutrophils and dramatically improves the pathogenesis and inflammation-related organ damage in models of sickle cell disease or endotoxin-induced septic shock. These results thus identify an unprecedented role for the microbiota in regulating a disease-promoting neutrophil subset. PMID:26374999

  9. Staphylococcal Superantigen-Like Protein 1 and 5 (SSL1 & SSL5) Limit Neutrophil Chemotaxis and Migration through MMP-Inhibition

    PubMed Central

    Koymans, Kirsten J.; Bisschop, Adinda; Vughs, Mignon M.; van Kessel, Kok P. M.; de Haas, Carla J. C.; van Strijp, Jos A. G.

    2016-01-01

    Matrix metalloproteinases (MMPs) are endopeptidases that degrade components of the extracellular matrix, but also modulate inflammation. During bacterial infections, MMPs are important in the recruitment and migration of inflammatory cells. Besides facilitating cell migration by degrading extracellular matrix components, they potentiate the action of several inflammatory molecules, including cytokines, chemokines, and antimicrobial peptides. Staphylococcus aureus secretes an arsenal of immune evasion molecules that interfere with immune cell functioning and hamper proper immune responses. An earlier study identified staphylococcal superantigen-like protein 5 (SSL5) as an MMP9 inhibitor. Since multiple MMPs are involved in neutrophil recruitment, we set up an in-depth search for additional MMP inhibitors by testing a panel of over 70 secreted staphylococcal proteins on the inhibition of the two main neutrophil MMPs: MMP8 (neutrophil collagenase) and MMP9 (neutrophil gelatinase B). We identified SSL1 and SSL5 as potent inhibitors of both neutrophil MMPs and show that they are actually broad range MMP inhibitors. SSL1 and SSL5 prevent MMP-induced cleavage and potentiation of IL-8 and inhibit the migration of neutrophils through collagen. Thus, through MMP-inhibition, SSL1 and SSL5 interfere with neutrophil activation, chemotaxis, and migration, all vital neutrophil functions in bacterial clearance. Studies on MMP-SSL interactions can have therapeutic potential and SSL based derivatives might prove useful in treatment of cancer and destructive inflammatory diseases. PMID:27399672

  10. Nicotine is Chemotactic for Neutrophils and Enhances Neutrophil Responsiveness to Chemotactic Peptides

    NASA Astrophysics Data System (ADS)

    Totti, Noel; McCusker, Kevin T.; Campbell, Edward J.; Griffin, Gail L.; Senior, Robert M.

    1984-01-01

    Neutrophils contribute to chronic bronchitis and pulmonary emphysema associated with cigarette smoking. Nicotine was found to be chemotactic for human neutrophils but not monocytes, with a peak activity at ~ 31 micromolar. In lower concentrations (comparable to those in smokers' plasma), nicotine enhanced the response of neutrophils to two chemotactic peptides. In contrast to most other chemoattractants for neutrophils, however, nicotine did not affect degranulation or superoxide production. Nicotine thus may promote inflammation and consequent lung injury in smokers.

  11. Biomaterial surface-dependent neutrophil mobility.

    PubMed

    Zhou, Yue; Doerschuk, Claire M; Anderson, James M; Marchant, Roger E

    2004-06-15

    Compromised neutrophil function in the presence of an implanted biomaterial may represent an important mechanism that allows for the development of implant-associated infections. Here, human neutrophil mobility has been investigated on a polyurethane (ChronoFlex AR), a hydrophobic surface consisting of an octadecyltrichlorosilane (OTS) self-assembled monolayer, and a glass reference material. Neutrophil mobility was quantified, based on cell movement speed and persistence time obtained from time-lapse optical microscopy, while neutrophil cytoskeletal structures and morphology were visualized using confocal microscopy and atomic force microscopy. Our results show that material surface properties affect neutrophil-surface interactions, as reflected by morphological changes, and the mobility of neutrophils stimulated by N-formylmethionyl-leucyl-phenylalanine (fMLP). In the absence of adsorbed plasma proteins, the mobility of stimulated neutrophils increased with increasing material hydrophobicity from glass, to polyurethane, to OTS. The opposite trend was observed in the presence of adsorbed plasma proteins, such that neutrophil mobility increased with decreasing material hydrophobicity. Analysis of the results showed that the mobility of fMLP-stimulated neutrophils cells was inversely related to the extent of cell spreading on the materials. PMID:15162402

  12. Two neutrophilic dermatoses captured simultaneously on histology

    PubMed Central

    Wlodek, Christina; Bhatt, Nidhi; Kennedy, Cameron

    2016-01-01

    A number of neutrophilic dermatoses are associated with malignancies and their treatment. These rarely occur together in the same patient. A Caucasian 72-year-old male was treated for acute myeloid leukemia (AML) with chemotherapy including daunorubicin and cytarabine. Within 48 hours of commencing treatment, he developed pyrexia and, two days later, disseminated non-tender pink plaques on the limbs and trunk. A skin biopsy showed a dermal interstitial infiltrate of lymphocytes, histiocytoid cells and predominantly neutrophils. This extended into the subcutis, where a neutrophilic lobular panniculitis was seen. These findings are consistent with Sweet’s syndrome. In addition, a neutrophilic and lymphocytic infiltrate was also present around eccrine coils and lower ducts. The eccrine epithelium showed squamous metaplasia with dyskeratosis and sloughing into the lumen. These latter findings are consistent with neutrophilic eccrine hidradenitis (NEH). These two histologically distinct entities form part of the neutrophilic dermatoses that have been described in oncology patients with reports of concurrent or sequential occurrence of various neutrophilic dermatoses in the same patient. Ours, however, is only the second reported case of simultaneously captured Sweet’s and NEH in the setting of AML. The most likely explanation is that of an epiphenomenon, whereby the neutrophilic infiltrate extended around the sweat glands in the context of the neutrophilic dermatosis. PMID:27648385

  13. Two neutrophilic dermatoses captured simultaneously on histology.

    PubMed

    Wlodek, Christina; Bhatt, Nidhi; Kennedy, Cameron

    2016-07-01

    A number of neutrophilic dermatoses are associated with malignancies and their treatment. These rarely occur together in the same patient. A Caucasian 72-year-old male was treated for acute myeloid leukemia (AML) with chemotherapy including daunorubicin and cytarabine. Within 48 hours of commencing treatment, he developed pyrexia and, two days later, disseminated non-tender pink plaques on the limbs and trunk. A skin biopsy showed a dermal interstitial infiltrate of lymphocytes, histiocytoid cells and predominantly neutrophils. This extended into the subcutis, where a neutrophilic lobular panniculitis was seen. These findings are consistent with Sweet's syndrome. In addition, a neutrophilic and lymphocytic infiltrate was also present around eccrine coils and lower ducts. The eccrine epithelium showed squamous metaplasia with dyskeratosis and sloughing into the lumen. These latter findings are consistent with neutrophilic eccrine hidradenitis (NEH). These two histologically distinct entities form part of the neutrophilic dermatoses that have been described in oncology patients with reports of concurrent or sequential occurrence of various neutrophilic dermatoses in the same patient. Ours, however, is only the second reported case of simultaneously captured Sweet's and NEH in the setting of AML. The most likely explanation is that of an epiphenomenon, whereby the neutrophilic infiltrate extended around the sweat glands in the context of the neutrophilic dermatosis. PMID:27648385

  14. Early infection during burn-induced inflammatory response results in increased mortality and p38-mediated neutrophil dysfunction.

    PubMed

    Adediran, Samuel G; Dauplaise, Derrick J; Kasten, Kevin R; Tschöp, Johannes; Dattilo, Jonathan; Goetzman, Holly S; England, Lisa G; Cave, Cindy M; Robinson, Chad T; Caldwell, Charles C

    2010-09-01

    Following burn injury, the host is susceptible to bacterial infections normally cleared by healthy patients. We hypothesized that during the systemic immune response that follows scald injury, the host's altered immune status increases infection susceptibility. Using a murine model of scald injury under inhaled anesthesia followed by intraperitoneal infection, we observed increased neutrophil numbers and function at postburn day (PBD) 1 compared with sham-burned and PBD4 mice. Further, increased mortality, bacteremia, and serum IL-6 were observed in PBD1 mice after Pseudomonas aeruginosa (PA) infection compared with sham-burned and PBD4 mice infected with PA. To examine these disparate responses, we investigated neutrophils isolated at 5 and 24 h following PA infection from PBD1 and sham-burned mice. Five hours after infection, there was no significant difference in number of recruited neutrophils; however, neutrophils from injured mice had decreased activation, active-p38, and oxidative burst compared with sham-burned mice. In direct contrast, 24 h after infection, we observed increased numbers, active-p38, and oxidative burst of neutrophils from PBD1 mice. Finally, we demonstrated that in neutrophils isolated from PBD1 mice, the observed increase in oxidative burst was p38 dependent. Altogether, neutrophil activation and function from thermally injured mice are initially delayed and later exacerbated by a p38-dependent mechanism. This mechanism is likely key to the observed increase in bacterial load and mortality of PBD1 mice infected with PA.

  15. Blockade of p-selectin reduces neutrophil infiltration into the murine testis after ischemia-reperfusion-injury.

    PubMed

    Celebi, M; Paul, A G A

    2008-12-01

    Germ cell specific apoptosis after ischemia-reperfusion (I/R) induced testicular injury is dependent on neutrophil recruitment to the testis. Intravascular adhesion molecules like the P- and E- selectins play an important role in this recruitment.The purpose of this study was to inhibit neutrophil recruitment in I/R induced testicular injury by using a function-blocking monoclonal anti-mouse P-selectin antibody. Adult mice were subjected to a 2 h period of testicular torsion (ischemia) followed by detorsion (reperfusion).Ten minutes after the onset of reperfusion, mice received either 100 microg of a function-blocking monoclonal P-selectin antibody (FBMAB group) or isotype-matched control antibody (IMCA group). Separate groups of mice underwent sham-operation (SO group) or received 500 ng of TNFalpha (IF group) to induce inflammation. Mice were sacrificed 24 h after reperfusion and testiscular interstitial cells were isolated and analyzed for the presence of neutrophils by means of flow cytometry. The function-blocking monoclonal P-selectin antibody reduced neutrophil recruitment in I/R induced testicular injury significantly (FBMAB group as compared to the IMCA group 26 +/- 4 vs. 52 +/- 10% Gr-1 +CD11 b+ of total leucocytes; P < 0.001). Therefore, blocking P-selectin may be therapeutically beneficial to protect postischemic testis.

  16. Bacterial lipopolysaccharide stimulates bovine neutrophil production of TNF-alpha, IL-1beta, IL-12 and IFN-gamma.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Rapid recruitment and bacterial phagocytosis and killing by neutrophils (PMN) are the most effective defenses against establishment of bacterial infection. In addition to their phagocytic and bactericidal properties, PMN may play a key supportive role through secretion of cytokines during the innat...

  17. Transendothelial migration enhances integrin-dependent human neutrophil chemokinesis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transendothelial migration of neutrophils induces phenotypic changes that influence the interactions of neutrophils with extravascular tissue components. To assess the influence of transmigration on neutrophil chemokinetic motility, we used polyethylene glycol hydrogels covalently modified with spec...

  18. The regulatory role of serum response factor pathway in neutrophil inflammatory response

    PubMed Central

    Taylor, Ashley; Halene, Stephanie

    2015-01-01

    Purpose of review Neutrophils rapidly migrate to sites of injury and infection. Egress of neutrophils from the circulation into tissues is a highly regulated process involving several distinct steps. Cell–cell interactions mediated by selectins and integrins and reorganization of the actin cytoskeleton are key mechanisms facilitating appropriate neutrophil recruitment. Neutrophil function is impaired in inherited and acquired disorders, such as leukocyte adhesion deficiency and myelodysplasia. Since the discovery that deletion of all or part of chromosome 5 is the most common genetic aberration in myelodysplasia, the roles of several of the deleted genes have been investigated in hematopoiesis. Several genes encoding proteins of the serum response factor (SRF) pathway are located on 5q. This review focuses, in particular, on the role of SRF in myeloid maturation and neutrophil function. Recent findings SRF and its pathway fulfill multiple complex roles in the regulation of the innate and adaptive immune system. Loss of SRF leads to defects in B-cell and T-cell development. SRF-deficient macrophages fail to spread, transmigrate, and phagocytose bacteria, and SRF-deficient neutrophils show defective chemotaxis in vitro and in vivo with failure of inside-out activation and trafficking of the Mac1 integrin complex. Loss of the formin mammalian Diaphanous 1, a regulator of linear actin polymerization and mediator of Ras homolog family member A signaling to SRF, results in aberrant myeloid differentiation and hyperactivity of the immune system. Summary SRF is an essential transcription factor in hematopoiesis and mature myeloid cell function. SRF regulates neutrophil migration, integrin activation, and trafficking. Disruption of the SRF pathway results in myelodysplasia and immune dysfunction. PMID:25402621

  19. Breast cancer associated a2 isoform vacuolar ATPase immunomodulates neutrophils: potential role in tumor progression.

    PubMed

    Ibrahim, Safaa A; Katara, Gajendra K; Kulshrestha, Arpita; Jaiswal, Mukesh K; Amin, Magdy A; Beaman, Kenneth D

    2015-10-20

    In invasive breast cancer, tumor associated neutrophils (TAN) represent a significant portion of the tumor mass and are associated with increased angiogenesis and metastasis. Identifying the regulatory factors that control TAN behavior will help in developing ideal immunotherapies. Vacuolar ATPases (V-ATPases), multi-subunit proton pumps, are highly expressed in metastatic breast cancer cells. A cleaved peptide from a2 isoform V-ATPase (a2NTD) has immunomodulatory role in tumor microenvironment. Here, we report for the first time the role of V-ATPase in neutrophils modulation. In invasive breast cancer cells, a2NTD was detected and a2V was highly expressed on the surface. Immunohistochemical analysis of invasive breast cancer tissues revealed that increased neutrophil recruitment and blood vessel density correlated with increased a2NTD levels. In order to determine the direct regulatory role of a2NTD on neutrophils, recombinant a2NTD was used for the treatment of neutrophils isolated from the peripheral blood of healthy volunteers. Neutrophils treated with a2NTD (a2Neuɸ) showed increased secretion of IL-1RA, IL-10, CCL-2 and IL-6 that are important mediators in cancer related inflammation. Moreover, a2Neuɸ exhibited an increased production of protumorigenic factors including IL-8, matrix metaloprotinase-9 and vascular endothelial growth factor. Further, functional characterization of a2Neuɸ revealed that a2Neuɸ derived products induce in vitro angiogenesis as well as increase the invasiveness of breast cancer cells. This study establishes the modulatory effect of breast cancer associated a2V on neutrophils, by the action of a2NTD, which has a positive impact on tumor progression, supporting that a2V can be a potential selective target for breast cancer therapy.

  20. Essential Role for Neutrophils in Pathogenesis and Adaptive Immunity in Chlamydia caviae Ocular Infections ▿

    PubMed Central

    Lacy, H. Marie; Bowlin, Anne K.; Hennings, Leah; Scurlock, Amy M.; Nagarajan, Uma M.; Rank, Roger G.

    2011-01-01

    Trachoma, the world's leading cause of preventable blindness, is produced by chronic ocular infection with Chlamydia trachomatis, an obligate intracellular bacterium. While many studies have focused on immune mechanisms for trachoma during chronic stages of infection, less research has targeted immune mechanisms in primary ocular infections, events that could impact chronic responses. The goal of this study was to investigate the function of neutrophils during primary chlamydial ocular infection by using the guinea pig model of Chlamydia caviae inclusion conjunctivitis. We hypothesized that neutrophils help modulate the adaptive response and promote host tissue damage. To test these hypotheses, guinea pigs with primary C. caviae ocular infections were depleted of neutrophils by using rabbit antineutrophil antiserum, and immune responses and immunopathology were evaluated during the first 7 days of infection. Results showed that neutrophil depletion dramatically decreased ocular pathology, both clinically and histologically. The adaptive response was also altered, with increased C. caviae-specific IgA titers in tears and serum and decreased numbers of CD4+ and CD8+ T cells in infected conjunctivae. Additionally, there were changes in conjunctival chemokines and cytokines, such as increased expression of IgA-promoting interleukin-5 and anti-inflammatory transforming growth factor β, along with decreased expression of T cell-recruiting CCL5 (RANTES). This study, the first to investigate the role of neutrophils in primary chlamydial ocular infection, indicates a previously unappreciated role for neutrophils in modulating the adaptive response and suggests a prominent role for neutrophils in chlamydia-associated ocular pathology. PMID:21402767

  1. Essential role for neutrophils in pathogenesis and adaptive immunity in Chlamydia caviae ocular infections.

    PubMed

    Lacy, H Marie; Bowlin, Anne K; Hennings, Leah; Scurlock, Amy M; Nagarajan, Uma M; Rank, Roger G

    2011-05-01

    Trachoma, the world's leading cause of preventable blindness, is produced by chronic ocular infection with Chlamydia trachomatis, an obligate intracellular bacterium. While many studies have focused on immune mechanisms for trachoma during chronic stages of infection, less research has targeted immune mechanisms in primary ocular infections, events that could impact chronic responses. The goal of this study was to investigate the function of neutrophils during primary chlamydial ocular infection by using the guinea pig model of Chlamydia caviae inclusion conjunctivitis. We hypothesized that neutrophils help modulate the adaptive response and promote host tissue damage. To test these hypotheses, guinea pigs with primary C. caviae ocular infections were depleted of neutrophils by using rabbit antineutrophil antiserum, and immune responses and immunopathology were evaluated during the first 7 days of infection. Results showed that neutrophil depletion dramatically decreased ocular pathology, both clinically and histologically. The adaptive response was also altered, with increased C. caviae-specific IgA titers in tears and serum and decreased numbers of CD4(+) and CD8(+) T cells in infected conjunctivae. Additionally, there were changes in conjunctival chemokines and cytokines, such as increased expression of IgA-promoting interleukin-5 and anti-inflammatory transforming growth factor β, along with decreased expression of T cell-recruiting CCL5 (RANTES). This study, the first to investigate the role of neutrophils in primary chlamydial ocular infection, indicates a previously unappreciated role for neutrophils in modulating the adaptive response and suggests a prominent role for neutrophils in chlamydia-associated ocular pathology. PMID:21402767

  2. Necrostatin-1 enhances the resolution of inflammation by specifically inducing neutrophil apoptosis

    PubMed Central

    Jie, Hongyu; He, Yi; Huang, Xuechan; Zhou, Qingyou; Han, Yanping; Li, Xing; Bai, Yongkun; Sun, Erwei

    2016-01-01

    Neutrophils play a central role in innate immunity and are rapidly recruited to sites of infection and injury. Neutrophil apoptosis is essential for the successful resolution of inflammation. Necrostatin-1 (Nec-1,methyl-thiohydantoin-tryptophan (MTH-Trp)), is a potent and specific inhibitor of necroptosis[1] (a newly identified type of cell death representing a form of programmed necrosis or regulated non apoptotic cell death) by inhibiting the receptor interacting protein 1(RIP1) kinase. Here we report that Nec-1 specifically induces caspase-dependent neutrophils apoptosis and overrides powerful anti-apoptosis signaling from survival factors such as GM-CSF and LPS. We showed that Nec-1 markedly enhanced the resolution of established neutrophil-dependent inflammation in LPS-induced acute lung injury in mice. We also provided evidence that Nec-1 promoted apoptosis by reducing the expression of the anti-apoptotic protein Mcl-1 and increasing the expression of pro-apoptotic protein Bax. Thus, Nec-1 is not only an inhibitor of necroptosis, but also a promoter of apoptosis, of neutrophils, enhancing the resolution of established inflammation by inducing apoptosis of inflammatory cells. Our results suggest that Nec-1 may have potential roles for the treatment of diseases with increased or persistent inflammatory responses. PMID:27027357

  3. Involvement of purinergic signaling on nitric oxide production by neutrophils stimulated with Trichomonas vaginalis.

    PubMed

    Frasson, Amanda Piccoli; De Carli, Geraldo Attilio; Bonan, Carla Denise; Tasca, Tiana

    2012-03-01

    Trichomonas vaginalis is a parasite from the human urogenital tract that causes trichomonosis, the most prevalent non-viral sexually transmitted disease. The neutrophil infiltration has been considered to be primarily responsible for cytological changes observed at infection site, and the chemoattractants can play an important role in this leukocytic recruitment. Nitric oxide (NO) is one of the most widespread mediator compounds, and it is implicated in modulation of immunological mechanisms. Extracellular nucleotides and nucleosides are signaling molecules involved in several processes, including immune responses and control of leukocyte trafficking. Ectonucleoside triphosphate diphosphohydrolase members, ecto-5'-nucleotidase, and adenosine deaminase (ectoADA) have been characterized in T. vaginalis. Herein, we investigated the effects of purinergic system on NO production by neutrophils stimulated with T. vaginalis. The trophozoites were able to induce a high NO synthesis by neutrophils through iNOS pathway. The extracellular nucleotides ATP, ADP, and ATPγS (a non-hydrolyzable ATP analog) showed no significant change in NO secretion. In contrast, adenosine and its degradation product, inosine, promoted a low production of the compound. The immunosuppressive effect of adenosine upon NO release by neutrophils occurred due to adenosine A(2A) receptor activation. The ecto-5'-nucleotidase activity displayed by T. vaginalis was shown to be important in adenosine generation, indicating the efficiency of purinergic cascade. Our data suggest the influence of purinergic signaling, specifically adenosinergic system, on NO production by neutrophils in T. vaginalis infection, contributing to the immunological aspects of disease.

  4. Superoxide Induces Neutrophil Extracellular Trap Formation in a TLR-4 and NOX-Dependent Mechanism

    PubMed Central

    Al-Khafaji, Ahmed B; Tohme, Samer; Yazdani, Hamza Obaid; Miller, David; Huang, Hai; Tsung, Allan

    2016-01-01

    Neutrophils constitute the early innate immune response to perceived infectious and sterile threats. Neutrophil extracellular traps (NETs) are a novel mechanism to counter pathogenic invasion and sequelae of ischemia, including cell death and oxidative stress. Superoxide is a radical intermediate of oxygen metabolism produced by parenchymal and nonparenchymal hepatic cells, and is a hallmark of oxidative stress after liver ischemia-reperfusion (I/R). While extracellular superoxide recruits neutrophils to the liver and initiates sterile inflammatory injury, it is unknown whether superoxide induces the formation of NETs. We hypothesize that superoxide induces NET formation through a signaling cascade involving Toll-like receptor 4 (TLR-4) and neutrophil NADPH oxidase (NOX). We treated neutrophils with extracellular superoxide and observed NET DNA release, histone H3 citrullination and increased levels of MPO-DNA complexes occurring in a TLR-4–dependent manner. Inhibition of superoxide generation by allopurinol and inhibition of NOX by diphenyleneiodonium prevented NET formation. When mice were subjected to warm liver I/R, we found significant NET formation associated with liver necrosis and increased serum ALT in TLR-4 WT but not TLR-4 KO mice. To reduce circulating superoxide, we pretreated mice undergoing I/R with allopurinol and N-acetylcysteine, which resulted in decreased NETs and ameliorated liver injury. Our study demonstrates a requirement for TLR-4 and NOX in superoxide-induced NETs, and suggests involvement of superoxide-induced NETs in pathophysiologic settings. PMID:27453505

  5. DAP12 expression in lung macrophages mediates ischemia/reperfusion injury by promoting neutrophil extravasation.

    PubMed

    Spahn, Jessica H; Li, Wenjun; Bribriesco, Alejandro C; Liu, Jie; Shen, Hua; Ibricevic, Aida; Pan, Jie-Hong; Zinselmeyer, Bernd H; Brody, Steven L; Goldstein, Daniel R; Krupnick, Alexander S; Gelman, Andrew E; Miller, Mark J; Kreisel, Daniel

    2015-04-15

    Neutrophils are critical mediators of innate immune responses and contribute to tissue injury. However, immune pathways that regulate neutrophil recruitment to injured tissues during noninfectious inflammation remain poorly understood. DAP12 is a cell membrane-associated protein that is expressed in myeloid cells and can either augment or dampen innate inflammatory responses during infections. To elucidate the role of DAP12 in pulmonary ischemia/reperfusion injury (IRI), we took advantage of a clinically relevant mouse model of transplant-mediated lung IRI. This technique allowed us to dissect the importance of DAP12 in tissue-resident cells and those that infiltrate injured tissue from the periphery during noninfectious inflammation. Macrophages in both mouse and human lungs that have been subjected to cold ischemic storage express DAP12. We found that donor, but not recipient, deficiency in DAP12 protected against pulmonary IRI. Analysis of the immune response showed that DAP12 promotes the survival of tissue-resident alveolar macrophages and contributes to local production of neutrophil chemoattractants. Intravital imaging demonstrated a transendothelial migration defect into DAP12-deficient lungs, which can be rescued by local administration of the neutrophil chemokine CXCL2. We have uncovered a previously unrecognized role for DAP12 expression in tissue-resident alveolar macrophages in mediating acute noninfectious tissue injury through regulation of neutrophil trafficking.

  6. CXCL1/KC and CXCL5/LIX are selectively produced by corneal fibroblasts and mediate neutrophil infiltration to the corneal stroma in LPS keratitis

    PubMed Central

    Lin, Michelle; Carlson, Eric; Diaconu, Eugenia; Pearlman, Eric

    2012-01-01

    The severity of corneal inflammation depends on the activity of infiltrating neutrophils responding to chemotactic factors such as CXC chemokines. This study examines the relative contribution of CXCL1/keratinocyte-derived chemokine (KC), CXCL2/monocyte-inhibitory protein-2 (MIP-2), and CXCL5/LPS-induced chemokine (LIX) in neutrophil recruitment to the corneal stroma during LPS keratitis, where neutrophils infiltrate the corneal stroma at 6 h after LPS injection and peak at 24 h. Consistent with this timeframe, KC was detected after 3 h, reached peak levels at 24 h, and decreased thereafter. In contrast, LIX production was not detected until 8 h after injection and peaked at 24 h. MIP-2 was detected at 3 h but did not reach the levels of KC and LIX. Cell types associated with corneal inflammation produced markedly different chemokines in vitro: Murine corneal fibroblasts (MK/T-1) produced LIX and KC in response to LPS but did not produce MIP-2, whereas peritoneal macrophages and neutrophils produced MIP-2 and KC but did not produce LIX. To determine the role of these chemokines in neutrophil recruitment to the cornea, anti-LIX, anti-KC, or anti-MIP-2 was injected into the corneal stroma of enhanced GFP chimeric mice prior to LPS, and total cell and neutrophil infiltration was examined. Antibody to LIX and KC, injected individually or in combination, significantly inhibited neutrophil recruitment to the cornea, whereas anti-MIP-2 had no inhibitory effect. Together, these findings demonstrate cell-specific production of CXC chemokines and show that LIX and KC mediate neutrophil recruitment into the cornea during LPS keratitis. PMID:17110418

  7. The Novel Functions of the PLC/PKC/PKD Signaling Axis in G Protein-Coupled Receptor-Mediated Chemotaxis of Neutrophils

    PubMed Central

    Xu, Xuehua; Jin, Tian

    2015-01-01

    Chemotaxis, a directional cell migration guided by extracellular chemoattractant gradients, plays an essential role in the recruitment of neutrophils to sites of inflammation. Chemotaxis is mediated by the G protein-coupled receptor (GPCR) signaling pathway. Extracellular stimuli trigger activation of the PLC/PKC/PKD signaling axis, which controls several signaling pathways. Here, we concentrate on the novel functions of PLC/PKC/PKD signaling in GPCR-mediated chemotaxis of neutrophils. PMID:26605346

  8. A Radical Break: Restraining Neutrophil Migration.

    PubMed

    Renkawitz, Jörg; Sixt, Michael

    2016-09-12

    When neutrophils infiltrate a site of inflammation, they have to stop at the right place to exert their effector function. In this issue of Developmental Cell, Wang et al. (2016) show that neutrophils sense reactive oxygen species via the TRPM2 channel to arrest migration at their target site. PMID:27623379

  9. Mechanotransduction in neutrophil activation and deactivation.

    PubMed

    Ekpenyong, Andrew E; Toepfner, Nicole; Chilvers, Edwin R; Guck, Jochen

    2015-11-01

    Mechanotransduction refers to the processes through which cells sense mechanical stimuli by converting them to biochemical signals and, thus, eliciting specific cellular responses. Cells sense mechanical stimuli from their 3D environment, including the extracellular matrix, neighboring cells and other mechanical forces. Incidentally, the emerging concept of mechanical homeostasis,long term or chronic regulation of mechanical properties, seems to apply to neutrophils in a peculiar manner, owing to neutrophils' ability to dynamically switch between the activated/primed and deactivated/deprimed states. While neutrophil activation has been known for over a century, its deactivation is a relatively recent discovery. Even more intriguing is the reversibility of neutrophil activation and deactivation. We review and critically evaluate recent findings that suggest physiological roles for neutrophil activation and deactivation and discuss possible mechanisms by which mechanical stimuli can drive the oscillation of neutrophils between the activated and resting states. We highlight several molecules that have been identified in neutrophil mechanotransduction, including cell adhesion and transmembrane receptors, cytoskeletal and ion channel molecules. The physiological and pathophysiological implications of such mechanically induced signal transduction in neutrophils are highlighted as a basis for future work. This article is part of a Special Issue entitled: Mechanobiology. PMID:26211453

  10. Neutrophil survival in the death zone.

    PubMed

    Croker, Ben A

    2014-01-16

    In this issue of Blood, Thompson et al reveal a key role for hypoxia-inducible factor (HIF)-2a in the adaptation of neutrophils to hypoxia. Tissue hypoxia is a common feature of trauma and inflammation. Infiltrating neutrophils must adapt to this low-oxygen environment to satisfy the metabolic and functional demands of an immune response.

  11. Chronic neutrophilic leukaemia and plasma cell-related neutrophilic leukaemoid reactions.

    PubMed

    Bain, Barbara J; Ahmad, Shahzaib

    2015-11-01

    Many cases reported as 'chronic neutrophilic leukaemia' have had an associated plasma cell neoplasm. Recent evidence suggests that the great majority of such cases represent a neutrophilic leukaemoid reaction to the underlying multiple myeloma or monoclonal gammopathy of undetermined significance. We have analysed all accessible reported cases to clarify the likely diagnosis and to ascertain whether toxic granulation, Döhle bodies and an increased neutrophil alkaline phosphatase score were useful in making a distinction between chronic neutrophilic leukaemia and a neutrophilic leukaemoid reaction. We established that all these changes occur in both conditions. Toxic granulation and Döhle bodies are more consistently present in leukaemoid reactions but also occur quite frequently in chronic neutrophilic leukaemia. The neutrophil alkaline phosphatase score is increased in both conditions and is of no value in making a distinction.

  12. How Neutrophils Shape Adaptive Immune Responses

    PubMed Central

    Leliefeld, Pieter H. C.; Koenderman, Leo; Pillay, Janesh

    2015-01-01

    Neutrophils are classically considered as cells pivotal for the first line of defense against invading pathogens. In recent years, evidence has accumulated that they are also important in the orchestration of adaptive immunity. Neutrophils rapidly migrate in high numbers to sites of inflammation (e.g., infection, tissue damage, and cancer) and are subsequently able to migrate to draining lymph nodes (LNs). Both at the site of inflammation as well as in the LNs, neutrophils can engage with lymphocytes and antigen-presenting cells. This crosstalk occurs either directly via cell–cell contact or via mediators, such as proteases, cytokines, and radical oxygen species. In this review, we will discuss the current knowledge regarding locations and mechanisms of interaction between neutrophils and lymphocytes in the context of homeostasis and various pathological conditions. In addition, we will highlight the complexity of the microenvironment that is involved in the generation of suppressive or stimulatory neutrophil phenotypes. PMID:26441976

  13. How Neutrophils Shape Adaptive Immune Responses.

    PubMed

    Leliefeld, Pieter H C; Koenderman, Leo; Pillay, Janesh

    2015-01-01

    Neutrophils are classically considered as cells pivotal for the first line of defense against invading pathogens. In recent years, evidence has accumulated that they are also important in the orchestration of adaptive immunity. Neutrophils rapidly migrate in high numbers to sites of inflammation (e.g., infection, tissue damage, and cancer) and are subsequently able to migrate to draining lymph nodes (LNs). Both at the site of inflammation as well as in the LNs, neutrophils can engage with lymphocytes and antigen-presenting cells. This crosstalk occurs either directly via cell-cell contact or via mediators, such as proteases, cytokines, and radical oxygen species. In this review, we will discuss the current knowledge regarding locations and mechanisms of interaction between neutrophils and lymphocytes in the context of homeostasis and various pathological conditions. In addition, we will highlight the complexity of the microenvironment that is involved in the generation of suppressive or stimulatory neutrophil phenotypes. PMID:26441976

  14. Neutrophil uptake of vaccinia virus in vitro

    SciTech Connect

    West, B.C.; Eschete, M.L.; Cox, M.E.; King, J.W.

    1987-10-01

    We studied human neutrophils for uptake of vaccinia virus. Uptake was determined radiometrically and by electron microscopy. Vaccinia virus was labeled with /sup 14/C or /sup 3/H, incubated with neutrophils, and quantified in neutrophil pellets in a new radiometric phagocytosis assay. Better results were obtained from assays of (/sup 3/H)thymidine-labeled virus; uptake increased through 1 hr and then plateaued. Phagocytosis of 3H-labeled Staphylococcus aureus was normal. Uptake of virus was serum dependent. Hexose monophosphate shunt activity was measured by two methods. No /sup 14/CO/sub 2/ from (/sup 14/C)1-glucose accompanied uptake of vaccinia virus, in contrast to the respiratory burst accompanying bacterial phagocytosis. Electron microscopy showed intact to slightly digested intraphagolysosomal vaccinia virus. Pock reduction assay showed a decrease in viral content due to neutrophils until 6 hr of incubation, when a modest but significant increase was observed. Thus, neutrophil uptake of vaccinia virus is distinguished from bacterial phagocytosis.

  15. Dynamic interactions of neutrophils and biofilms

    PubMed Central

    Hirschfeld, Josefine

    2014-01-01

    Background The majority of microbial infections in humans are biofilm-associated and difficult to treat, as biofilms are highly resistant to antimicrobial agents and protect themselves from external threats in various ways. Biofilms are tenaciously attached to surfaces and impede the ability of host defense molecules and cells to penetrate them. On the other hand, some biofilms are beneficial for the host and contain protective microorganisms. Microbes in biofilms express pathogen-associated molecular patterns and epitopes that can be recognized by innate immune cells and opsonins, leading to activation of neutrophils and other leukocytes. Neutrophils are part of the first line of defense and have multiple antimicrobial strategies allowing them to attack pathogenic biofilms. Objective/design In this paper, interaction modes of neutrophils with biofilms are reviewed. Antimicrobial strategies of neutrophils and the counteractions of the biofilm communities, with special attention to oral biofilms, are presented. Moreover, possible adverse effects of neutrophil activity and their biofilm-promoting side effects are discussed. Results/conclusion Biofilms are partially, but not entirely, protected against neutrophil assault, which include the processes of phagocytosis, degranulation, and formation of neutrophil extracellular traps. However, virulence factors of microorganisms, microbial composition, and properties of the extracellular matrix determine whether a biofilm and subsequent microbial spread can be controlled by neutrophils and other host defense factors. Besides, neutrophils may inadvertently contribute to the physical and ecological stability of biofilms by promoting selection of more resistant strains. Moreover, neutrophil enzymes can degrade collagen and other proteins and, as a result, cause harm to the host tissues. These parameters could be crucial factors in the onset of periodontal inflammation and the subsequent tissue breakdown. PMID:25523872

  16. Human neutrophils in auto-immunity.

    PubMed

    Thieblemont, Nathalie; Wright, Helen L; Edwards, Steven W; Witko-Sarsat, Véronique

    2016-04-01

    Human neutrophils have great capacity to cause tissue damage in inflammatory diseases via their inappropriate activation to release reactive oxygen species (ROS), proteases and other tissue-damaging molecules. Furthermore, activated neutrophils can release a wide variety of cytokines and chemokines that can regulate almost every element of the immune system. In addition to these important immuno-regulatory processes, activated neutrophils can also release, expose or generate neoepitopes that have the potential to break immune tolerance and result in the generation of autoantibodies, that characterise a number of human auto-immune diseases. For example, in vasculitis, anti-neutrophil cytoplasmic antibodies (ANCA) that are directed against proteinase 3 or myeloperoxidase are neutrophil-derived autoantigens and activated neutrophils are the main effector cells of vascular damage. In other auto-immune diseases, these neutrophil-derived neoepitopes may arise from a number of processes that include release of granule enzymes and ROS, changes in the properties of components of their plasma membrane as a result of activation or apoptosis, and via the release of Neutrophil Extracellular Traps (NETs). NETs are extracellular structures that contain chromatin that is decorated with granule enzymes (including citrullinated proteins) that can act as neo-epitopes to generate auto-immunity. This review therefore describes the processes that can result in neutrophil-mediated auto-immunity, and the role of neutrophils in the molecular pathologies of auto-immune diseases such as vasculitis, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). We discuss the potential role of NETs in these processes and some of the debate in the literature regarding the role of this phenomenon in microbial killing, cell death and auto-immunity. PMID:27036091

  17. Neutrophils establish rapid and robust WAVE complex polarity in an actin-dependent fashion

    PubMed Central

    Millius, Arthur; Dandekar, Sheel N.; Houk, Andrew R.; Weiner, Orion D.

    2009-01-01

    Asymmetric intracellular signals enable cells to migrate in response to external cues. The multiprotein WAVE (SCAR/WASF) complex activates the actin-nucleating Arp2/3 complex [1-4] and localizes to propagating “waves”, which direct actin assembly during neutrophil migration [5, 6]. Here, we observe similar WAVE complex dynamics in other mammalian cells and analyze WAVE complex dynamics during the establishment of neutrophil polarity. Earlier models proposed that either spatially-biased generation [7] or selection of protrusions [8] enables chemotaxis. These models require existing morphological polarity to control protrusions. Similar spatially-biased generation and selection of WAVE complex recruitment occur in morphologically unpolarized neutrophils during the development of their first protrusions. Additionally, several mechanisms limit WAVE complex recruitment during polarization and movement: intrinsic cues restrict WAVE complex distribution during the establishment of polarity, and asymmetric intracellular signals constrain WAVE complex distribution in morphologically polarized cells. External gradients can overcome both intrinsic biases and control WAVE complex localization. Following latrunculin-mediated inhibition of actin polymerization, addition and removal of agonist gradients globally recruits and releases the WAVE complex from the membrane. Under these conditions the WAVE complex no longer polarizes, despite the presence of strong external gradients. Thus, actin polymer and the WAVE complex reciprocally interact during polarization. PMID:19200726

  18. Heart-resident CCR2+ macrophages promote neutrophil extravasation through TLR9/MyD88/CXCL5 signaling

    PubMed Central

    Li, Wenjun; Higashikubo, Ryuji; Saunders, Brian T.; Bharat, Ankit; Goldstein, Daniel R.; Krupnick, Alexander S.; Gelman, Andrew E.; Lavine, Kory J.

    2016-01-01

    It is well established that maladaptive innate immune responses to sterile tissue injury represent a fundamental mechanism of disease pathogenesis. In the context of cardiac ischemia reperfusion injury, neutrophils enter inflamed heart tissue, where they play an important role in potentiating tissue damage and contributing to contractile dysfunction. The precise mechanisms that govern how neutrophils are recruited to and enter the injured heart are incompletely understood. Using a model of cardiac transplant–mediated ischemia reperfusion injury and intravital 2-photon imaging of beating mouse hearts, we determined that tissue-resident CCR2+ monocyte–derived macrophages are essential mediators of neutrophil recruitment into ischemic myocardial tissue. Our studies revealed that neutrophil extravasation is mediated by a TLR9/MyD88/CXCL5 pathway. Intravital 2-photon imaging demonstrated that CXCL2 and CXCL5 play critical and nonredundant roles in guiding neutrophil adhesion and crawling, respectively. Together, these findings uncover a specific role for a tissue-resident monocyte-derived macrophage subset in sterile tissue inflammation and support the evolving concept that macrophage ontogeny is an important determinant of function. Furthermore, our results provide the framework for targeting of cell-specific signaling pathways in myocardial ischemia reperfusion injury. PMID:27536731

  19. Proliferating cell nuclear antigen in neutrophil fate.

    PubMed

    Witko-Sarsat, Véronique; Ohayon, Delphine

    2016-09-01

    The life span of a neutrophil is a tightly regulated process as extended survival is beneficial for pathogen elimination and cell death necessary to prevent cytotoxic content release from activated neutrophils at the inflammatory site. Therefore, the control between survival and death must be a dynamic process. We have previously described that proliferating cell nuclear antigen (PCNA) which is known as a nuclear protein pivotal in DNA synthesis, is a key element in controlling neutrophil survival through its association with procaspases. Contrary to the dogma which asserted that PCNA has a strictly nuclear function, in mature neutrophils, PCNA is present exclusively within the cytosol due to its nuclear export at the end of the granulocytic differentiation. More recent studies are consistent with the notion that the cytosolic scaffold of PCNA is aimed at modulating neutrophil fate rather than simply preventing death. Ultimately, targeting neutrophil survival might have important applications not just in the field of immunology and inflammation, but also in hematology and transfusion. The neutrophil emerges as a unique and powerful cellular model to unravel the basic mechanisms governing the cell cycle-independent functions of PCNA and should be considered as a leader of the pack. PMID:27558345

  20. Candida albicans escapes from mouse neutrophils.

    PubMed

    Ermert, David; Niemiec, Maria J; Röhm, Marc; Glenthøj, Andreas; Borregaard, Niels; Urban, Constantin F

    2013-08-01

    Candida albicans, the most commonly isolated human fungal pathogen, is able to grow as budding yeasts or filamentous forms, such as hyphae. The ability to switch morphology has been attributed a crucial role for the pathogenesis of C. albicans. To mimic disseminated candidiasis in humans, the mouse is the most widely used model organism. Neutrophils are essential immune cells to prevent opportunistic mycoses. To explore potential differences between the rodent infection model and the human host, we compared the interactions of C. albicans with neutrophil granulocytes from mice and humans. We revealed that murine neutrophils exhibited a significantly lower ability to kill C. albicans than their human counterparts. Strikingly, C. albicans yeast cells formed germ tubes upon internalization by murine neutrophils, eventually rupturing the neutrophil membrane and thereby, killing the phagocyte. On the contrary, growth and subsequent escape of C. albicans are blocked inside human neutrophils. According to our findings, this blockage in human neutrophils might be a result of higher levels of MPO activity and the presence of α-defensins. We therefore outline differences in antifungal immune defense between humans and mouse strains, which facilitates a more accurate interpretation of in vivo results.

  1. NET formation induced by Pseudomonas aeruginosa cystic fibrosis isolates measured as release of myeloperoxidase-DNA and neutrophil elastase-DNA complexes.

    PubMed

    Yoo, Dae-goon; Floyd, Madison; Winn, Matthew; Moskowitz, Samuel M; Rada, Balázs

    2014-08-01

    Cystic fibrosis (CF) airway disease is characterized by Pseudomonas aeruginosa infection and recruitment of neutrophil granulocytes. Neutrophil granule components (myeloperoxidase (MPO), human neutrophil elastase (HNE)), extracellular DNA and P. aeruginosa can all be found in the CF respiratory tract and have all been associated with worsening CF lung function. Pseudomonas-induced formation of neutrophil extracellular traps (NETs) offers a likely mechanism for release of MPO, HNE and DNA from neutrophils. NETs are composed of a DNA backbone decorated with granule proteins like MPO and HNE. Here we sought to examine whether CF clinical isolates of Pseudomonas are capable of inducing NET release from human neutrophil granulocytes. We used two methods to quantify NETs. We modified a previously employed ELISA that detects MPO-DNA complexes and established a new HNE-DNA ELISA. We show that these methods reliably quantify MPO-DNA and HNE-DNA complexes, measures of NET formation. We have found that CF isolates of P. aeruginosa stimulate robust respiratory burst and NET release in human neutrophils. By comparing paired "early" and "late" bacterial isolates obtained from the same CF patient we have found that early isolates induced significantly more NET release than late isolates. Our data support that Pseudomonas-induced NET release represents an important mechanism for release of neutrophil-derived CF inflammatory mediators, and confirm that decreased induction of NET formation is required for long-term adaptation of P. aeruginosa to CF airways.

  2. Platelet–neutrophil interactions under thromboinflammatory conditions

    PubMed Central

    Li, Jing; Kim, Kyungho; Barazia, Andrew; Tseng, Alan

    2015-01-01

    Platelets primarily mediate hemostasis and thrombosis, whereas leukocytes are responsible for immune responses. Since platelets interact with leukocytes at the site of vascular injury, thrombosis and vascular inflammation are closely intertwined and occur consecutively. Recent studies using real-time imaging technology demonstrated that platelet–neutrophil interactions on the activated endothelium are an important determinant of microvascular occlusion during thromboinflammatory disease in which inflammation is coupled to thrombosis. Although the major receptors and counter receptors have been identified, it remains poorly understood how heterotypic platelet–neutrophil interactions are regulated under disease conditions. This review discusses our current understanding of the regulatory mechanisms of platelet– neutrophil interactions in thromboinflammatory disease. PMID:25650236

  3. Neutrophils Directly Recognize Group B Streptococci and Contribute to Interleukin-1β Production during Infection

    PubMed Central

    Mohammadi, Nastaran; Midiri, Angelina; Mancuso, Giuseppe; Patanè, Francesco; Venza, Mario; Venza, Isabella; Passantino, Annamaria; Galbo, Roberta; Teti, Giuseppe; Beninati, Concetta; Biondo, Carmelo

    2016-01-01

    Previous studies have shown that the pro-inflammatory cytokine IL-1β has a crucial role in host defenses against group B streptococcus (GBS), a frequent human pathogen, by recruiting neutrophils to infection sites. We examined here the cell types and mechanisms involved in IL-1β production during infection. Using a GBS-induced peritonitis model in mice, we first found that a large proportion of exudate cells contain intracellular IL-1β by immunofluorescence. Of the IL-1β positive cells, 82 and 7% were neutrophils and macrophages, respectively, suggesting that the former cell type might significantly contribute to IL-1β production. Accordingly, depletion of neutrophils with anti-Ly6G antibodies resulted in a significant reduction in the levels of IL-1β, but not of TNF-α or IL-6. We next found that neutrophils are capable of releasing mature IL-1β and TNF-α directly in response to in vitro stimulation with GBS. The production of pro-IL-1β and TNF-α in these cells required the Toll-like receptor (TLR) adaptor MyD88 and the chaperone protein UNC93B1, which is involved in mobilization of a subfamily of TLRs to the endosomes. Moreover, pro-IL-1β processing and IL-1β release was triggered by GBS hemolysin and required components of the canonical inflammasome, including caspase-1, ASC and NLRP3. Collectively our findings indicate that neutrophils make a significant contribution to IL-1β production during GBS infection, thereby amplifying their own recruitment. These cells directly recognize GBS by means of endosomal TLRs and cytosolic sensors, leading to activation of the caspase-1 inflammasome. PMID:27509078

  4. Neutrophils Directly Recognize Group B Streptococci and Contribute to Interleukin-1β Production during Infection.

    PubMed

    Mohammadi, Nastaran; Midiri, Angelina; Mancuso, Giuseppe; Patanè, Francesco; Venza, Mario; Venza, Isabella; Passantino, Annamaria; Galbo, Roberta; Teti, Giuseppe; Beninati, Concetta; Biondo, Carmelo

    2016-01-01

    Previous studies have shown that the pro-inflammatory cytokine IL-1β has a crucial role in host defenses against group B streptococcus (GBS), a frequent human pathogen, by recruiting neutrophils to infection sites. We examined here the cell types and mechanisms involved in IL-1β production during infection. Using a GBS-induced peritonitis model in mice, we first found that a large proportion of exudate cells contain intracellular IL-1β by immunofluorescence. Of the IL-1β positive cells, 82 and 7% were neutrophils and macrophages, respectively, suggesting that the former cell type might significantly contribute to IL-1β production. Accordingly, depletion of neutrophils with anti-Ly6G antibodies resulted in a significant reduction in the levels of IL-1β, but not of TNF-α or IL-6. We next found that neutrophils are capable of releasing mature IL-1β and TNF-α directly in response to in vitro stimulation with GBS. The production of pro-IL-1β and TNF-α in these cells required the Toll-like receptor (TLR) adaptor MyD88 and the chaperone protein UNC93B1, which is involved in mobilization of a subfamily of TLRs to the endosomes. Moreover, pro-IL-1β processing and IL-1β release was triggered by GBS hemolysin and required components of the canonical inflammasome, including caspase-1, ASC and NLRP3. Collectively our findings indicate that neutrophils make a significant contribution to IL-1β production during GBS infection, thereby amplifying their own recruitment. These cells directly recognize GBS by means of endosomal TLRs and cytosolic sensors, leading to activation of the caspase-1 inflammasome. PMID:27509078

  5. Critical role of the C5a-activated neutrophils in high-fat diet-induced vascular inflammation

    PubMed Central

    Osaka, Mizuko; Ito, Shunsuke; Honda, Masaki; Inomata, Yukihiro; Egashira, Kensuke; Yoshida, Masayuki

    2016-01-01

    Exceed and chronic high-fat diet (HFD) contributes to the diagnosis and development of atherosclerosis, obesity, and metabolic syndrome. However, the key molecular component(s) triggered by HFD responsible for initiating vascular inflammation remain unknown. We observed that feeding HFD for 4 weeks is sufficient to induce leukocyte recruitment in the femoral artery of wild-type mice. Neutrophil- and monocyte-depletion analyses confirmed the preferential recruitment of neutrophils in these mice. Protein analysis of sera from HFD-fed mice revealed a marked elevation of complement component C5a levels. Exogenous C5a alone induced leukocyte recruitment, which was abolished by a C5a-receptor antagonist. We also examined the role of neutrophil-derived MCP-1 in accumulation of leukocytes in the artery. These results demonstrated a previously unrecognized role for C5a and neutrophils in the early onset of HFD-induced vascular inflammation. Further study may help in elucidating a novel regulatory pathway to control diet-induced inflammation such as that in case of atherosclerosis. PMID:26893238

  6. Intravital Imaging of Neutrophil Priming Using IL-1β Promoter-driven DsRed Reporter Mice.

    PubMed

    Yao, Yi; Liu, Yun; Takashima, Akira

    2016-01-01

    Neutrophils are the most abundant leukocytes in human blood circulation and are quickly recruited to inflammatory sites. Priming is a critical event that enhances the phagocytic functionality of neutrophils. Although extensive studies have unveiled the existence and importance of neutrophil priming during infection and injury, means of visualizing this process in vivo have been unavailable. The protocol provided enables monitoring of the dynamic process of neutrophil priming in living animals by combining three methodologies: 1) DsRed reporter signal - used as a measure of priming 2) in vivo neutrophil labeling - achieved by injection of fluorescence-conjugated anti-lymphocyte antigen 6G (Ly6G) monoclonal antibody (mAb) and 3) intravital confocal imaging. Several critical steps are involved in this protocol: oxazolone-induced mouse ear skin inflammation, appropriate sedation of animals, repeated injections of anti-Ly6G mAb, and prevention of focus drift during imaging. Although a few limitations have been observed, such as the limit of continuous imaging time (~ 8 hr) in one mouse and the leakage of fluorescein isothiocyanate-dextran from blood vessels in the inflammatory state, this protocol provides a fundamental framework for intravital imaging of primed neutrophil behavior and function, which can easily be expanded to examination of other immune cells in mouse inflammation models. PMID:27403648

  7. ICAM-1-dependent and ICAM-1-independent neutrophil lung infiltration by porcine reproductive and respiratory syndrome virus infection.

    PubMed

    Liu, Jie; Hou, Make; Yan, Meiping; Lü, Xinhui; Gu, Wei; Zhang, Songlin; Gao, Jianfeng; Liu, Bang; Wu, Xiaoxiong; Liu, Guoquan

    2015-08-01

    Neutrophils are innate immune cells that play a crucial role in the first line of host defense. It is also known that neutrophil lung recruitment and infiltration may cause lung injury. The roles of neutrophils in virus infection-induced lung injury are not clear. We explore the mechanisms of neutrophil lung infiltration and the potential biomarkers for lung injury in a swine model of lung injury caused by natural or experimental porcine reproductive and respiratory syndrome virus (PRRSV) infection. Neutrophil lung infiltration was determined by measurement of myeloperoxidase expression and enzyme activity of lung tissues. Myeloperoxidase expression and enzyme activity were dramatically increased in the naturally and experimentally infected lung tissues. Chemokine analysis by quantitative PCR and ELISA showed that IL-8 expression was increased in both infections, while monocyte chemoattractant protein-1 expression was increased only in experimentally infected lung tissues. Expression of the cell adhesion molecules VCAM-1 and ICAM-1 was measured by quantitative PCR and Western blotting. VCAM-1 expression was increased in experimentally and naturally infected lungs, whereas ICAM-1 expression was increased only in the naturally infected lung samples. Our results suggest that neutrophil lung infiltrations in the infected animals are both ICAM-1- and -independent and that combined expression of VCAM-1 and IL-8 may serve as the biomarker for lung injury induced by virus infection.

  8. The hepatitis B virus e antigen suppresses the respiratory burst and mobility of human monocytes and neutrophils.

    PubMed

    Leu, Chuen-Miin; Lu, Yong-Chen; Peng, Wei-Li; Chu, Hsin-Tzu; Hu, Cheng-po

    2014-11-01

    The Hepatitis B virus (HBV) e antigen (HBeAg) is a secretory, non-structural protein, and associated with persistent infection of HBV. Previous studies indicate that HBeAg is able to regulate T cell-mediated responses, however, the interaction between HBeAg and the innate immune system is poorly understood. In this study, we demonstrated that recombinant HBeAg (rHBe) bound to human peripheral blood monocytes, neutrophils, and B lymphocytes but not to T lymphocytes. We focused on investigating the effects of HBeAg on monocytes and neutrophils and found that rHBe decreased the respiratory burst in both types of cells. Furthermore, we observed that cell migration in monocytes and neutrophils was suppressed by rHBe in a transwell assay. The attenuation of rHBe was not caused by a general cytotoxic effect because rHBe treatment stimulated low levels of cytokine and chemokine production by monocytes and it promoted neutrophil survival. Since the recruitment of monocytes and neutrophils to the infected site is crucial for the initiation of inflammation, HBeAg may modulate innate immune responses by diminishing the respiratory burst and migration of monocytes and neutrophils, which might interfere with the subsequent innate and adaptive immune responses against HBV, leading to the establishment of chronic infection.

  9. The Neutrophil's Eye-View: Inference and Visualisation of the Chemoattractant Field Driving Cell Chemotaxis In Vivo

    PubMed Central

    Kadirkamanathan, Visakan; Anderson, Sean R.; Billings, Stephen A.; Zhang, Xiliang; Holmes, Geoffrey R.; Reyes-Aldasoro, Constantino C.; Elks, Philip M.; Renshaw, Stephen A.

    2012-01-01

    As we begin to understand the signals that drive chemotaxis in vivo, it is becoming clear that there is a complex interplay of chemotactic factors, which changes over time as the inflammatory response evolves. New animal models such as transgenic lines of zebrafish, which are near transparent and where the neutrophils express a green fluorescent protein, have the potential to greatly increase our understanding of the chemotactic process under conditions of wounding and infection from video microscopy data. Measurement of the chemoattractants over space (and their evolution over time) is a key objective for understanding the signals driving neutrophil chemotaxis. However, it is not possible to measure and visualise the most important contributors to in vivo chemotaxis, and in fact the understanding of the main contributors at any particular time is incomplete. The key insight that we make in this investigation is that the neutrophils themselves are sensing the underlying field that is driving their action and we can use the observations of neutrophil movement to infer the hidden net chemoattractant field by use of a novel computational framework. We apply the methodology to multiple in vivo neutrophil recruitment data sets to demonstrate this new technique and find that the method provides consistent estimates of the chemoattractant field across the majority of experiments. The framework that we derive represents an important new methodology for cell biologists investigating the signalling processes driving cell chemotaxis, which we label the neutrophils eye-view of the chemoattractant field. PMID:22563379

  10. Recruitment in Radiotherapy

    ERIC Educational Resources Information Center

    Deeley, T. J.; And Others

    1976-01-01

    The Faculty Board of Radiotherapy and Oncology of the Royal College of Radiobiologists surveyed the factors thought to influence recruitment into the specialty. Possible factors listed in replies of 36 questionnaires are offered. (LBH)

  11. Epithelial neutrophil activating peptide-78: a novel chemotactic cytokine for neutrophils in arthritis.

    PubMed Central

    Koch, A E; Kunkel, S L; Harlow, L A; Mazarakis, D D; Haines, G K; Burdick, M D; Pope, R M; Walz, A; Strieter, R M

    1994-01-01

    We and others have shown that cells obtained from inflamed joints of rheumatoid arthritis (RA) patients produce interleukin-8, a potent chemotactic cytokine for neutrophils (PMNs). However, IL-8 accounted for only 40% of the chemotactic activity for PMNs found in these synovial fluids. Currently, we have examined the production of the novel PMN chemotactic cytokine, epithelial neutrophil activating peptide-78 (ENA-78), using peripheral blood, synovial fluid, and synovial tissue from 70 arthritic patients. RA ENA-78 levels were greater in RA synovial fluid (239 +/- 63 ng/ml) compared with synovial fluid from other forms of arthritis (130 +/- 118 ng/ml) or osteoarthritis (2.6 +/- 1.8 ng/ml) (P < 0.05). RA peripheral blood ENA-78 levels (70 +/- 26 ng/ml) were greater than normal peripheral blood levels (0.12 +/- 0.04 ng/ml) (P < 0.05). Anti-ENA-78 antibodies neutralized 42 +/- 9% (mean +/- SE) of the chemotactic activity for PMNs found in RA synovial fluids. Isolated RA synovial tissue fibroblasts in vitro constitutively produced significant levels of ENA-78, and this production was further augmented when stimulated with tumor necrosis factor-alpha (TNF-alpha). In addition RA and osteoarthritis synovial tissue fibroblasts as well as RA synovial tissue macrophages were found to constitutively produce ENA-78. RA synovial fluid mononuclear cells spontaneously produced ENA-78, which was augmented in the presence of lipopolysaccharide. Immunohistochemical localization of ENA-78 from the synovial tissue of patients with arthritis or normal subjects showed that the predominant cellular source of this chemokine was synovial lining cells, followed by macrophages, endothelial cells, and fibroblasts. Synovial tissue macrophages and fibroblasts were more ENA-78 immunopositive in RA than in normal synovial tissue (P < 0.05). These results, which are the first demonstration of ENA-78 in a human disease state, suggest that ENA-78 may play an important role in the recruitment of PMNs

  12. Leukocyte Recruitment and Ischemic Brain Injury

    PubMed Central

    Yilmaz, Gokhan

    2010-01-01

    Leukocytes are recruited into the cerebral microcirculation following an ischemic insult. The leukocyte–endothelial cell adhesion manifested within a few hours after ischemia (followed by reperfusion, I/R) largely reflects an infiltration of neutrophils, while other leukocyte populations appear to dominate the adhesive interactions with the vessel wall at 24 h of reperfusion. The influx of rolling and adherent leukocytes is accompanied by the recruitment of adherent platelets, which likely enhances the cytotoxic potential of the leukocytes to which they are attached. The recruitment of leukocytes and platelets in the postischemic brain is mediated by specific adhesion glycoproteins expressed by the activated blood cells and on cerebral microvascular endothelial cells. This process is also modulated by different signaling pathways (e.g., CD40/CD40L, Notch) and cytokines (e.g., RANTES) that are activated/released following I/R. Some of the known risk factors for cardiovascular disease, including hypercholesterolemia and obesity appear to exacerbate the leukocyte and platelet recruitment elicited by brain I/R. Although lymphocyte–endothelial cell and –platelet interactions in the postischemic cerebral microcirculation have not been evaluated to date, recent evidence in experimental animals implicate both CD4+ and CD8+ T-lymphocytes in the cerebral microvascular dysfunction, inflammation, and tissue injury associated with brain I/R. Evidence implicating regulatory T-cells as cerebroprotective modulators of the inflammatory and tissue injury responses to brain I/R support a continued focus on leukocytes as a target for therapeutic intervention in ischemic stroke. PMID:19579016

  13. Tumor Necrosis Factor, but Not Neutrophils, Alters the Metabolic Profile in Acute Experimental Arthritis.

    PubMed

    Oliveira, Marina C; Tavares, Luciana P; Vago, Juliana P; Batista, Nathália V; Queiroz-Junior, Celso M; Vieira, Angelica T; Menezes, Gustavo B; Sousa, Lirlândia P; van de Loo, Fons A J; Teixeira, Mauro M; Amaral, Flávio A; Ferreira, Adaliene V M

    2016-01-01

    Metabolic alterations are associated with arthritis apart from obesity. However, it is still unclear which is the underlying process behind these metabolic changes. Here, we investigate the role of tumor necrosis factor (TNF) in this process in an acute model of antigen-induced arthritis (AIA). Immunized male BALB/c mice received an intra-articular injection of PBS (control) or methylated bovine serum albumin (mBSA) into their knees, and were also pre-treated with different drugs: Etanercept, an anti-TNF drug, DF2156A, a CXCR1/2 receptor antagonist, or a monoclonal antibody RB6-8C5 to deplete neutrophils. Local challenge with mBSA evoked an acute neutrophil influx into the knee joint, and enhanced the joint nociception, along with a transient systemic metabolic alteration (higher levels of glucose and lipids, and altered adipocytokines). Pre-treatment with the conventional biological Etanercept, an inhibitor of TNF action, ameliorated the nociception and the acute joint inflammation dominated by neutrophils, and markedly improved many of the altered systemic metabolites (glucose and lipids), adipocytokines and PTX3. However, the lessening of metabolic changes was not due to diminished accumulation of neutrophils in the joint by Etanercept. Reduction of neutrophil recruitment by pre-treating AIA mice with DF2156A, or even the depletion of these cells by using RB6-8C5 reduced all of the inflammatory parameters and hypernociception developed after AIA challenge, but could not prevent the metabolic changes. Therefore, the induction of joint inflammation provoked acute metabolic alterations which were involved with TNF. We suggest that the role of TNF in arthritis-associated metabolic changes is not due to local neutrophils, which are the major cells present in this model, but rather due to cytokines. PMID:26742100

  14. Effects of Porphyromonas gingivalis LipopolysaccharideTolerized Monocytes on Inflammatory Responses in Neutrophils

    PubMed Central

    Chen, Yang; Cheng, Xiao-fan; Qiu, Jia-ying; Xu, Yan; Sun, Ying

    2016-01-01

    Periodontitis is a chronic inflammatory disease induced by bacteria. Exposure of the host to periodontal pathogens and their virulence factors induces a state of hyporesponsiveness to subsequent stimulations, which is termed endotoxin tolerance. The role and mechanism of lipopolysaccharide (LPS)–tolerized monocytes in inflammatory responses in neutrophils are currently unclear. Here, conditioned supernatants were collected from THP-1 cells treated with or without repeated 1 μg/ml Porphyromonas gingivalis (P.gingivalis) LPS. The chemotactic response of freshly isolated neutrophils recruited by supernatants was determined by a transwell migration assay, which demonstrated a reduced migration of neutrophils stimulated with supernatants from tolerized THP-1 cells in comparison to non-tolerized THP-1 cells. In addition, there was a marked increase in reactive oxygen species (ROS) generation and a significant decrease in Caspase 3 activities in neutrophils treated with supernatants from THP-1 cells that were treated repeatedly with P.gingivalis LPS in comparison to single treatment. A cytokine antibody array was then used to assess cytokine expression patterns in THP-1 cells. In tolerized THP-1 cells, 43 cytokine (43/170) expression levels were decreased, including chemokine ligand 23 (CCL23) and IFN-γ, while 11 cytokine (11/170) expression levels were increased, such as death receptor 6 (DR6). Furthermore, there was decreased production of IFN-γ and epithelial neutrophil activating peptide-78 (ENA-78) in THP-1 cells after stimulation with repeated P. gingivalis LPS in comparison to single challenge, which was confirmed by ELISA. Therefore, P.gingivalis LPS- tolerized THP-1 cells were able to depress neutrophil chemotaxis and apoptosis, and contribute to respiratory burst, which might be related to the changes in cytokine expression patterns in THP-1 cells. PMID:27536946

  15. Effects of Porphyromonas gingivalis LipopolysaccharideTolerized Monocytes on Inflammatory Responses in Neutrophils.

    PubMed

    Zhu, Xiang-Qing; Lu, Wei; Chen, Yang; Cheng, Xiao-Fan; Qiu, Jia-Ying; Xu, Yan; Sun, Ying

    2016-01-01

    Periodontitis is a chronic inflammatory disease induced by bacteria. Exposure of the host to periodontal pathogens and their virulence factors induces a state of hyporesponsiveness to subsequent stimulations, which is termed endotoxin tolerance. The role and mechanism of lipopolysaccharide (LPS)-tolerized monocytes in inflammatory responses in neutrophils are currently unclear. Here, conditioned supernatants were collected from THP-1 cells treated with or without repeated 1 μg/ml Porphyromonas gingivalis (P.gingivalis) LPS. The chemotactic response of freshly isolated neutrophils recruited by supernatants was determined by a transwell migration assay, which demonstrated a reduced migration of neutrophils stimulated with supernatants from tolerized THP-1 cells in comparison to non-tolerized THP-1 cells. In addition, there was a marked increase in reactive oxygen species (ROS) generation and a significant decrease in Caspase 3 activities in neutrophils treated with supernatants from THP-1 cells that were treated repeatedly with P.gingivalis LPS in comparison to single treatment. A cytokine antibody array was then used to assess cytokine expression patterns in THP-1 cells. In tolerized THP-1 cells, 43 cytokine (43/170) expression levels were decreased, including chemokine ligand 23 (CCL23) and IFN-γ, while 11 cytokine (11/170) expression levels were increased, such as death receptor 6 (DR6). Furthermore, there was decreased production of IFN-γ and epithelial neutrophil activating peptide-78 (ENA-78) in THP-1 cells after stimulation with repeated P. gingivalis LPS in comparison to single challenge, which was confirmed by ELISA. Therefore, P.gingivalis LPS- tolerized THP-1 cells were able to depress neutrophil chemotaxis and apoptosis, and contribute to respiratory burst, which might be related to the changes in cytokine expression patterns in THP-1 cells. PMID:27536946

  16. Tumor Necrosis Factor, but Not Neutrophils, Alters the Metabolic Profile in Acute Experimental Arthritis

    PubMed Central

    Oliveira, Marina C.; Tavares, Luciana P.; Vago, Juliana P.; Batista, Nathália V.; Queiroz-Junior, Celso M.; Vieira, Angelica T.; Menezes, Gustavo B.; Sousa, Lirlândia P.; van de Loo, Fons A. J.; Teixeira, Mauro M.; Amaral, Flávio A.; Ferreira, Adaliene V. M.

    2016-01-01

    Metabolic alterations are associated with arthritis apart from obesity. However, it is still unclear which is the underlying process behind these metabolic changes. Here, we investigate the role of tumor necrosis factor (TNF) in this process in an acute model of antigen-induced arthritis (AIA). Immunized male BALB/c mice received an intra-articular injection of PBS (control) or methylated bovine serum albumin (mBSA) into their knees, and were also pre-treated with different drugs: Etanercept, an anti-TNF drug, DF2156A, a CXCR1/2 receptor antagonist, or a monoclonal antibody RB6-8C5 to deplete neutrophils. Local challenge with mBSA evoked an acute neutrophil influx into the knee joint, and enhanced the joint nociception, along with a transient systemic metabolic alteration (higher levels of glucose and lipids, and altered adipocytokines). Pre-treatment with the conventional biological Etanercept, an inhibitor of TNF action, ameliorated the nociception and the acute joint inflammation dominated by neutrophils, and markedly improved many of the altered systemic metabolites (glucose and lipids), adipocytokines and PTX3. However, the lessening of metabolic changes was not due to diminished accumulation of neutrophils in the joint by Etanercept. Reduction of neutrophil recruitment by pre-treating AIA mice with DF2156A, or even the depletion of these cells by using RB6-8C5 reduced all of the inflammatory parameters and hypernociception developed after AIA challenge, but could not prevent the metabolic changes. Therefore, the induction of joint inflammation provoked acute metabolic alterations which were involved with TNF. We suggest that the role of TNF in arthritis-associated metabolic changes is not due to local neutrophils, which are the major cells present in this model, but rather due to cytokines. PMID:26742100

  17. N-Acylhomoserine lactones are potent neutrophil chemoattractants that act via calcium mobilization and actin remodeling.

    PubMed

    Karlsson, Thommie; Musse, Farah; Magnusson, Karl-Eric; Vikström, Elena

    2012-01-01

    In gram-negative bacteria, cell-cell communication based on HSL QS molecules is known to coordinate the production of virulence factors and biofilms. These bacterial signals can also modulate human immune cell behavior. Using a Transwell migration assay, we found that human primary neutrophils are strongly stimulated by 3O-C(12)-HSL and -C(10)-HSL but not C(4)-HSL in a concentration-dependent manner. Moreover, 3O-C(12)-HSL and -C(10)-HSL activate PLCγ1 but not -γ2, mobilize intracellular calcium, and up-regulate IP(3)R. These changes were paralleled by F-actin accumulation, primarily in the leading edge of neutrophils, as evidenced by phalloidin staining and confocal microscopy. F- and G-actin isolation and quantification by immunoblotting revealed that the F/G-actin ratio was increased significantly after treatment with all three HSLs. Furthemore, 3O-C(12)-HSL- and 3O-C(10)-HSL treatment resulted in phosphorylation of Rac1 and Cdc42. In contrast, C(4)-HSL had negligible influence on the phosphorylation status of PLC and Rac1/Cdc42 and failed to attract neutrophils and induce calcium release. The calcium inhibitor thapsigargin, which blocks ER calcium uptake, strongly prevented neutrophil migration toward 3O-C(12)-HSL and -C(10)-HSL. These findings show that the bacterial QS molecules 3O-C(12)-HSL and -C(10)-HSL may attract human neutrophils to the sites of bacterial infection and developing biofilms. Indeed, recognition of HSL QS signals by neutrophils may play a critical role in their recruitment during infections.

  18. Store-operated calcium signaling in neutrophils.

    PubMed

    Clemens, Regina A; Lowell, Clifford A

    2015-10-01

    Calcium signals in neutrophils are initiated by a variety of cell-surface receptors, including formyl peptide and other GPCRs, FcRs, and integrins. The predominant pathway by which calcium enters immune cells is termed SOCE, whereby plasma membrane CRAC channels allow influx of extracellular calcium into the cytoplasm when intracellular ER stores are depleted. The identification of 2 key families of SOCE regulators, STIM calcium "sensors" and ORAI calcium channels, has allowed for genetic manipulation of SOCE pathways and provided valuable insight into the molecular mechanism of calcium signaling in immune cells, including neutrophils. This review focuses on our current knowledge of the molecules involved in neutrophil SOCE and how study of these molecules has further informed our understanding of the role of calcium signaling in neutrophil activation.

  19. [Effect of erythromycin on neutrophil adhesion molecules].

    PubMed

    Kusano, S; Mukae, H; Morikawa, T; Asai, T; Sawa, H; Morikawa, N; Oda, H; Sakito, O; Shukuwa, C; Senju, R

    1993-01-01

    The mechanisms of erythromycin (EM) in chronic lower respiratory tract diseases including diffuse panbronchiolitis (DPB) has been reported. In this study we investigated the effect of EM on peripheral neutrophil adhesion molecules such as LFA-1 and Mac-1 obtained from six healthy subjects. Pretreatment of neutrophils with each concentration (10 ng/ml approximately 100 micrograms/ml) of EM resulted in no significant reduction in the expression of LFA-1 alpha, beta and Mac-1. Moreover, EM had no capability of reducing these expressions even when neutrophils were pretreated with 1 microgram/ml of EM at time from 0 to 60 min. These findings indicate that EM does not directly reduce the expression of LFA-1 alpha, beta and Mac-1 on peripheral neutrophil obtained from healthy subjects. PMID:8450276

  20. Neutrophils in asthma--a review.

    PubMed

    Ciepiela, Olga; Ostafin, Magdalena; Demkow, Urszula

    2015-04-01

    Asthma is a chronic inflammatory disease, with an array of cells involved in the pathogenesis of the disease. The role of neutrophils in the development of bronchial asthma is found to be complex, as they may trigger activation of immunocompetent cells and are a potent source of free oxygen radicals and enzymes participating in airway remodeling. The review highlights the role of neutrophils in bronchial asthma.

  1. Expression of IL-17A concentration and effector functions of peripheral blood neutrophils in food allergy hypersensitivity patients.

    PubMed

    Żbikowska-Gotz, Magdalena; Pałgan, Krzysztof; Gawrońska-Ukleja, Ewa; Kuźmiński, Andrzej; Przybyszewski, Michał; Socha, Ewa; Bartuzi, Zbigniew

    2016-03-01

    Lymphocytes Th17 and other types of immune system cells produce IL17. By induction of cytokines and chemokines, the IL17 cytokine is involved in mechanisms of allergic reaction with participation of neutrophil granulocytes. It affects activation, recruitment, and migration of neutrophils to the tissues, regulating inflammatory reaction intensity. Excited neutrophils secrete inter alia elastase and reactive oxygen species (ROS)--significant mediators of inflammation process responsible for tissues damage.The aim of the study was to evaluate the concentrations of serum interleukin 17A, serum neutrophil elastase, and ROS production by neutrophils in patients with food allergy.The study included 30 patients with food allergy diagnosed based on interview, clinical symptoms, positive SPT, placebo controlled double-blind oral provocation trial, and the presence of asIgE in blood serum against selected food allergens using fluoro-immuno-enzymatic method FEIA UNICap 100. The control group consisted of 10 healthy volunteers. The concentrations of IL17A were determined in all patients using ELISA method with eBioscience kits, and elastase using BenderMed Systems kits. Chemiluminescence of non-stimulated neutrophils was evaluated using luminol-dependent kinetic method for 40 min on Luminoskan (Labsystems luminometer).The results of serum IL-17A concentrations and the values of chemiluminescence obtained by non-activated neutrophils, as well as elastase concentrations, were higher in patients with food allergic hypersensitivity compared to healthy volunteers.This study demonstrates a significance of IL-17A and activated neutrophil granulocytes in the course of diseases with food allergic hypersensitivity. PMID:26684636

  2. MBL-Mediated Opsonophagocytosis of Candida albicans by Human Neutrophils Is Coupled with Intracellular Dectin-1-Triggered ROS Production

    PubMed Central

    Tong, Zhongsheng; Wang, Qinning; Liu, Weihuang; Wang, Yan; Liu, Wei; Chen, Jinbo; Xu, Li; Chen, Liuqing; Duan, Yiqun

    2012-01-01

    Mannan-binding lectin (MBL), a lectin homologous to C1q, greatly facilitates C3/C4-mediated opsonophagocytosis of Candida albicans (C. albicans) by human neutrophils, and has the capacity to bind to CR1 (CD35) expressed on circulating neutrophils. The intracellular pool of neutrophil Dectin-1 plays a critical role in stimulating the reactive oxygen species (ROS) generation through recognition of β-1,3-glucan component of phagocytized zymosan or yeasts. However, little is known about whether MBL can mediate the opsonophagocytosis of Candida albicans by neutrophils independent of complement activation, and whether MBL-mediated opsonophagocytosis influence the intracellular expression of Dectin-1 and ROS production. Here we showed that the inhibited phagocytic efficiency of neutrophils as a result of blockage of Dectin-1 was compensated by exogenous MBL alone in a dose-dependent manner. Furthermore, the expressions of Dectin-1 at mRNA and intracellular protein levels were significantly up-regulated in neutrophils stimulated by MBL-pre-incubated C. albicans, while the expression of surface Dectin-1 remained almost unchanged. Nevertheless, the stimulated ROS production in neutrophils was partly and irreversibly inhibited by blockage of Dectin-1 in the presence of exogenous MBL. Confocal microscopy examination showed that intracellular Dectin-1 was recruited and co-distributed with ROS on the surface of some phagocytized yeasts. The β-1,3-glucanase digestion test further suggested that the specific recognition and binding site of human Dectin-1 is just the β-1,3-glucan moiety on the cell wall of C. albicans. These data demonstrate that MBL has an ability to mediate the opsonophagocytosis of Candida albicans by human neutrophils independent of complement activation, which is coupled with intracellular Dectin-1-triggered ROS production. PMID:23239982

  3. Differential expression of pentraxin 3 in neutrophils.

    PubMed

    Razvina, Olga; Jiang, Shuying; Matsubara, Koichi; Ohashi, Riuko; Hasegawa, Go; Aoyama, Takashi; Daigo, Kenji; Kodama, Tatsuhiko; Hamakubo, Takao; Naito, Makoto

    2015-02-01

    Pentraxins belong to the superfamily of conserved proteins that are characterized by a cyclic multimeric structure. Pentraxin 3 (PTX3) is a long pentraxin which can be produced by different cell types upon exposure to various inflammatory signals. Inside the neutrophil PTX3 is stored in form of granules localized in the cytoplasm. Neutrophilic granules are divided into three types: azurophilic (primary) granules, specific (secondary) granules and gelatinase (tertiary) granules. PTX3 has been considered to be localized in specific (secondary) granules. Immunofluorescent analyses using confocal laser microscopic examination were performed to clarify the localization of all three groups of granules within the cytoplasm of the mature neutrophils and neutrophils stimulated with IL-8. Furthermore, PTX3 was localized in primary granules of promyelocyte cell line HL-60. As a result, we suggest that PTX3 is localized not only in specific granules, but is also partly expressed in primary and tertiary granules. After the stimulation with IL-8, irregular reticular structures called neutrophil extracellular traps (NETs) were formed, three types of granules were trapped by NETs and PTX3 showed partial colocalization with these granular components. PTX3 localized in all three types of granules in neutrophils may play important roles in host defense.

  4. Macrophage Recruitment Contributes to Regeneration of Mechanosensory Hair Cells in the Zebrafish Lateral Line.

    PubMed

    Carrillo, Simón A; Anguita-Salinas, Consuelo; Peña, Oscar A; Morales, Rodrigo A; Muñoz-Sánchez, Salomé; Muñoz-Montecinos, Carlos; Paredes-Zúñiga, Susana; Tapia, Karina; Allende, Miguel L

    2016-08-01

    In vertebrates, damage to mechanosensory hair cells elicits an inflammatory response, including rapid recruitment of macrophages and neutrophils. While hair cells in amniotes usually become permanently lost, they readily regenerate in lower vertebrates such as fish. Damage to hair cells of the fish lateral line is followed by inflammation and rapid regeneration; however the role of immune cells in this process remains unknown. Here, we show that recruited macrophages are required for normal regeneration of lateral line hair cells after copper damage. We found that genetic ablation or local ablation using clodronate liposomes of macrophages recruited to the site of injury, significantly delays hair cell regeneration. Neutrophils, on the other hand, are not needed for this process. We anticipate our results to be a starting point for a more detailed description of extrinsic signals important for regeneration of mechanosensory cells in vertebrates. J. Cell. Biochem. 117: 1880-1889, 2016. © 2016 Wiley Periodicals, Inc.

  5. IL-1 Coordinates the Neutrophil Response to C. albicans in the Oral Mucosa.

    PubMed

    Altmeier, Simon; Toska, Albulena; Sparber, Florian; Teijeira, Alvaro; Halin, Cornelia; LeibundGut-Landmann, Salomé

    2016-09-01

    Mucosal infections with Candida albicans belong to the most frequent forms of fungal diseases. Host protection is conferred by cellular immunity; however, the induction of antifungal immunity is not well understood. Using a mouse model of oropharyngeal candidiasis (OPC) we show that interleukin-1 receptor (IL-1R) signaling is critical for fungal control at the onset of infection through its impact on neutrophils at two levels. We demonstrate that both the recruitment of circulating neutrophils to the site of infection and the mobilization of newly generated neutrophils from the bone marrow depended on IL-1R. Consistently, IL-1R-deficient mice displayed impaired chemokine production at the site of infection and defective secretion of granulocyte colony-stimulating factor (G-CSF) in the circulation in response to C. albicans. Strikingly, endothelial cells were identified as the primary cellular source of G-CSF during OPC, which responded to IL-1α that was released from keratinocytes in the infected tissue. The IL-1-dependent crosstalk between two different cellular subsets of the nonhematopoietic compartment was confirmed in vitro using a novel murine tongue-derived keratinocyte cell line and an established endothelial cell line. These data establish a new link between IL-1 and granulopoiesis in the context of fungal infection. Together, we identified two complementary mechanisms coordinating the neutrophil response in the oral mucosa, which is critical for preventing fungal growth and dissemination, and thus protects the host from disease. PMID:27632536

  6. Theophylline therapy inhibits neutrophil and mononuclear cell chemotaxis from chronic asthmatic children.

    PubMed Central

    Condino-Neto, A; Vilela, M M; Cambiucci, E C; Ribeiro, J D; Guglielmi, A A; Magna, L A; De Nucci, G

    1991-01-01

    1. Theophylline is commonly used to relieve symptoms of chronic asthma. Since neutrophil and mononuclear cell activation are associated with late phase asthmatic reactions, effects of theophylline on these cells may be of importance. 2. In the present investigation we compared neutrophil and mononuclear cell chemotaxis from chronic asthmatic children during and after theophylline therapy. 3. Thirty patients were recruited for the study. Each patient received theophylline orally for 10 days. The theophylline dose was 20 mg kg-1 day-1 given in four divided doses. On the tenth day, blood was collected into heparinized (100 u ml-1) and siliconized tubes 2 h after the last theophylline dose for chemotactic assays, cAMP and theophylline plasma determinations. When clinical conditions allowed, theophylline was discontinued for 7 days and the chemotactic assays, cAMP and theophylline plasma concentrations repeated. Serum complement and IgE levels were also determined. 4. Theophylline therapy clearly inhibited both spontaneous and stimulated neutrophil and mononuclear cell chemotaxis. Twenty-seven patients had therapeutic plasma concentrations of theophylline (5-20 micrograms ml-1). Discontinuation of theophylline therapy caused a significant decrease in plasma cAMP levels (44 and 31 pmol ml-1 respectively during and after treatment, n = 30, P less than 0.001). 5. The inhibition of neutrophil and mononuclear cell migration by theophylline therapy in chronic asthmatic children may be beneficial for the control of the inflammatory response observed in these patients. PMID:1659436

  7. Neutrophil Extracellular Traps Enhance Early Inflammatory Response in Sendai Virus-Induced Asthma Phenotype.

    PubMed

    Akk, Antonina; Springer, Luke E; Pham, Christine T N

    2016-01-01

    Paramyxoviral infection in childhood has been linked to a significant increased rate of asthma development. In mice, paramyxoviral infection with the mouse parainfluenza virus type I, Sendai virus (Sev), causes a limited bronchiolitis followed by persistent asthma traits. We have previously shown that the absence of cysteine protease dipeptidyl peptidase I (DPPI) dampened the acute lung inflammatory response and the subsequent asthma phenotype induced by Sev. Adoptive transfer of wild-type neutrophils into DPPI-deficient mice restored leukocyte influx, the acute cytokine response, and the subsequent mucous cell metaplasia that accompanied Sev-induced asthma phenotype. However, the exact mechanism by which DPPI-sufficient neutrophils promote asthma development following Sev infection is still unknown. We hypothesize that neutrophils recruited to the alveolar space following Sev infection elaborate neutrophil extracellular traps (NETs) that propagate the inflammatory cascade, culminating in the eventual asthma phenotype. Indeed, we found that Sev infection was associated with NET formation in the lung and release of cell-free DNA complexed to myeloperoxidase in the alveolar space and plasma that peaked on day 2 post infection. Absence of DPPI significantly attenuated Sev-induced NET formation in vivo and in vitro. Furthermore, concomitant administration of DNase 1, which dismantled NETs, or inhibition of peptidylarginine deiminase 4 (PAD4), an essential mediator of NET formation, suppressed the early inflammatory responses to Sev infection. Lastly, NETs primed bone marrow-derived cells to release cytokines that can amplify the inflammatory cascade. PMID:27617014

  8. Neutrophil Extracellular Traps Enhance Early Inflammatory Response in Sendai Virus-Induced Asthma Phenotype

    PubMed Central

    Akk, Antonina; Springer, Luke E.; Pham, Christine T. N.

    2016-01-01

    Paramyxoviral infection in childhood has been linked to a significant increased rate of asthma development. In mice, paramyxoviral infection with the mouse parainfluenza virus type I, Sendai virus (Sev), causes a limited bronchiolitis followed by persistent asthma traits. We have previously shown that the absence of cysteine protease dipeptidyl peptidase I (DPPI) dampened the acute lung inflammatory response and the subsequent asthma phenotype induced by Sev. Adoptive transfer of wild-type neutrophils into DPPI-deficient mice restored leukocyte influx, the acute cytokine response, and the subsequent mucous cell metaplasia that accompanied Sev-induced asthma phenotype. However, the exact mechanism by which DPPI-sufficient neutrophils promote asthma development following Sev infection is still unknown. We hypothesize that neutrophils recruited to the alveolar space following Sev infection elaborate neutrophil extracellular traps (NETs) that propagate the inflammatory cascade, culminating in the eventual asthma phenotype. Indeed, we found that Sev infection was associated with NET formation in the lung and release of cell-free DNA complexed to myeloperoxidase in the alveolar space and plasma that peaked on day 2 post infection. Absence of DPPI significantly attenuated Sev-induced NET formation in vivo and in vitro. Furthermore, concomitant administration of DNase 1, which dismantled NETs, or inhibition of peptidylarginine deiminase 4 (PAD4), an essential mediator of NET formation, suppressed the early inflammatory responses to Sev infection. Lastly, NETs primed bone marrow-derived cells to release cytokines that can amplify the inflammatory cascade.

  9. Alveolar macrophage-stimulated neutrophil and monocyte migration: effects of in vitro ozone exposure

    SciTech Connect

    Driscoll, K.E.; Schlesinger, R.B.

    1988-04-01

    The ability of rabbit alveolar macrophages (AM) to release factors which stimulate the migration of peripheral blood neutrophils and monocytes was examined, and the influence of in vitro ozone exposure on this secretory activity was investigated. To evaluate the ability of AM to release leukocyte chemotactic activity, AM obtained by bronchoalveolar lavage were established in monolayer or suspension culture, with and without added zymosan, for 2 and 6 hr. The resulting macrophage-conditioned medium was tested for chemotactic activity using modified Boyden-type chambers and rabbit peripheral blood neutrophils or monocytes as the responding cells. The results demonstrate that substrate attachment (monolayer culture) and/or zymosan phagocytosis can stimulate AM to release chemoattractants for monocytes and neutrophils. Additionally, the results suggest that AM are constitutively producing low levels of monocyte chemotactic factors. The effects of in vitro ozone exposure on the secretion of chemotactic activity was investigated by exposing monolayer cultures of AM to air, 0.1, 0.3, or 1.2 ppm ozone for 2 hr. Macrophage-conditioned medium was harvested immediately, 2 and 6 hr postexposure, and tested for chemotactic activity. Exposure to 0.3 and 1.2 ppm ozone significantly increased the AM secretion of factors which stimulated neutrophil migration; additionally, the results strongly suggest that ozone can augment the ability of AM to stimulate monocyte migration. These results imply a role for the AM in the recruitment of inflammatory cells after ozone inhalation.

  10. IL-1 Coordinates the Neutrophil Response to C. albicans in the Oral Mucosa

    PubMed Central

    Altmeier, Simon; Toska, Albulena; Sparber, Florian; Teijeira, Alvaro; Halin, Cornelia; LeibundGut-Landmann, Salomé

    2016-01-01

    Mucosal infections with Candida albicans belong to the most frequent forms of fungal diseases. Host protection is conferred by cellular immunity; however, the induction of antifungal immunity is not well understood. Using a mouse model of oropharyngeal candidiasis (OPC) we show that interleukin-1 receptor (IL-1R) signaling is critical for fungal control at the onset of infection through its impact on neutrophils at two levels. We demonstrate that both the recruitment of circulating neutrophils to the site of infection and the mobilization of newly generated neutrophils from the bone marrow depended on IL-1R. Consistently, IL-1R-deficient mice displayed impaired chemokine production at the site of infection and defective secretion of granulocyte colony-stimulating factor (G-CSF) in the circulation in response to C. albicans. Strikingly, endothelial cells were identified as the primary cellular source of G-CSF during OPC, which responded to IL-1α that was released from keratinocytes in the infected tissue. The IL-1-dependent crosstalk between two different cellular subsets of the nonhematopoietic compartment was confirmed in vitro using a novel murine tongue-derived keratinocyte cell line and an established endothelial cell line. These data establish a new link between IL-1 and granulopoiesis in the context of fungal infection. Together, we identified two complementary mechanisms coordinating the neutrophil response in the oral mucosa, which is critical for preventing fungal growth and dissemination, and thus protects the host from disease. PMID:27632536

  11. Neutrophil Extracellular Traps Enhance Early Inflammatory Response in Sendai Virus-Induced Asthma Phenotype

    PubMed Central

    Akk, Antonina; Springer, Luke E.; Pham, Christine T. N.

    2016-01-01

    Paramyxoviral infection in childhood has been linked to a significant increased rate of asthma development. In mice, paramyxoviral infection with the mouse parainfluenza virus type I, Sendai virus (Sev), causes a limited bronchiolitis followed by persistent asthma traits. We have previously shown that the absence of cysteine protease dipeptidyl peptidase I (DPPI) dampened the acute lung inflammatory response and the subsequent asthma phenotype induced by Sev. Adoptive transfer of wild-type neutrophils into DPPI-deficient mice restored leukocyte influx, the acute cytokine response, and the subsequent mucous cell metaplasia that accompanied Sev-induced asthma phenotype. However, the exact mechanism by which DPPI-sufficient neutrophils promote asthma development following Sev infection is still unknown. We hypothesize that neutrophils recruited to the alveolar space following Sev infection elaborate neutrophil extracellular traps (NETs) that propagate the inflammatory cascade, culminating in the eventual asthma phenotype. Indeed, we found that Sev infection was associated with NET formation in the lung and release of cell-free DNA complexed to myeloperoxidase in the alveolar space and plasma that peaked on day 2 post infection. Absence of DPPI significantly attenuated Sev-induced NET formation in vivo and in vitro. Furthermore, concomitant administration of DNase 1, which dismantled NETs, or inhibition of peptidylarginine deiminase 4 (PAD4), an essential mediator of NET formation, suppressed the early inflammatory responses to Sev infection. Lastly, NETs primed bone marrow-derived cells to release cytokines that can amplify the inflammatory cascade. PMID:27617014

  12. The PSGL-1–L-selectin signaling complex regulates neutrophil adhesion under flow

    PubMed Central

    Stadtmann, Anika; Germena, Giulia; Block, Helena; Boras, Mark; Rossaint, Jan; Sundd, Prithu; Lefort, Craig; Fisher, Charles I.; Buscher, Konrad; Gelschefarth, Bernadette; Urzainqui, Ana; Gerke, Volker; Ley, Klaus

    2013-01-01

    Neutrophils are recruited from the blood to sites of inflammation, where they contribute to immune defense but may also cause tissue damage. During inflammation, neutrophils roll along the microvascular endothelium before arresting and transmigrating. Arrest requires conformational activation of the integrin lymphocyte function–associated antigen 1 (LFA-1), which can be induced by selectin engagement. Here, we demonstrate that a subset of P-selectin glycoprotein ligand-1 (PSGL-1) molecules is constitutively associated with L-selectin. Although this association does not require the known lectin-like interaction between L-selectin and PSGL-1, the signaling output is dependent on this interaction and the cytoplasmic tail of L-selectin. The PSGL-1–L-selectin complex signals through Src family kinases, ITAM domain–containing adaptor proteins, and other kinases to ultimately result in LFA-1 activation. The PSGL-1–L-selectin complex–induced signaling effects on neutrophil slow rolling and recruitment in vivo demonstrate the functional importance of this pathway. We conclude that this is a signaling complex specialized for sensing adhesion under flow. PMID:24127491

  13. Researching participant recruitment times.

    PubMed

    O'Brien, Rachel; Black, Polly

    2015-11-01

    Conducting research in emergency departments is relatively new, and there are a number of ethical and practical challenges to recruiting patients in these settings. In 2008, the Emergency Medicine Research Group Edinburgh (EMERGE) was set up at the Royal Infirmary of Edinburgh emergency department to support researchers and encourage the growth of research in emergency medicine. As part of a review of their working methods, the group's clinical nurse researchers undertook a small study to identify participant recruitment times. The results showed a significant difference between perceived and actual recruitment times, which has implications for planning staff numbers and budgets. This article describes the evaluation process and methods of data collection, and discusses the results. PMID:26542924

  14. Superoxide Anion Production by Human Neutrophils Activated by Trichomonas vaginalis

    PubMed Central

    Song, Hyun-Ouk

    2013-01-01

    Neutrophils are the predominant inflammatory cells found in vaginal discharges of patients infected with Trichomonas vaginalis. In this study, we examined superoxide anion (O2.-) production by neutrophils activated by T. vaginalis. Human neutrophils produced superoxide anions when stimulated with either a lysate of T. vaginalis, its membrane component (MC), or excretory-secretory product (ESP). To assess the role of trichomonad protease in production of superoxide anions by neutrophils, T. vaginalis lysate, ESP, and MC were each pretreated with a protease inhibitor cocktail before incubation with neutrophils. Superoxide anion production was significantly decreased by this treatment. Trichomonad growth was inhibited by preincubation with supernatants of neutrophils incubated for 3 hr with T. vaginalis lysate. Furthermore, myeloperoxidase (MPO) production by neutrophils was stimulated by live trichomonads. These results indicate that the production of superoxide anions and MPO by neutrophils stimulated with T. vaginalis may be a part of defense mechanisms of neutrophils in trichomoniasis. PMID:24039294

  15. Superoxide anion production by human neutrophils activated by Trichomonas vaginalis.

    PubMed

    Song, Hyun-Ouk; Ryu, Jae-Sook

    2013-08-01

    Neutrophils are the predominant inflammatory cells found in vaginal discharges of patients infected with Trichomonas vaginalis. In this study, we examined superoxide anion (O2 (.-)) production by neutrophils activated by T. vaginalis. Human neutrophils produced superoxide anions when stimulated with either a lysate of T. vaginalis, its membrane component (MC), or excretory-secretory product (ESP). To assess the role of trichomonad protease in production of superoxide anions by neutrophils, T. vaginalis lysate, ESP, and MC were each pretreated with a protease inhibitor cocktail before incubation with neutrophils. Superoxide anion production was significantly decreased by this treatment. Trichomonad growth was inhibited by preincubation with supernatants of neutrophils incubated for 3 hr with T. vaginalis lysate. Furthermore, myeloperoxidase (MPO) production by neutrophils was stimulated by live trichomonads. These results indicate that the production of superoxide anions and MPO by neutrophils stimulated with T. vaginalis may be a part of defense mechanisms of neutrophils in trichomoniasis. PMID:24039294

  16. Superoxide anion production by human neutrophils activated by Trichomonas vaginalis.

    PubMed

    Song, Hyun-Ouk; Ryu, Jae-Sook

    2013-08-01

    Neutrophils are the predominant inflammatory cells found in vaginal discharges of patients infected with Trichomonas vaginalis. In this study, we examined superoxide anion (O2 (.-)) production by neutrophils activated by T. vaginalis. Human neutrophils produced superoxide anions when stimulated with either a lysate of T. vaginalis, its membrane component (MC), or excretory-secretory product (ESP). To assess the role of trichomonad protease in production of superoxide anions by neutrophils, T. vaginalis lysate, ESP, and MC were each pretreated with a protease inhibitor cocktail before incubation with neutrophils. Superoxide anion production was significantly decreased by this treatment. Trichomonad growth was inhibited by preincubation with supernatants of neutrophils incubated for 3 hr with T. vaginalis lysate. Furthermore, myeloperoxidase (MPO) production by neutrophils was stimulated by live trichomonads. These results indicate that the production of superoxide anions and MPO by neutrophils stimulated with T. vaginalis may be a part of defense mechanisms of neutrophils in trichomoniasis.

  17. GPCR-mediated PLCβγ/PKCβ/PKD signaling pathway regulates the cofilin phosphatase slingshot 2 in neutrophil chemotaxis

    PubMed Central

    Xu, Xuehua; Gera, Nidhi; Li, Hongyan; Yun, Michelle; Zhang, Liyong; Wang, Youhong; Wang, Q. Jane; Jin, Tian

    2015-01-01

    Chemotaxis requires precisely coordinated polymerization and depolymerization of the actin cytoskeleton at leading fronts of migrating cells. However, GPCR activation-controlled F-actin depolymerization remains largely elusive. Here, we reveal a novel signaling pathway, including Gαi, PLC, PKCβ, protein kinase D (PKD), and SSH2, in control of cofilin phosphorylation and actin cytoskeletal reorganization, which is essential for neutrophil chemotaxis. We show that PKD is essential for neutrophil chemotaxis and that GPCR-mediated PKD activation depends on PLC/PKC signaling. More importantly, we discover that GPCR activation recruits/activates PLCγ2 in a PI3K-dependent manner. We further verify that PKCβ specifically interacts with PKD1 and is required for chemotaxis. Finally, we identify slingshot 2 (SSH2), a phosphatase of cofilin (actin depolymerization factor), as a target of PKD1 that regulates cofilin phosphorylation and remodeling of the actin cytoskeleton during neutrophil chemotaxis. PMID:25568344

  18. Neutrophils and neutrophil extracellular traps orchestrate initiation and resolution of inflammation.

    PubMed

    Hahn, Jonas; Knopf, Jasmin; Maueröder, Christian; Kienhöfer, Deborah; Leppkes, Moritz; Herrmann, Martin

    2016-01-01

    Neutrophils, the most abundant leukocytes in the human body, are considered to be the first line of defense in the fight against microorganisms. In this fight neutrophils employ weaponry such as reactive oxygen species produced via the NADPH oxidase complex 2 together with the release of intracellular granules containing antimicrobial agents. The discovery that activated neutrophils release decondensed chromatin as DNase-sensitive neutrophil extracellular traps (NETs) lead to a renewed interest in these leukocytes and the function of NETs in vivo. In this review, we will focus on desirable as well as detrimental features of NETs by the example of gout and pancreatitis. In our models we observed that neutrophils drive the initiation of inflammation and are required for the resolution of inflammation. PMID:27586795

  19. eHealth Recruitment Challenges

    ERIC Educational Resources Information Center

    Thompson, Debbe; Canada, Ashanti; Bhatt, Riddhi; Davis, Jennifer; Plesko, Lisa; Baranowski, Tom; Cullen, Karen; Zakeri, Issa

    2006-01-01

    Little is known about effective eHealth recruitment methods. This paper presents recruitment challenges associated with enrolling African-American girls aged 8-10 years in an eHealth obesity prevention program, their effect on the recruitment plan, and potential implications for eHealth research. Although the initial recruitment strategy was…

  20. TLR2 and TLR9 contribute to alcohol-mediated liver injury through induction of CXCL1 and neutrophil infiltration.

    PubMed

    Roh, Yoon Seok; Zhang, Bi; Loomba, Rohit; Seki, Ekihiro

    2015-07-01

    Although previous studies reported the involvement of the TLR4-TRIF pathway in alcohol-induced liver injury, the role of TLR2 and TLR9 signaling in alcohol-mediated neutrophil infiltration and liver injury has not been elucidated. Since alcohol binge drinking is recognized to induce more severe form of alcohol liver disease, we used a chronic-binge ethanol-feeding model as a mouse model for early stage of alcoholic hepatitis. Whereas a chronic-binge ethanol feeding induced alcohol-mediated liver injury in wild-type mice, TLR2- and TLR9-deficient mice showed reduced liver injury. Induction of neutrophil-recruiting chemokines, including Cxcl1, Cxcl2, and Cxcl5, and hepatic neutrophil infiltration were increased in wild-type mice, but not in TLR2- and TLR9-deficient mice. In vivo depletion of Kupffer cells (KCs) by liposomal clodronate reduced liver injury and the expression of Il1b, but not Cxcl1, Cxcl2, and Cxcl5, suggesting that KCs are partly associated with liver injury, but not neutrophil recruitment, in a chronic-binge ethanol-feeding model. Notably, hepatocytes and hepatic stellate cells (HSCs) produce high amounts of CXCL1 in ethanol-treated mice. The treatment with TLR2 and TLR9 ligands synergistically upregulated CXCL1 expression in hepatocytes. Moreover, the inhibitors for CXCR2, a receptor for CXCL1, and MyD88 suppressed neutrophil infiltration and liver injury induced by chronic-binge ethanol treatment. Consistent with the above findings, hepatic CXCL1 expression was highly upregulated in patients with alcoholic hepatitis. In a chronic-binge ethanol-feeding model, the TLR2 and TLR9-dependent MyD88-dependent pathway mediates CXCL1 production in hepatocytes and HSCs; the CXCL1 then promotes neutrophil infiltration into the liver via CXCR2, resulting in the development of alcohol-mediated liver injury.

  1. Recruiting Adult Education Students.

    ERIC Educational Resources Information Center

    Learning Resources Network, Manhattan, KS.

    This document is the first nationwide compilation of successful recruiting techniques for students in adult basic education, literacy, General Educational Development classes, and adult high school degree programs. Information for the publication was gathered from a literature search and other sources, especially "Reaching the Least Educated," a…

  2. Recruitment and Information Program

    ERIC Educational Resources Information Center

    Liebergott, Harvey

    1976-01-01

    The Bureau of Education for the Handicapped's Recruitment and Information Program provides parents and other interested individuals with information on the educational needs of handicapped children through such activities as the National Information Center for the Handicapped ("Closer Look"), pamphlets on various subjects, and media campaigns that…

  3. Recruiting Seminary Trustees.

    ERIC Educational Resources Information Center

    Warford, Malcolm

    1985-01-01

    The important role played by trustees of seminaries and the recruitment of these leaders are considered. The serious financial problems facing the board of directors has focused attention on the role and scope of seminary trusteeship. Leadership is needed by trustees to address issues of declining enrollments, curricula that address theological…

  4. Graduate Student Recruitment.

    ERIC Educational Resources Information Center

    Baron, Patricia B.

    1987-01-01

    A study of graduate student recruitment practices was conducted in the spring of 1986 to determine the current practice of graduate schools and to determine the extent to which they are using marketing techniques. The members of the Council of Graduate Schools were surveyed; 250 graduate schools responded (69% response rate). Questions concerned…

  5. Recruitment. Hello, goodbye.

    PubMed

    Moore, Alison

    2008-07-10

    The UK is "moving to a policy of self-sufficiency" according to the Department of Health. The numbers of new overseas entrants into healthcare, including doctors, nurses and midwives has slumped. Several other countries, including Canada, the US and Australia, are aggressively recruiting from overseas, including from the UK. There is an increasing perception the UK does not want overseas staff.

  6. Neutrophil Leukocyte: Combustive Microbicidal Action and Chemiluminescence.

    PubMed

    Allen, Robert C

    2015-01-01

    Neutrophil leukocytes protect against a varied and complex array of microbes by providing microbicidal action that is simple, potent, and focused. Neutrophils provide such action via redox reactions that change the frontier orbitals of oxygen (O2) facilitating combustion. The spin conservation rules define the symmetry barrier that prevents direct reaction of diradical O2 with nonradical molecules, explaining why combustion is not spontaneous. In burning, the spin barrier is overcome when energy causes homolytic bond cleavage producing radicals capable of reacting with diradical O2 to yield oxygenated radical products that further participate in reactive propagation. Neutrophil mediated combustion is by a different pathway. Changing the spin quantum state of O2 removes the symmetry restriction to reaction. Electronically excited singlet molecular oxygen ((1)O2(*)) is a potent electrophilic reactant with a finite lifetime that restricts its radius of reactivity and focuses combustive action on the target microbe. The resulting exergonic dioxygenation reactions produce electronically excited carbonyls that relax by light emission, that is, chemiluminescence. This overview of neutrophil combustive microbicidal action takes the perspectives of spin conservation and bosonic-fermionic frontier orbital considerations. The necessary principles of particle physics and quantum mechanics are developed and integrated into a fundamental explanation of neutrophil microbicidal metabolism.

  7. Exercise, training and neutrophil microbicidal activity.

    PubMed

    Smith, J A; Telford, R D; Mason, I B; Weidemann, M J

    1990-06-01

    The concentration in human plasma of putative neutrophil-"priming" cytokines like endogenous pyrogens is known to increase significantly in response to moderate exercise (11). This is characteristic of an acute-phase response. The ability of blood neutrophils isolated from both trained and untrained human subjects (n = 11, 9) to produce microbicidal reactive oxygen species was determined using luminol-enhanced chemiluminescence both before and after one hour of aerobic exercise at 60% VO2max. Irrespective of training and stimulus concentration, exercise nearly always caused significant "priming" of the capacity of neutrophils to produce H2O2 and HOCl upon stimulation with opsonized zymosan (P less than 0.01); however, compared to their untrained counterparts, the activity of cells isolated from trained individuals was depressed about 50% at unit stimulus concentration, both before and after exercise (P less than 0.075), whilst remaining unaltered at saturating concentrations. Although neutrophil oxygenation activity is only one parameter that contributes to immunological status, regular episodes of moderate exercise may increase resistance to infection by priming the "killing capacity" of neutrophils. In contrast, prolonged periods of intensive training may lead to increased susceptibility to common infections by diminishing this activity. PMID:2115507

  8. Methoxatin (PQQ) in guinea-pig neutrophils.

    PubMed

    Bishop, A; Paz, M A; Gallop, P M; Karnovsky, M L

    1994-10-01

    PQQ, also called methoxatin, has been isolated from guinea-pig neutrophils. The organic cations diphenyleneiodonium (DPI) and diphenyliodonium (BPI) and the aromatic o-diamine 4,5-dimethylphenylenediamine (DIMPDA) sequester synthetic PQQ and inhibit its redox-cycling activity in a model system. Standards were made of adducts of tritiated PQQ with unlabeled DIMPDA and of unlabeled PQQ with tritiated DPI or DIMPDA. PQQ adducts were isolated from guinea-pig neutrophils with each of the tritiated inhibitors. They were separated and defined by high-performance liquid chromatography (HPLC). Tiron, a disodium benzene disulphonic acid, broke the DPI-PQQ adduct isolated from neutrophils and released free PQQ. Both DPI and DIMPDA, as well as BPI, blocked O2.- release by stimulated neutrophils. The blockade exerted by these inhibitors was released by the addition of PQQ to the cell suspensions. The data demonstrate the presence of PQQ in guinea-pig neutrophils and suggest that it has a possible role, direct or indirect, in the O2.(-)-producing respiratory burst.

  9. Stimulation of neutrophils by tumor necrosis factor

    SciTech Connect

    Klebanoff, S.J.; Vadas, M.A.; Harlan, J.M.; Sparks, L.H.; Gamble, J.R.; Agosti, J.M.; Waltersdorph, A.M.

    1986-06-01

    Human recombinant tumor necrosis factor (TNF) was shown to be a weak direct stimulus of the neutrophil respiratory burst and degranulation. The stimulation, as measured by iodination, H/sub 2/O/sub 2/ production, and lysozyme release, was considerably increased by the presence of unopsonized zymosan in the reaction mixture, an effect which was associated with the increased ingestion of the zymosan. TNF does not act as an opsonin but, rather, reacts with the neutrophil to increase its phagocytic activity. TNF-dependent phagocytosis, as measured indirectly by iodination, is inhibited by monoclonal antibodies (Mab) 60.1 and 60.3, which recognize different epitopes on the C3bi receptor/adherence-promoting surface glycoprotein of neutrophils. Other neutrophil stimulants, namely N-formyl-methionyl-leucyl-phenylalanine, the Ca2+ ionophore A23187, and phorbol myristic acetate, also increase iodination in the presence of zymosan; as with TNF, the effect of these stimulants is inhibited by Mab 60.1 and 60.3, whereas, in contrast to that of TNF, their stimulation of iodination is unaffected by an Mab directed against TNF. TNF may be a natural stimulant of neutrophils which promotes adherence to endothelial cells and to particles, leading to increased phagocytosis, respiratory burst activity, and degranulation.

  10. Neutrophil Leukocyte: Combustive Microbicidal Action and Chemiluminescence

    PubMed Central

    Allen, Robert C.

    2015-01-01

    Neutrophil leukocytes protect against a varied and complex array of microbes by providing microbicidal action that is simple, potent, and focused. Neutrophils provide such action via redox reactions that change the frontier orbitals of oxygen (O2) facilitating combustion. The spin conservation rules define the symmetry barrier that prevents direct reaction of diradical O2 with nonradical molecules, explaining why combustion is not spontaneous. In burning, the spin barrier is overcome when energy causes homolytic bond cleavage producing radicals capable of reacting with diradical O2 to yield oxygenated radical products that further participate in reactive propagation. Neutrophil mediated combustion is by a different pathway. Changing the spin quantum state of O2 removes the symmetry restriction to reaction. Electronically excited singlet molecular oxygen (1O2*) is a potent electrophilic reactant with a finite lifetime that restricts its radius of reactivity and focuses combustive action on the target microbe. The resulting exergonic dioxygenation reactions produce electronically excited carbonyls that relax by light emission, that is, chemiluminescence. This overview of neutrophil combustive microbicidal action takes the perspectives of spin conservation and bosonic-fermionic frontier orbital considerations. The necessary principles of particle physics and quantum mechanics are developed and integrated into a fundamental explanation of neutrophil microbicidal metabolism. PMID:26783542

  11. Neutrophils alter the inflammatory milieu by signal-dependent translation of constitutive messenger RNAs

    NASA Astrophysics Data System (ADS)

    Lindemann, Stephan W.; Yost, Christian C.; Denis, Melvin M.; McIntyre, Thomas M.; Weyrich, Andrew S.; Zimmerman, Guy A.

    2004-05-01

    The mechanisms by which neutrophils, key effector cells of the innate immune system, express new gene products in inflammation are largely uncharacterized. We found that they rapidly translate constitutive mRNAs when activated, a previously unrecognized response. One of the proteins synthesized without a requirement for transcription is the soluble IL-6 receptor , which translocates to endothelial cells and induces a temporal switch to mononuclear leukocyte recruitment. Its synthesis is regulated by a specialized translational control pathway that is inhibited by rapamycin, a bacterial macrolide with therapeutic efficacy in transplantation, inflammatory syndromes, and neoplasia. Signal-dependent translation in activated neutrophils may be a critical mechanism for alteration of the inflammatory milieu and a therapeutic target.

  12. Predator nonconsumptive effects on prey recruitment weaken with recruit density.

    PubMed

    Ellrich, Julius A; Scrosati, Ricardo A; Molis, Markus

    2015-03-01

    We investigated the nonconsumptive effects (NCEs) of predatory dogwhelks (Nucella lapillus) on intertidal barnacle (Semibalanus balanoides) recruitment through field experiments on the Gulf of St. Lawrence coast and the Atlantic coast of Nova Scotia, Canada. We studied the recruitment seasons (May-June) of 2011 and 2013. In 2011, the Gulf coast had five times more nearshore phytoplankton (food for barnacle larvae and recruits) during the recruitment season and yielded a 58% higher barnacle recruit density than the Atlantic coast at the end of the recruitment season. In 2013, phytoplankton levels and barnacle recruit density were similar on both coasts and also lower than for the Gulf coast in 2011. Using the comparative-experimental method, the manipulation of dogwhelk presence (without allowing physical contact with prey) revealed that dogwhelk cues limited barnacle recruitment under moderate recruit densities (Atlantic 2011/2013 and Gulf 2013) but had no effect under a high recruit density (Gulf 2011). Barnacle recruits attract settling larvae through chemical cues. Thus, the highest recruit density appears to have neutralized dogwhelk effects. This study suggests that the predation risk perceived by settling larvae may decrease with increasing recruit density and that prey food supply may indirectly influence predator NCEs on prey recruitment. PMID:26236858

  13. Decreased apoptosis of beta 2- integrin-deficient bovine neutrophils.

    PubMed

    Nagahata, Hajime; Higuchi, Hidetoshi; Teraoka, Hiroki; Takahashi, Kenji; Takahashi, Kensi; Kuwabara, Mikinori; Inanami, Osamu; Kuwabara, Mikwori

    2004-02-01

    Stimulant-induced viability of neutrophils, nuclear-fragmentation, increase in intracellular calcium ([Ca2+]i), expression of annexin V on neutrophils and proteolysis of a fluorogenic peptide substrate Ac-DEVD-MCA (acetyl Asp-Glu-Val-Asp alpha-[4-methyl-coumaryl-7-amide]) by neutrophil lysates from five normal calves and three calves with leucocyte adhesion deficiency were determined to evaluate the apoptosis of normal and CD18-deficient neutrophils. Viability was markedly decreased in control neutrophils stimulated with opsonized zymosan (OPZ), compared to CD18-deficient neutrophils at 37 degrees C after incubation periods of 6 and 24 hours. The rate of apoptosis of control neutrophils stimulated with OPZ increased significantly depending on the incubation time, whereas no apparent increase in apoptosis was found in CD18-deficient neutrophils under the same conditions. Aggregated bovine (Agg) IgG-induced apoptosis of control neutrophils was not significantly different from that of CD18-deficient neutrophils. The expression of annexin V on OPZ-stimulated control neutrophils was greater than that of unstimulated ones 6 h after stimulation. No apparent increase in annexin V expression on CD18-deficient neutrophils was found with OPZ stimulation. A delay in apoptosis was demonstrated in CD18-deficient bovine neutrophils and this appeared to be closely associated with lowered signalling via [Ca2+]i, diminished annexin V expression on the cell surface, and decreased caspase 3 activity in lysates. PMID:14984592

  14. Neutrophil activation by Candida glabrata but not Candida albicans promotes fungal uptake by monocytes.

    PubMed

    Duggan, Seána; Essig, Fabian; Hünniger, Kerstin; Mokhtari, Zeinab; Bauer, Laura; Lehnert, Teresa; Brandes, Susanne; Häder, Antje; Jacobsen, Ilse D; Martin, Ronny; Figge, Marc Thilo; Kurzai, Oliver

    2015-09-01

    Candida albicans and Candida glabrata account for the majority of candidiasis cases worldwide. Although both species are in the same genus, they differ in key virulence attributes. Within this work, live cell imaging was used to examine the dynamics of neutrophil activation after confrontation with either C. albicans or C. glabrata. Analyses revealed higher phagocytosis rates of C. albicans than C. glabrata that resulted in stronger PMN (polymorphonuclear cells) activation by C. albicans. Furthermore, we observed differences in the secretion of chemokines, indicating chemotactic differences in PMN signalling towards recruitment of further immune cells upon confrontation with Candida spp. Supernatants from co-incubations of neutrophils with C. glabrata primarily attracted monocytes and increased the phagocytosis of C. glabrata by monocytes. In contrast, PMN activation by C. albicans resulted in recruitment of more neutrophils. Two complex infection models confirmed distinct targeting of immune cell populations by the two Candida spp.: In a human whole blood infection model, C. glabrata was more effectively taken up by monocytes than C. albicans and histopathological analyses of murine model infections confirmed primarily monocytic infiltrates in C. glabrata kidney infection in contrast to PMN-dominated infiltrates in C. albicans infection. Taken together, our data demonstrate that the human opportunistic fungi C. albicans and C. glabrata are differentially recognized by neutrophils and one outcome of this differential recognition is the preferential uptake of C. glabrata by monocytes.

  15. Dermal neutrophil, macrophage and dendritic cell responses to Yersinia pestis transmitted by fleas.

    PubMed

    Shannon, Jeffrey G; Bosio, Christopher F; Hinnebusch, B Joseph

    2015-03-01

    Yersinia pestis, the causative agent of plague, is typically transmitted by the bite of an infected flea. Many aspects of mammalian innate immune response early after Y. pestis infection remain poorly understood. A previous study by our lab showed that neutrophils are the most prominent cell type recruited to the injection site after intradermal needle inoculation of Y. pestis, suggesting that neutrophil interactions with Y. pestis may be important in bubonic plague pathogenesis. In the present study, we developed new tools allowing for intravital microscopy of Y. pestis in the dermis of an infected mouse after transmission by its natural route of infection, the bite of an infected flea. We found that uninfected flea bites typically induced minimal neutrophil recruitment. The magnitude of neutrophil response to flea-transmitted Y. pestis varied considerably and appeared to correspond to the number of bacteria deposited at the bite site. Macrophages migrated towards flea bite sites and interacted with small numbers of flea-transmitted bacteria. Consistent with a previous study, we observed minimal interaction between Y. pestis and dendritic cells; however, dendritic cells did consistently migrate towards flea bite sites containing Y. pestis. Interestingly, we often recovered viable Y. pestis from the draining lymph node (dLN) 1 h after flea feeding, indicating that the migration of bacteria from the dermis to the dLN may be more rapid than previously reported. Overall, the innate cellular host responses to flea-transmitted Y. pestis differed from and were more variable than responses to needle-inoculated bacteria. This work highlights the importance of studying the interactions between fleas, Y. pestis and the mammalian host to gain a better understanding of the early events in plague pathogenesis.

  16. Dermal Neutrophil, Macrophage and Dendritic Cell Responses to Yersinia pestis Transmitted by Fleas

    PubMed Central

    Shannon, Jeffrey G.; Bosio, Christopher F.; Hinnebusch, B. Joseph

    2015-01-01

    Yersinia pestis, the causative agent of plague, is typically transmitted by the bite of an infected flea. Many aspects of mammalian innate immune response early after Y. pestis infection remain poorly understood. A previous study by our lab showed that neutrophils are the most prominent cell type recruited to the injection site after intradermal needle inoculation of Y. pestis, suggesting that neutrophil interactions with Y. pestis may be important in bubonic plague pathogenesis. In the present study, we developed new tools allowing for intravital microscopy of Y. pestis in the dermis of an infected mouse after transmission by its natural route of infection, the bite of an infected flea. We found that uninfected flea bites typically induced minimal neutrophil recruitment. The magnitude of neutrophil response to flea-transmitted Y. pestis varied considerably and appeared to correspond to the number of bacteria deposited at the bite site. Macrophages migrated towards flea bite sites and interacted with small numbers of flea-transmitted bacteria. Consistent with a previous study, we observed minimal interaction between Y. pestis and dendritic cells; however, dendritic cells did consistently migrate towards flea bite sites containing Y. pestis. Interestingly, we often recovered viable Y. pestis from the draining lymph node (dLN) 1 h after flea feeding, indicating that the migration of bacteria from the dermis to the dLN may be more rapid than previously reported. Overall, the innate cellular host responses to flea-transmitted Y. pestis differed from and were more variable than responses to needle-inoculated bacteria. This work highlights the importance of studying the interactions between fleas, Y. pestis and the mammalian host to gain a better understanding of the early events in plague pathogenesis. PMID:25781984

  17. Clinical Microfluidics for Neutrophil Genomics and Proteomics

    PubMed Central

    Kotz, Kenneth T.; Xiao, Wenzong; Miller-Graziano, Carol; Qian, Wei-Jun; Russom, Aman; Warner, Elizabeth A.; Moldawer, Lyle L.; De, Asit; Bankey, Paul E.; Petritis, Brianne O.; Camp, David G.; Rosenbach, Alan E.; Goverman, Jeremy; Fagan, Shawn P.; Brownstein, Bernard H.; Irimia, Daniel; Xu, Weihong; Wilhelmy, Julie; Mindrinos, Michael N.; Smith, Richard D.; Davis, Ronald W.; Tompkins, Ronald G.; Toner, Mehmet

    2010-01-01

    Neutrophils play critical roles in modulating the immune response. We present a robust methodology for rapidly isolating neutrophils directly from whole blood and develop ‘on-chip’ processing for mRNA and protein isolation for genomics and proteomics. We validate this device with an ex vivo stimulation experiment and by comparison with standard bulk isolation methodologies. Lastly, we implement this tool as part of a near patient blood processing system within a multi-center clinical study of the immune response to severe trauma and burn injury. The preliminary results from a small cohort of patients in our study and healthy controls show a unique time-dependent gene expression pattern clearly demonstrating the ability of this tool to discriminate temporal transcriptional events of neutrophils within a clinical setting. PMID:20802500

  18. Clinical microfluidics for neutrophil genomics and proteomics.

    PubMed

    Kotz, Kenneth T; Xiao, Wenzong; Miller-Graziano, Carol; Qian, Wei-Jun; Russom, Aman; Warner, Elizabeth A; Moldawer, Lyle L; De, Asit; Bankey, Paul E; Petritis, Brianne O; Camp, David G; Rosenbach, Alan E; Goverman, Jeremy; Fagan, Shawn P; Brownstein, Bernard H; Irimia, Daniel; Xu, Weihong; Wilhelmy, Julie; Mindrinos, Michael N; Smith, Richard D; Davis, Ronald W; Tompkins, Ronald G; Toner, Mehmet

    2010-09-01

    Neutrophils have key roles in modulating the immune response. We present a robust methodology for rapidly isolating neutrophils directly from whole blood with 'on-chip' processing for mRNA and protein isolation for genomics and proteomics. We validate this device with an ex vivo stimulation experiment and by comparison with standard bulk isolation methodologies. Last, we implement this tool as part of a near-patient blood processing system within a multi-center clinical study of the immune response to severe trauma and burn injury. The preliminary results from a small cohort of subjects in our study and healthy controls show a unique time-dependent gene expression pattern clearly demonstrating the ability of this tool to discriminate temporal transcriptional events of neutrophils within a clinical setting.

  19. Granulopoiesis and granules of human neutrophils.

    PubMed

    Cowland, Jack B; Borregaard, Niels

    2016-09-01

    Granules are essential for the ability of neutrophils to fulfill their role in innate immunity. Granule membranes contain proteins that react to environmental cues directing neutrophils to sites of infection and initiate generation of bactericidal oxygen species. Granules are densely packed with proteins that contribute to microbial killing when liberated to the phagosome or extracellularly. Granules are, however, highly heterogeneous and are traditionally subdivided into azurophil granules, specific granules, and gelatinase granules in addition to secretory vesicles. This review will address issues pertinent to formation of granules, which is a process intimately connected to maturation of neutrophils from their precursors in the bone marrow. We further discuss possible mechanisms by which decisions are made regarding sorting of proteins to constitutive secretion or storage in granules and how degranulation of granule subsets is regulated. PMID:27558325

  20. Defective neutrophil chemotaxis in juvenile periodontitis.

    PubMed Central

    Clark, R A; Page, R C; Wilde, G

    1977-01-01

    Neutrophil chemotaxis was evaluated in nine patients with juvenile periodontitis, with normal subjects and patients with the adult form of periodontitis as controls. Defective chemotactic responses were observed in neutrophils from seven of nine juvenile patients, and a reduced level of complement-derived chemotactic activity was demonstrated in serum from four patients. These determinations were normal in all the patients with adult periodontitis. Serum from five of the juvenile patients contained a heat-stable, non-dialyzable factor that markedly inhibited the chemotaxis of normal neutrophils. Thus the characteristic tissue destruction seen in juvenile periodontitis may be, at least in part, a consequence of a failure of host defense mechanisms. PMID:591063

  1. [Ultrastructural location of enzymes in peripheral blood neutrophils and in cerebrospinal fluid neutrophils in neuroinfections].

    PubMed

    Skotarczak, B

    1993-01-01

    Using cytochemical methods the location and activity were determined of alkaline phosphatase, ATP-ase and succinate dehydrogenase as representative enzymes for the metabolic processes in neutrophils isolated from blood and cerebrospinal fluid (CSF) of patients with meningococcal meningoencephalitis as compared with peripheral blood neutrophils in a control group. The study showed presence of phosphatase on the membranes of many intracellular structures. The activity of the enzymes was higher than in the control group in the membranes of neutrophils in blood and CSF. This is explained as an effect of action of the chemotactic factor on the cell membrane and activation of the cell to movements and phagocytosis. ATP-ase activity in peripheral blood neutrophils in controls was found in all membranous structures in the cell. However, in peripheral blood neutrophils and CSF neutrophils in the acute stage of the disease the active enzyme was noted, in the first place, in cell membranes and digesting vacuoles, which reflected probably the direction of metabolic processes for phagocytosis and destroying of bacteria. The activity of succinate dehydrogenase was found in mitochondrial membranes. Peripheral blood and CSF neutrophils showed a high activity of the enzyme. In the CSF cells in acute phase atypical sites of succinate dehydrogenase activity were noted, which was explained as a sign of cell destruction.

  2. Mannheimia haemolytica and its leukotoxin cause neutrophil extracellular trap formation by bovine neutrophils.

    PubMed

    Aulik, Nicole A; Hellenbrand, Katrina M; Klos, Heather; Czuprynski, Charles J

    2010-11-01

    Mannheimia haemolytica is an important member of the bovine respiratory disease complex, which is characterized by abundant neutrophil infiltration into the alveoli and fibrin deposition. Recently several authors have reported that human neutrophils release neutrophil extracellular traps (NETs), which are protein-studded DNA matrices capable of trapping and killing pathogens. Here, we demonstrate that the leukotoxin (LKT) of M. haemolytica causes NET formation by bovine neutrophils in a CD18-dependent manner. Using an unacylated, noncytotoxic pro-LKT produced by an ΔlktC mutant of M. haemolytica, we show that binding of unacylated pro-LKT stimulates NET formation despite a lack of cytotoxicity. Inhibition of LKT binding to the CD18 chain of lymphocyte function-associated antigen 1 (LFA-1) on bovine neutrophils reduced NET formation in response to LKT or M. haemolytica cells. Further investigation revealed that NETs formed in response to M. haemolytica are capable of trapping and killing a portion of the bacterial cells. NET formation was confirmed by confocal microscopy and by scanning and transmission electron microscopy. Prior exposure of bovine neutrophils to LKT enhanced subsequent trapping and killing of M. haemolytica cells in bovine NETs. Understanding NET formation in response to M. haemolytica and its LKT provides a new perspective on how neutrophils contribute to the pathogenesis of bovine respiratory disease. PMID:20823211

  3. Streptococcal M1 protein triggers chemokine formation, neutrophil infiltration, and lung injury in an NFAT-dependent manner.

    PubMed

    Zhang, Songen; Zhang, Su; Garcia-Vaz, Eliana; Herwald, Heiko; Gomez, Maria F; Thorlacius, Henrik

    2015-06-01

    Streptococcus pyogenes of the M1 serotype can cause STSS, which is associated with significant morbidity and mortality. The purpose of the present study was to examine the role of NFAT signaling in M1 protein-induced lung injury. NFAT-luc mice were treated with the NFAT inhibitor A-285222 before administration of the M1 protein. Neutrophil infiltration, edema, and CXC chemokines were quantified in the lung, 4 h after challenge with the M1 protein. Flow cytometry was used to determine Mac-1 expression. Challenge with the M1 protein increased NFAT-dependent transcriptional activity in the lung, spleen, and liver in NFAT-luc mice. Administration of the NFAT inhibitor A-285222 abolished M1 protein-evoked NFAT activation in the lung, spleen, and liver. M1 protein challenge induced neutrophil recruitment, edema, and CXC chemokine production in the lung, as well as up-regulation of Mac-1 on circulating neutrophils. Inhibition of NFAT activity attenuated M1 protein-induced neutrophil infiltration by 77% and edema formation by 50% in the lung. Moreover, administration of A-285222 reduced M1 protein-evoked pulmonary formation of CXC chemokine >80%. In addition, NFAT inhibition decreased M1 protein-triggered Mac-1 up-regulation on neutrophils. These findings indicate that NFAT signaling controls pulmonary infiltration of neutrophils in response to streptococcal M1 protein via formation of CXC chemokines and neutrophil expression of Mac-1. Thus, the targeting of NFAT activity might be a useful way to ameliorate lung injury in streptococcal infections.

  4. Neutrophils Select Hypervirulent CovRS Mutants of M1T1 Group A Streptococcus during Subcutaneous Infection of Mice

    PubMed Central

    Li, Jinquan; Liu, Guanghui; Feng, Wenchao; Zhou, Yang; Liu, Mengyao; Wiley, James A.

    2014-01-01

    Pathogen mutants arise during infections. Mechanisms of selection for pathogen variants are poorly understood. We tested whether neutrophils select mutations in the two-component regulatory system CovRS of group A Streptococcus (GAS) during infection using the lack of production of the protease SpeB (SpeB activity negative [SpeBA−]) as a marker. Depletion of neutrophils by antibodies RB6-8C5 and 1A8 reduced the percentage of SpeBA− variants (SpeBA−%) recovered from mice infected with GAS strain MGAS2221 by >76%. Neutrophil recruitment and SpeBA−% among recovered GAS were reduced by 95% and 92%, respectively, in subcutaneous MGAS2221 infection of CXCR2−/− mice compared with control mice. In air sac infection with MGAS2221, levels of neutrophils and macrophages in lavage fluid were reduced by 49% and increased by 287%, respectively, in CXCR2−/− mice compared with control mice, implying that macrophages play an insignificant role in the reduction of selection for SpeBA− variants in CXCR2−/− mice. One randomly chosen SpeBA− mutant outcompeted MGAS2221 in normal mice but was outcompeted by MGAS2221 in neutropenic mice and had enhancements in expression of virulence factors, innate immune evasion, skin invasion, and virulence. This and nine other SpeBA− variants from a mouse all had nonsynonymous covRS mutations that resulted in the SpeBA− phenotype and enhanced expression of the CovRS-controlled secreted streptococcal esterase (SsE). Our findings are consistent with a model that neutrophils select spontaneous covRS mutations that maximize the potential of GAS to evade neutrophil responses, resulting in variants with enhanced survival and virulence. To our knowledge, this is the first report of the critical contribution of neutrophils to the selection of pathogen variants. PMID:24452689

  5. Porphyromonas gingivalis Participates in Pathogenesis of Human Abdominal Aortic Aneurysm by Neutrophil Activation. Proof of Concept in Rats

    PubMed Central

    Delbosc, Sandrine; Alsac, Jean-Marc; Journe, Clement; Louedec, Liliane; Castier, Yves; Bonnaure-Mallet, Martine; Ruimy, Raymond; Rossignol, Patrick; Bouchard, Philippe; Michel, Jean-Baptiste; Meilhac, Olivier

    2011-01-01

    Background Abdominal Aortic Aneurysms (AAAs) represent a particular form of atherothrombosis where neutrophil proteolytic activity plays a major role. We postulated that neutrophil recruitment and activation participating in AAA growth may originate in part from repeated episodes of periodontal bacteremia. Methods and Findings Our results show that neutrophil activation in human AAA was associated with Neutrophil Extracellular Trap (NET) formation in the IntraLuminal Thrombus, leading to the release of cell-free DNA. Human AAA samples were shown to contain bacterial DNA with high frequency (11/16), and in particular that of Porphyromonas gingivalis (Pg), the most prevalent pathogen involved in chronic periodontitis, a common form of periodontal disease. Both DNA reflecting the presence of NETs and antibodies to Pg were found to be increased in plasma of patients with AAA. Using a rat model of AAA, we demonstrated that repeated injection of Pg fostered aneurysm development, associated with pathological characteristics similar to those observed in humans, such as the persistence of a neutrophil-rich luminal thrombus, not observed in saline-injected rats in which a healing process was observed. Conclusions Thus, the control of periodontal disease may represent a therapeutic target to limit human AAA progression. PMID:21533243

  6. Understanding the Mechanisms Controlling Leishmania amazonensis Infection In Vitro: The Role of LTB4 Derived From Human Neutrophils

    PubMed Central

    Tavares, Natalia Machado; Araújo-Santos, Théo; Afonso, Lilian; Nogueira, Paula Monalisa; Lopes, Ulisses Gazos; Soares, Rodrigo Pedro; Bozza, Patrícia Torres; Bandeira-Melo, Christianne; Borges, Valeria Matos; Brodskyn, Cláudia

    2014-01-01

    Neutrophils are rapidly recruited to the site of Leishmania infection and play an active role in capturing and killing parasites. They are the main source of leukotriene B4 (LTB4), a potent proinflammatory lipid mediator. However, the role of LTB4 in neutrophil infection by Leishmania amazonensis is not clear. In this study, we show that L. amazonensis or its lipophosphoglycan can induce neutrophil activation, degranulation, and LTB4 production. Using pharmacological inhibitors of leukotriene synthesis, our findings reveal an LTB4-driven autocrine/paracrine regulatory effect. In particular, neutrophil-derived LTB4 controls L. amazonensis killing, degranulation, and reactive oxygen species production. In addition, L. amazonensis infection induces an early increase in Toll-like receptor 2 expression, which facilitates parasite internalization. Nuclear factor kappa B (NFkB) pathway activation represents a required upstream event for L. amazonensis–induced LTB4 synthesis. These leishmanicidal mechanisms mediated by neutrophil-derived LTB4 act through activation of its receptor, B leukotriene receptor 1 (BLT1). PMID:24634497

  7. Inhibition of Nicotinamide Phosphoribosyltransferase Reduces Neutrophil-Mediated Injury in Myocardial Infarction

    PubMed Central

    Bauer, Inga; Braunersreuther, Vincent; Bruzzone, Santina; Akhmedov, Alexander; Lüscher, Thomas F.; Speer, Timo; Poggi, Alessandro; Mannino, Elena; Pelli, Graziano; Galan, Katia; Bertolotto, Maria; Lenglet, Sébastien; Garuti, Anna; Montessuit, Christophe; Lerch, René; Pellieux, Corinne; Vuilleumier, Nicolas; Dallegri, Franco; Mage, Jacqueline; Sebastian, Carlos; Mostoslavsky, Raul; Gayet-Ageron, Angèle; Patrone, Franco; Mach, François; Nencioni, Alessio

    2013-01-01

    Abstract Aims: Nicotinamide phosphoribosyltransferase (Nampt) is a key enzyme for nicotinamide adenine dinucleotide (NAD+) biosynthesis, and recent evidence indicates its role in inflammatory processes. Here, we investigated the potential effects of pharmacological Nampt inhibition with FK866 in a mouse myocardial ischemia/reperfusion model. In vivo and ex vivo mouse myocardial ischemia/reperfusion procedures were performed. Results: Treatment with FK866 reduced myocardial infarct size, neutrophil infiltration, and reactive oxygen species (ROS) generation within infarcted hearts in vivo in a mouse model of ischemia and reperfusion. The benefit of FK866 was not shown in the Langendorff model (ex vivo model of working heart without circulating leukocytes), suggesting a direct involvement of these cells in cardiac injury. Sera from FK866-treated mice showed reduced circulating levels of the neutrophil chemoattractant CXCL2 and impaired capacity to prime migration of these cells in vitro. The release of CXCL8 (human homolog of murine chemokine CXCL2) by human peripheral blood mononuclear cells (PBMCs) and Jurkat cells was also reduced by FK866, as well as by sirtuin (SIRT) inhibitors and SIRT6 silencing, implying a pivotal role for this NAD+-dependent deacetylase in the production of this chemokine. Innovation: The pharmacological inhibition of Nampt might represent an effective approach to reduce neutrophilic inflammation- and oxidative stress-mediated tissue damage in early phases of reperfusion after a myocardial infarction. Conclusions: Nampt inhibition appears as a new strategy to dampen CXCL2-induced neutrophil recruitment and thereby reduce neutrophil-mediated tissue injury in mice. Antioxid. Redox Signal. 18, 630–641. PMID:22452634

  8. Ozone inhalation stimulates expression of a neutrophil chemotactic protein, macrophage inflammatory protein 2

    SciTech Connect

    Driscoll, K.E.; Simpson, L.; Carter, J.; Hassenbein, D.; Leikauf, G.D. )

    1993-04-01

    Short-term exposure of humans and animals to ozone results in increased lung neutrophils; however, the mechanisms underlying this response are not completely understood. We examined the potential involvement of the neutrophil chemotactic factor, macrophage inflammatory protein 2 (MIP-2), in ozone-induced inflammation. Exposure-response relationships for ozone and MIP-2 expression were characterized by exposing C57B1/6 mice to 0.1-2 ppm ozone for 3 hr and determining lung levels of MIP-2 mRNA 6 hr after exposure. Temporal relationships between ozone and MIP-2 were determined by exposing mice (2 ppm ozone x 3 hr) and characterizing MIP-2 mRNA expression 0, 2, 6, and 24 hr after exposure. Neutrophils in lung lavage fluid were determined in both exposure-response and time course studies. Ozone concentrations > or = 1.0 ppm increased MIP-2 mRNA and this increase corresponded with recruitment of neutrophils. MIP-2 mRNA was increased immediately after ozone exposure and decreased to control levels by 24 hr. To examine the role of direct oxidant effects in ozone-induced MIP-2 expression, alveolar macrophages were exposed in vitro for 4 hr to 10(-10)-10(-5) M hydrogen peroxide and MIP-2 expression was characterized. MIP-2 mRNA levels in lung macrophages were increased by > or = 10(-9) M hydrogen peroxide. In summary, our findings suggest the chemotactic protein MIP-2 may be responsible, at least in part, for ozone-induced increases in lung neutrophils and indicate that direct exposure of alveolar macrophages to an oxidant is sufficient to induce MIP-2 expression.

  9. Interaction of porcine neutrophils with different strains of enterotoxigenic Escherichia coli.

    PubMed

    Ondrackova, Petra; Alexa, Pavel; Matiasovic, Jan; Volf, Jiri; Faldyna, Martin

    2012-11-01

    Enterotoxigenic Escherichia coli (ETEC) is one of the most important causes of post-weaning diarrhea in piglets. Whilst serotype O149:F4 is frequently associated with hemorrhagic gastroenteritis, other serotypes have been found to be associated with mild or moderate enteritis. As neutrophils are recruited to sites of inflammation, the aim of this study was to ascertain whether or not there is any difference in the in vitro interaction between neutrophils and two different ETEC serotypes: O149:F4 and O147:F18. The association of bacteria with neutrophils was evaluated by flow cytometry. The respiratory burst was measured by the fluorescent probe dichlorofluorescein diacetate using flow cytometry and by L012-amplified chemiluminescence. The titers of antibodies against ETEC present in cultivation sera were assessed by agglutination. The viability of E. coli was ascertained by cultivation. It was found that the strains of O149 serotype were more frequently associated with neutrophils and induced a more intensive respiratory burst compared to the strains of O147 serotype. These differences might be due to the presence of different types of fimbriae on the surface of the strains tested and by the presence of anti-fimbrial antibodies in the porcine plasma. However, the intensive interaction between E. coli and the neutrophils and respiratory burst induced by the O149 strain did not lead to more efficient killing of the bacteria. It is suggested that a stronger respiratory burst may be an important factor causing severe clinical signs of post-weaning diarrhea in piglets. PMID:22704243

  10. Metabolic requirements for neutrophil extracellular traps formation

    PubMed Central

    Rodríguez-Espinosa, Oscar; Rojas-Espinosa, Oscar; Moreno-Altamirano, María Maximina Bertha; López-Villegas, Edgar Oliver; Sánchez-García, Francisco Javier

    2015-01-01

    As part of the innate immune response, neutrophils are at the forefront of defence against infection, resolution of inflammation and wound healing. They are the most abundant leucocytes in the peripheral blood, have a short lifespan and an estimated turnover of 1010 to 1011 cells per day. Neutrophils efficiently clear microbial infections by phagocytosis and by oxygen-dependent and oxygen-independent mechanisms. In 2004, a new neutrophil anti-microbial mechanism was described, the release of neutrophil extracellular traps (NETs) composed of DNA, histones and anti-microbial peptides. Several microorganisms, bacterial products, as well as pharmacological stimuli such as PMA, were shown to induce NETs. Neutrophils contain relatively few mitochondria, and derive most of their energy from glycolysis. In this scenario we aimed to analyse some of the metabolic requirements for NET formation. Here it is shown that NETs formation is strictly dependent on glucose and to a lesser extent on glutamine, that Glut-1, glucose uptake, and glycolysis rate increase upon PMA stimulation, and that NET formation is inhibited by the glycolysis inhibitor, 2-deoxy-glucose, and to a lesser extent by the ATP synthase inhibitor oligomycin. Moreover, when neutrophils were exposed to PMA in glucose-free medium for 3 hr, they lost their characteristic polymorphic nuclei but did not release NETs. However, if glucose (but not pyruvate) was added at this time, NET release took place within minutes, suggesting that NET formation could be metabolically divided into two phases; the first, independent from exogenous glucose (chromatin decondensation) and, the second (NET release), strictly dependent on exogenous glucose and glycolysis. PMID:25545227

  11. Neutrophil haptotaxis induced by the lectin KM+.

    PubMed

    Ganiko, L; Martins, A R; Espreáfico, E M; Roque-Barreira, M C

    1998-05-01

    KM+ is a D-mannose binding lectin from Artocarpus integrifolia that induces neutrophil migration in vitro and in vivo. This attractant activity was shown to be caused by haptotaxis rather than chemotaxis. The inhibition by D-mannose of the neutrophil attraction exerted by KM+, both in vitro and in vivo, supports the idea that haptotaxis is triggered in vivo by the sugar binding sites interacting with glycoconjugates located on the neutrophil surface and in the extracellular matrix. In the present study an in vivo haptotaxis assay was performed by intradermally (i.d.) injecting 125I-KM+ (200 ng), which led to a selective staining of loose connective tissue and vascular endothelium. The radiolabelled area exhibited a maximum increase (five-fold) in neutrophil infiltration 3 h after injection, relative to i.d. 200 ng 125I-BSA. We characterized the ex vivo binding of KM+ to tissue elements by immunohistochemistry, using paraformaldehyde-fixed, paraffin-embedded, untreated rat skin. Bound KM+ was detected with an affinity-purified rabbit IgG anti-KM+ and visualized with an alkaline phosphatase based system. KM+ binding to connective tissue and vascular endothelium was inhibited by preincubating KM+ with 0.4 mM D-mannose and was potentiated by heparan sulfate (100 microg ml(-1)). An in vitro assay carried out in a Boyden microchamber showed that heparan sulfate potentiated the attractant effect of 10 microg KM+ by 34%. The present data suggest that KM+ induces neutrophil migration in vivo by haptotaxis and that the haptotactic gradient could be provided by the interaction of the KM+ carbohydrate recognition site(s) with mannose-containing glycoconjugate(s) in vascular endothelium and connective tissue. Heparan sulfate would act as an accessory molecule, enhancing the KM+ tissue binding and potentiating the induced neutrophil haptotaxis.

  12. Dynamics of neutrophil migration in lymph nodes during infection

    PubMed Central

    Chtanova, Tatyana; Schaeffer, Marie; Han, Seong-Ji; van Dooren, Giel G.; Nollmann, Marcelo; Herzmark, Paul; Chan, Shiao Wei; Satija, Harshita; Camfield, Kristin; Aaron, Holly; Striepen, Boris; Robey, Ellen A.

    2008-01-01

    Summary While the signals that control neutrophil migration from the blood to sites of infection have been well characterized, little is known about their migration patterns within lymph nodes, or the strategies that neutrophils use to find their local sites of action. To address these questions, we used two-photon scanning laser microscopy (TPSLM) to examine neutrophil migration in intact lymph nodes during infection with an intracellular parasite, Toxoplasma gondii. We find that neutrophils form both small, transient or large, persistent swarms via a strikingly coordinated migration pattern. We provide evidence that cooperative action of neutrophils and parasite egress from host cells can trigger swarm formation. Neutrophil swarm formation coincides in space and time with the removal of macrophages that line the subcapsular sinus of the lymph node. Our data provide insights into the cellular mechanisms underlying neutrophil swarming and suggest new roles for neutrophils in shaping immune responses. PMID:18718768

  13. The Students-Recruiting-Students Undergraduate Engineering Recruiting Programme.

    ERIC Educational Resources Information Center

    Gattis, Carol; Nachtmann, Heather; Youngblood, Alisha D.

    2003-01-01

    Describes the Students-Recruiting-Students (SRS) program developed to recruit high school students into the Industrial Engineering Department at the University of Arkansas. Presents four phases of the program along with seven years of program results. Encourages successful development of similar recruiting programs. (KHR)

  14. The Recruiter's Guidebook for Identification and Recruitment. Revised.

    ERIC Educational Resources Information Center

    Wisconsin State Dept. of Public Instruction, Madison. Div. for Management and Planning Services.

    A uniform understanding of the identification and recruitment component of the Wisconsin Elementary and Secondary Education Act Title I Migrant Education Program is the purpose of this recruiter's guidebook which can be an aid to recruiters, administrators, site coordinators, teachers, and parents in meeting the special educational needs of…

  15. Recruiting and Selecting New Teachers: The Recruitment Budgeting Cycle.

    ERIC Educational Resources Information Center

    Beebe, Robert J.

    1998-01-01

    The recruitment budgeting cycle (RBC) places teacher-recruitment activities on a more systematic, professional plane. RBC action steps include reviewing and updating recruitment goals and activities, identifying the previous year's activities, defining outcome criteria for determining cost effectiveness, ranking activity costs, evaluating…

  16. Minimum recruitment frequency in plants with episodic recruitment.

    PubMed

    Wiegand, Kerstin; Jeltsch, Florian; Ward, David

    2004-10-01

    There is concern about the lack of recruitment of Acacia trees in the Negev desert of Israel. We have developed three models to estimate the frequency of recruitment necessary for long-term population survival (i.e. positive average population growth for 1,000 years and < 10% probability of extinction). Two models assume purely episodic recruitment based on the general notion that recruitment in arid environments is highly episodic. They differ in that the deterministic model investigates average dynamics while the stochastic model does not. Studies indicating that recruitment episodes in arid environments have been overemphasized motivated the development of the third model. This semi-stochastic model simulates a mixture of continuous and episodic recruitment. Model analysis was done analytically for the deterministic model and via running model simulations for the stochastic and semi-stochastic models. The deterministic and stochastic models predict that, on average, 2.2 and 3.7 recruitment events per century, respectively, are necessary to sustain the population. According to the semi-stochastic model, 1.6 large recruitment events per century and an annual probability of 50% that a small recruitment event occurs are needed. A consequence of purely episodic recruitment is that all recruitment episodes produce extremely large numbers of recruits (i.e. at odds with field observations), an evaluation that holds even when considering that rare events must be large. Thus, the semi-stochastic model appears to be the most realistic model. Comparing the prediction of the semi-stochastic model to field observations in the Negev desert shows that the absence of observations of extremely large recruitment events is no reason for concern. However, the almost complete absence of small recruitment events is a serious reason for concern. The lack of recruitment may be due to decreased densities of large mammalian herbivores and might be further exacerbated by possible changes

  17. Preparing Alumni for Student Recruitment.

    ERIC Educational Resources Information Center

    Mount, Brian

    As recruitment budgets continue to tighten and with fewer colleges reporting application increases for their freshmen classes, enrollment managers must continue to explore all potential sources of recruitment talent. Alumni are often an underutilized or sometimes poorly utilized resource in recruitment efforts. Younger alumni, for example, may…

  18. Pneumolysin activates neutrophil extracellular trap formation.

    PubMed

    G Nel, J; Theron, A J; Durandt, C; Tintinger, G R; Pool, R; Mitchell, T J; Feldman, C; Anderson, R

    2016-06-01

    The primary objective of the current study was to investigate the potential of the pneumococcal toxin, pneumolysin (Ply), to activate neutrophil extracellular trap (NET) formation in vitro. Isolated human blood neutrophils were exposed to recombinant Ply (5-20 ng ml(-1) ) for 30-90 min at 37°C and NET formation measured using the following procedures to detect extracellular DNA: (i) flow cytometry using Vybrant® DyeCycle™ Ruby; (ii) spectrofluorimetry using the fluorophore, Sytox(®) Orange (5 μM); and (iii) NanoDrop(®) technology. These procedures were complemented by fluorescence microscopy using 4', 6-diamino-2-phenylindole (DAPI) (nuclear stain) in combination with anti-citrullinated histone monoclonal antibodies to visualize nets. Exposure of neutrophils to Ply resulted in relatively rapid (detected within 30-60 min), statistically significant (P < 0·05) dose- and time-related increases in the release of cellular DNA impregnated with both citrullinated histone and myeloperoxidase. Microscopy revealed that NETosis appeared to be restricted to a subpopulation of neutrophils, the numbers of NET-forming cells in the control and Ply-treated systems (10 and 20 ng ml(-1) ) were 4·3 (4·2), 14.3 (9·9) and 16·5 (7·5), respectively (n = 4, P < 0·0001 for comparison of the control with both Ply-treated systems). Ply-induced NETosis occurred in the setting of retention of cell viability, and apparent lack of involvement of reactive oxygen species and Toll-like receptor 4. In conclusion, Ply induces vital NETosis in human neutrophils, a process which may either contribute to host defence or worsen disease severity, depending on the intensity of the inflammatory response during pneumococcal infection. PMID:26749379

  19. Changes in Neutrophil Functions in Astronauts

    NASA Technical Reports Server (NTRS)

    Kaur, Indreshpal; Simons, Elizabeth R.; Castro, Victoria; Pierson, Duane L.

    2002-01-01

    Neutrophil functions (phagocytosis, oxidative burst, degranulation) and expression of surface markers involved in these functions were studied in 25 astronauts before and after 4 space shuttle missions. Space flight duration ranged from 5 to 11 days. Blood specimens were obtained 10 days before launch (preflight or L-10), immediately after landing (landing or R+0), and again at 3 days after landing (postflight or R+3). Blood samples were also collected from 9 healthy low-stressed subjects at 3 time points simulating a 10-day shuttle mission. The number of neutrophils increased at landing by 85 percent when compared to the preflight numbers. Neutrophil functions were studied in whole blood using flow cytometric methods. Phagocytosis of E.coli-FITC and oxidative burst capacity of the neutrophils following the 9 to 11 day missions were lower at all three sampling points than the mean values for control subjects. Phagocytosis and oxidative burst capacity of the astronauts was decreased even 10-days before space flight. Mission duration appears to be a factor in phagocytic and oxidative functions. In contrast, following the short-duration (5-days) mission, these functions were unchanged from control values. No consistent changes in degranulation were observed following either short or medium length space missions. The expression of CD16, CD32, CD11a, CD11b, CD11c, L-selectin and CD36 was measured and found to be variable. Specifically, CD16 and CD32 did not correlate with the changes in oxidative burst and phagocytosis. We can conclude from this study that the stresses associated with space flight can alter the important functions of neutrophils.

  20. [Perfluorocarbon emulsions and other corpuscular systems influence on neutrophil activity].

    PubMed

    Shekhtman, D G; Safronova, V G; Sklifas, A N; Alovskaia, A A; Gapeev, A B; Obraztsov, V V; Chemeris, N K

    1997-01-01

    Influence of perfluorodecalin, perfluoromethilcyclohexylpiperidine, perfluorotributylamine emulsions on active oxygen form (AOF) generation by neutrophils has been studied. All investigated emulsions stabilized both proxanol 268 and egg yolk phospholipids inhibited PMA-stimulated neutrophil activity. Castor oil emulsion also inhibited the neutrophil activity. Neutrophil response for chemotactic peptide, was unchanged in the presence of all tested emulsions. We suppose that fast hydrophobic attachment of inert submicrone emulsion particles to cell surface provokes alteration of neutrophil plasma membrane function resulting in a decrease of AOF generation. PMID:9490112

  1. A Combretastatin-Mediated Decrease in Neutrophil Concentration in Peripheral Blood and the Impact on the Anti-Tumor Activity of This Drug in Two Different Murine Tumor Models

    PubMed Central

    Bohn, Anja Bille; Wittenborn, Thomas; Brems-Eskildsen, Anne Sofie; Laurberg, Tinne; Bertelsen, Lotte Bonde; Nielsen, Thomas; Stødkilde-Jørgensen, Hans; Møller, Bjarne Kuno; Horsman, Michael R.

    2014-01-01

    The vascular disrupting agent combretastatin A-4 disodium phosphate (CA4P) induces fluctuations in peripheral blood neutrophil concentration. Because neutrophils have the potential to induce both vascular damage and angiogenesis we analyzed neutrophil involvement in the anti-tumoral effects of CA4P in C3H mammary carcinomas in CDF1 mice and in SCCVII squamous cell carcinomas in C3H/HeN mice. Flow cytometry analyses of peripheral blood before and up to 144 h after CA4P administration (25 and 250 mg/kg) revealed a decrease 1 h after treatment, followed by an early (3–6 h) and a late (>72 h) increase in the granulocyte concentration. We suggest that the early increase (3–6 h) in granulocyte concentration was caused by the initial decrease at 1 h and found that the late increase was associated with tumor size, and hence independent of CA4P. No alterations in neutrophil infiltration into the C3H tumor after CA4P treatment (25 and 250 mg/kg) were found. Correspondingly, neutrophil depletion in vivo, using an anti-neutrophil antibody, followed by CA4P treatment (25 mg/kg) did not increase the necrotic fraction in C3H tumors significantly. However, by increasing the CA4P dose to 250 mg/kg we found a significant increase of 359% in necrotic fraction when compared to neutrophil-depleted mice; in mice with no neutrophil depletion CA4P induced an 89% change indicating that the presence of neutrophils reduced the effect of CA4P. In contrast, neither CA4P nor 1A8 affected the necrotic fraction in the SCCVII tumors significantly. Hence, we suggest that the initial decrease in granulocyte concentration was caused by non-tumor-specific recruitment of neutrophils and that neutrophils may attenuate CA4P-mediated anti-tumor effect in some tumor models. PMID:25299269

  2. Inhibition of Neutrophil Exocytosis Ameliorates Acute Lung Injury in Rats

    PubMed Central

    Uriarte, Silvia M.; Rane, Madhavi J.; Merchant, Michael L.; Jin, Shunying; Lentsch, Alex B.; Ward, Richard A.; McLeish, Kenneth R.

    2013-01-01

    Exocytosis of neutrophil granules contributes to acute lung injury (ALI) induced by infection or inflammation, suggesting that inhibition of neutrophil exocytosis in vivo could be a viable therapeutic strategy. This study was conducted to determine the effect of a cell-permeable fusion protein that inhibits neutrophil exocytosis (TAT-SNAP-23) on ALI using an immune complex deposition model in rats. The effect of inhibition of neutrophil exocytosis by intravenous administration of TAT-SNAP-23 on ALI was assessed by albumin leakage, neutrophil infiltration, lung histology, and proteomic analysis of bronchoalveolar lavage fluid (BALf). Administration of TAT-SNAP-23, but not TAT-Control, significantly reduced albumin leakage, total protein levels in the BALf, and intra-alveolar edema and hemorrhage. Evidence that TAT-SNAP-23 inhibits neutrophil exocytosis included a reduction in plasma membrane CD18 expression by BALf neutrophils and a decrease in neutrophil granule proteins in BALf. Similar degree of neutrophil accumulation in the lungs and/or BALf suggests that TAT-SNAP-23 did not alter vascular endothelial cell function. Proteomic analysis of BALf revealed that components of the complement and coagulation pathways were significantly reduced in BALf from TAT-SNAP-23-treated animals. Our results indicate that administration of a TAT-fusion protein that inhibits neutrophil exocytosis reduces in vivo ALI. Targeting neutrophil exocytosis is a potential therapeutic strategy to ameliorate ALI. PMID:23364427

  3. Endothelial cell phagocytosis of senescent neutrophils decreases procoagulant activity.

    PubMed

    Gao, Chunyan; Xie, Rui; Li, Wen; Zhou, Jin; Liu, Shuchuan; Cao, Fenglin; Liu, Yue; Ma, Ruishuang; Si, Yu; Liu, Yan; Bi, Yayan; Gilbert, Gary E; Shi, Jialan

    2013-06-01

    Abundant senescent neutrophils traverse the vascular compartment and may contribute to pathologic conditions. For example, they become procoagulant when undergoing apoptosis and may contribute to thrombosis or inflammation. Our previous studies demonstrated a dominant clearance pathway in which the neutrophils can be phagocytosed by liver macrophages. The aim of this study was to explore an alternate pathway of neutrophil clearance by endothelial cells. Phagocytosis of the neutrophils by endothelial cells was performed using various experimental approaches includingflow cytometry, confocal microscopy and electron microscopy assays in vitro and in vivo. Procoagulant activity of cultured neutrophils was evaluated by coagulation time, factor Xase and prothrombinase assays. Lactadherin functioned as a novel probe for the detection of phosphatidylserine on apoptotic cells, an opsonin (bridge) between apoptotic cell and phagocyte for promoting phagocytosis, and an efficient anticoagulant for inhibition of factor Xase and thrombin formation. When cultured, purified human neutrophils spontaneously entered apoptosis and developed procoagulant activity that was directly related to the degree of phosphatidylserine exposure. Co-culture of aged neutrophils and endothelial cells resulted in phagocytosis of the neutrophils and prolonged coagulation time. Lactadherin diminished the procoagulant activity and increased the rate of neutrophil clearance. In vivo, neutrophils were sequestered by endothelial cells after blockade of Kupffer cells, a process that was dependent upon both phosphatidylserine exposure and P-selectin expression. Thus, the ability of endothelial cells to clear senescent neutrophils may limit the procoagulant and/or inflammatory impact of these cells.

  4. Nichelle Nichols, NASA Recruiter

    NASA Technical Reports Server (NTRS)

    1977-01-01

    Actress Nichelle Nichols was born in Robbins, Illinois on December 29, 1936. She played Lieutenant Uhura the Communications Officer on the U.S.S. Enterprise in the original series, Star Trek. Nichols stayed with the show and has appeared in six Star Trek movies. Her portrayal of Uhura on Star Trek marked one of the first non-stereotypical roles assigned to an African-American actress. She also provided the voice for Lt. Uhura on the Star Trek animated series in 1974-75. Before joining the crew on Star Trek, she sang and danced with Duke Ellington's band. Nichols was always interested in space travel. She flew aboard the C-141 Astronomy Observatory, which analyzed the atmospheres of Mars and Saturn on an eight hour, high altitude mission. From the late 1970's until the late 1980's, NASA employed Nichelle Nichols to recruit new astronaut candidates. Many of her new recruits were women or members of racial and ethnic minorities, including Guion Bluford (the first African-American astronaut), Sally Ride (the first female American astronaut), Judith Resnik (one of the original set of female astronauts, who perished during the launch of the Challenger on January 28, 1986), and Ronald McNair (the second African-American astronaut, and another victim of the Challenger accident). Currently Nichelle Nichols is actively involved in movies and special appearances. She is also a spokesperson for her favorite charity, 'The Kwanzaa Foundation.'

  5. Neutrophil extracellular traps: Their role in periodontal disease

    PubMed Central

    Kolaparthy, Lakshmi Kanth; Sanivarapu, Sahitya; Swarna, Chakrapani; Devulapalli, Narasimha Swamy

    2014-01-01

    Neutrophils are the first line of innate immune defense against infectious diseases. Since their discovery, they have always been considered tissue-destructive cells responsible for inflammatory tissue damage occurring during infections. Extensive research in the field of neutrophil cell biology and their role skewing the immune response in various infections or inflammatory disorders revealed their importance in the regulation of immune response. Neutrophils also release neutrophil extracellular traps (NETs) for the containment of infection and inflammation along with other antimicrobial molecules. Activated neutrophils provide signals for the activation and maturation of macrophages as well as dendritic cells. Neutrophils are also involved in the regulation of T-cell immune response against various pathogens and tumor antigens. Thus, the present review is intended to highlight the emerging role of neutrophil extracellular trap production in the regulation of immune response and its role in periodontal disease. PMID:25624623

  6. Neutrophil extracellular traps: Their role in periodontal disease.

    PubMed

    Kolaparthy, Lakshmi Kanth; Sanivarapu, Sahitya; Swarna, Chakrapani; Devulapalli, Narasimha Swamy

    2014-01-01

    Neutrophils are the first line of innate immune defense against infectious diseases. Since their discovery, they have always been considered tissue-destructive cells responsible for inflammatory tissue damage occurring during infections. Extensive research in the field of neutrophil cell biology and their role skewing the immune response in various infections or inflammatory disorders revealed their importance in the regulation of immune response. Neutrophils also release neutrophil extracellular traps (NETs) for the containment of infection and inflammation along with other antimicrobial molecules. Activated neutrophils provide signals for the activation and maturation of macrophages as well as dendritic cells. Neutrophils are also involved in the regulation of T-cell immune response against various pathogens and tumor antigens. Thus, the present review is intended to highlight the emerging role of neutrophil extracellular trap production in the regulation of immune response and its role in periodontal disease. PMID:25624623

  7. The role of heparanase in pulmonary cell recruitment in response to an allergic but not non-allergic stimulus.

    PubMed

    Morris, Abigail; Wang, Bo; Waern, Ida; Venkatasamy, Radhakrishnan; Page, Clive; Schmidt, Eric P; Wernersson, Sara; Li, Jin-Ping; Spina, Domenico

    2015-01-01

    Heparanase is an endo-β-glucuronidase that specifically cleaves heparan sulfate proteoglycans in the extracellular matrix. Expression of this enzyme is increased in several pathological conditions including inflammation. We have investigated the role of heparanase in pulmonary inflammation in the context of allergic and non-allergic pulmonary cell recruitment using heparanase knockout (Hpa-/-) mice as a model. Following local delivery of LPS or zymosan, no significant difference was found in the recruitment of neutrophils to the lung between Hpa-/- and wild type (WT) control. Similarly neutrophil recruitment was not inhibited in WT mice treated with a heparanase inhibitor. However, in allergic inflammatory models, Hpa-/- mice displayed a significantly reduced eosinophil (but not neutrophil) recruitment to the airways and this was also associated with a reduction in allergen-induced bronchial hyperresponsiveness, indicating that heparanase expression is associated with allergic reactions. This was further demonstrated by pharmacological treatment with a heparanase inhibitor in the WT allergic mice. Examination of lung specimens from patients with different severity of chronic obstructive pulmonary disease (COPD) found increased heparanase expression. Thus, it is established that heparanase contributes to allergen-induced eosinophil recruitment to the lung and could provide a novel therapeutic target for the development of anti-inflammatory drugs for the treatment of asthma and other allergic diseases.

  8. Neutrophils mediate Salmonella Typhimurium clearance through the GBP4 inflammasome-dependent production of prostaglandins.

    PubMed

    Tyrkalska, Sylwia D; Candel, Sergio; Angosto, Diego; Gómez-Abellán, Victoria; Martín-Sánchez, Fátima; García-Moreno, Diana; Zapata-Pérez, Rubén; Sánchez-Ferrer, Álvaro; Sepulcre, María P; Pelegrín, Pablo; Mulero, Victoriano

    2016-07-01

    Inflammasomes are cytosolic molecular platforms that alert the immune system about the presence of infection. Here we report that zebrafish guanylate-binding protein 4 (Gbp4), an IFNγ-inducible GTPase protein harbouring a C-terminal CARD domain, is required for the inflammasome-dependent clearance of Salmonella Typhimurium (ST) by neutrophils in vivo. Despite the presence of the CARD domain, Gbp4 requires the universal inflammasome adaptor Asc for mediating its antibacterial function. In addition, the GTPase activity of Gbp4 is indispensable for inflammasome activation and ST clearance. Mechanistically, neutrophils are recruited to the infection site through the inflammasome-independent production of the chemokine (CXC motif) ligand 8 and leukotriene B4, and then mediate bacterial clearance through the Gbp4 inflammasome-dependent biosynthesis of prostaglandin D2. Our results point to GBPs as key inflammasome adaptors required for prostaglandin biosynthesis and bacterial clearance by neutrophils and suggest that transient activation of the inflammasome may be used to treat bacterial infections.

  9. Neutrophils mediate Salmonella Typhimurium clearance through the GBP4 inflammasome-dependent production of prostaglandins.

    PubMed

    Tyrkalska, Sylwia D; Candel, Sergio; Angosto, Diego; Gómez-Abellán, Victoria; Martín-Sánchez, Fátima; García-Moreno, Diana; Zapata-Pérez, Rubén; Sánchez-Ferrer, Álvaro; Sepulcre, María P; Pelegrín, Pablo; Mulero, Victoriano

    2016-01-01

    Inflammasomes are cytosolic molecular platforms that alert the immune system about the presence of infection. Here we report that zebrafish guanylate-binding protein 4 (Gbp4), an IFNγ-inducible GTPase protein harbouring a C-terminal CARD domain, is required for the inflammasome-dependent clearance of Salmonella Typhimurium (ST) by neutrophils in vivo. Despite the presence of the CARD domain, Gbp4 requires the universal inflammasome adaptor Asc for mediating its antibacterial function. In addition, the GTPase activity of Gbp4 is indispensable for inflammasome activation and ST clearance. Mechanistically, neutrophils are recruited to the infection site through the inflammasome-independent production of the chemokine (CXC motif) ligand 8 and leukotriene B4, and then mediate bacterial clearance through the Gbp4 inflammasome-dependent biosynthesis of prostaglandin D2. Our results point to GBPs as key inflammasome adaptors required for prostaglandin biosynthesis and bacterial clearance by neutrophils and suggest that transient activation of the inflammasome may be used to treat bacterial infections. PMID:27363812

  10. Neutrophils mediate Salmonella Typhimurium clearance through the GBP4 inflammasome-dependent production of prostaglandins

    PubMed Central

    Tyrkalska, Sylwia D.; Candel, Sergio; Angosto, Diego; Gómez-Abellán, Victoria; Martín-Sánchez, Fátima; García-Moreno, Diana; Zapata-Pérez, Rubén; Sánchez-Ferrer, Álvaro; Sepulcre, María P.; Pelegrín, Pablo; Mulero, Victoriano

    2016-01-01

    Inflammasomes are cytosolic molecular platforms that alert the immune system about the presence of infection. Here we report that zebrafish guanylate-binding protein 4 (Gbp4), an IFNγ-inducible GTPase protein harbouring a C-terminal CARD domain, is required for the inflammasome-dependent clearance of Salmonella Typhimurium (ST) by neutrophils in vivo. Despite the presence of the CARD domain, Gbp4 requires the universal inflammasome adaptor Asc for mediating its antibacterial function. In addition, the GTPase activity of Gbp4 is indispensable for inflammasome activation and ST clearance. Mechanistically, neutrophils are recruited to the infection site through the inflammasome-independent production of the chemokine (CXC motif) ligand 8 and leukotriene B4, and then mediate bacterial clearance through the Gbp4 inflammasome-dependent biosynthesis of prostaglandin D2. Our results point to GBPs as key inflammasome adaptors required for prostaglandin biosynthesis and bacterial clearance by neutrophils and suggest that transient activation of the inflammasome may be used to treat bacterial infections. PMID:27363812

  11. Imaging early pathogenesis of bubonic plague: are neutrophils commandeered for lymphatic transport of bacteria?

    PubMed

    Bland, David M; Anderson, Deborah M

    2013-11-05

    Vector-borne infections begin in the dermis when a pathogen is introduced by an arthropod during a blood meal. Several barriers separate an invading pathogen from its replicative niche, including phagocytic cells in the dermis that activate immunity by engulfing would-be pathogens and migrating to the lymph node. In addition, neutrophils circulating in the blood are rapidly recruited when the dermal barriers are penetrated. For flea-borne disease, no insect-encoded immune-suppressive molecules have yet been described that might influence the establishment of infection, leaving the bacteria on their own to defend against the mammalian immune system. Shortly after a flea transmits Yersinia pestis to a mammalian host, the bacteria are transported to the lymph node, where they grow logarithmically and later spread systemically. Even a single cell of Y. pestis can initiate a lethal case of plague. In their article, J. G. Shannon et al. [mBio 4(5):e00170-13, 2013, doi:10.1128/mBio.00170-13] used intravital microscopy to visualize trafficking of Y. pestis in transgenic mice in vivo, which allowed them to examine interactions between bacteria and specific immune cells. Bacteria appeared to preferentially interact with neutrophils but had no detectable interactions with dendritic cells. These findings suggest that Y. pestis infection of neutrophils not only prevents their activation but may even result in their return to circulation and migration to distal sites.

  12. Local Generation of Kynurenines Mediates Inhibition of Neutrophil Chemotaxis by Uropathogenic Escherichia coli

    PubMed Central

    Loughman, Jennifer A.; Yarbrough, Melanie L.; Tiemann, Kristin M.

    2016-01-01

    During epithelial infections, pathogenic bacteria employ an array of strategies to attenuate and evade host immune responses, including the influx of polymorphonuclear leukocytes (PMN; neutrophils). Among the most common bacterial infections in humans are those of the urinary tract, caused chiefly by uropathogenic Escherichia coli (UPEC). During the establishment of bacterial cystitis, UPEC suppresses innate responses via multiple independent strategies. We recently described UPEC attenuation of PMN trafficking to the urinary bladder through pathogen-specific local induction of indoleamine 2,3-dioxygenase (IDO), a tryptophan catabolic enzyme previously shown to have regulatory activity only in adaptive immunity. Here, we investigated the mechanism by which IDO induction attenuates PMN migration. Local tryptophan limitation, by which IDO is known to influence T cell longevity and proliferation, was not involved in its effect on PMN trafficking. Instead, metabolites in the IDO pathway, particularly l-kynurenine, directly suppressed PMN transepithelial migration and induced an attached, spread morphology in PMN both at rest and in the presence of chemotactic stimuli. Finally, kynurenines represent known ligands of the mammalian aryl hydrocarbon receptor (AHR), and UPEC infection of Ahr−/− mice recapitulated the derepressed PMN recruitment observed previously in Ido1−/− mice. UPEC therefore suppresses neutrophil migration early in bacterial cystitis by eliciting an IDO-mediated increase in local production of kynurenines, which act through the AHR to impair neutrophil chemotaxis. PMID:26857571

  13. Distinct Oral Neutrophil Subsets Define Health and Periodontal Disease States.

    PubMed

    Fine, N; Hassanpour, S; Borenstein, A; Sima, C; Oveisi, M; Scholey, J; Cherney, D; Glogauer, M

    2016-07-01

    Neutrophils exit the vasculature and swarm to sites of inflammation and infection. However, these cells are abundant in the healthy, inflammation-free human oral environment, suggesting a unique immune surveillance role within the periodontium. We hypothesize that neutrophils in the healthy oral cavity occur in an intermediary parainflammatory state that allows them to interact with and contain the oral microflora without eliciting a marked inflammatory response. Based on a high-throughput screen of neutrophil CD (cluster of differentiation) marker expression and a thorough literature review, we developed multicolor flow cytometry panels to determine the surface marker signatures of oral neutrophil subsets in periodontal health and disease. We define here 3 distinct neutrophil subsets: resting/naive circulatory neutrophils, parainflammatory neutrophils found in the healthy oral cavity, and proinflammatory neutrophils found in the oral cavity during chronic periodontal disease. Furthermore, parainflammatory neutrophils manifest as 2 distinct subpopulations-based on size, granularity, and expression of specific CD markers-and exhibit intermediate levels of activation as compared with the proinflammatory oral neutrophils. These intermediately activated parainflammatory populations occur in equal proportions in the healthy oral cavity, with a shift to one highly activated proinflammatory neutrophil population in chronic periodontal disease. This work is the first to identify and characterize oral parainflammatory neutrophils that interact with commensal biofilms without inducing an inflammatory response, thereby demonstrating that not all neutrophils trafficking through periodontal tissues are fully activated. In addition to establishing possible diagnostic and treatment monitoring biomarkers, this oral neutrophil phenotype model builds on existing literature suggesting that the healthy periodontium may be in a parainflammatory state. PMID:27270666

  14. Isolation and Characterization of Neutrophils with Anti-Tumor Properties.

    PubMed

    Sionov, Ronit Vogt; Assi, Simaan; Gershkovitz, Maya; Sagiv, Jitka Y; Polyansky, Lola; Mishalian, Inbal; Fridlender, Zvi G; Granot, Zvi

    2015-01-01

    Neutrophils, the most abundant of all white blood cells in the human circulation, play an important role in the host defense against invading microorganisms. In addition, neutrophils play a central role in the immune surveillance of tumor cells. They have the ability to recognize tumor cells and induce tumor cell death either through a cell contact-dependent mechanism involving hydrogen peroxide or through antibody-dependent cell-mediated cytotoxicity (ADCC). Neutrophils with anti-tumor activity can be isolated from peripheral blood of cancer patients and of tumor-bearing mice. These neutrophils are termed tumor-entrained neutrophils (TEN) to distinguish them from neutrophils of healthy subjects or naïve mice that show no significant tumor cytotoxic activity. Compared with other white blood cells, neutrophils show different buoyancy making it feasible to obtain a > 98% pure neutrophil population when subjected to a density gradient. However, in addition to the normal high-density neutrophil population (HDN), in cancer patients, in tumor-bearing mice, as well as under chronic inflammatory conditions, distinct low-density neutrophil populations (LDN) appear in the circulation. LDN co-purify with the mononuclear fraction and can be separated from mononuclear cells using either positive or negative selection strategies. Once the purity of the isolated neutrophils is determined by flow cytometry, they can be used for in vitro and in vivo functional assays. We describe techniques for monitoring the anti-tumor activity of neutrophils, their ability to migrate and to produce reactive oxygen species, as well as monitoring their phagocytic capacity ex vivo. We further describe techniques to label the neutrophils for in vivo tracking, and to determine their anti-metastatic capacity in vivo. All these techniques are essential for understanding how to obtain and characterize neutrophils with anti-tumor function. PMID:26132785

  15. Attenuation of human neutrophil migration and function by uropathogenic bacteria

    PubMed Central

    Loughman, Jennifer A.; Hunstad, David A.

    2011-01-01

    The establishment of bacterial infections at mucosal epithelial surfaces is determined by the balance of virulence attributes of the pathogen with the activity of innate host defenses. Polymorphonuclear leukocytes (PMN) are key responders in many bacterial infections, but the mechanisms by which pathogens subvert these early responses to establish infection are largely undefined. Here, we model early interactions between human PMN and the primary cause of urinary tract infections, namely uropathogenic Escherichia coli (UPEC). Our objective was to define virulence phenotypes of uropathogens that permit evasion of PMN activity. We show that UPEC strains, as compared with laboratory and commensal E. coli, resist phagocytic killing and dampen the production of antimicrobial reactive oxygen species by PMN. Analysis of the transcriptional responses of PMN to E. coli strains revealed that UPEC exposure downregulates the expression of PMN genes that direct proinflammatory signaling and PMN chemotaxis, adhesion, and migration. Consistent with these data, UPEC attenuated transepithelial neutrophil recruitment in an in vitro model of acute infection and in a murine model of bacterial cystitis. We propose that these UPEC strategies are important in the establishment of epithelial infection, and that the findings are germane to bacterial infections at other epithelial surfaces. PMID:21315174

  16. Pathogenic Neisseria hitchhike on the uropod of human neutrophils.

    PubMed

    Söderholm, Niklas; Vielfort, Katarina; Hultenby, Kjell; Aro, Helena

    2011-01-01

    Polymorphonuclear neutrophils (PMNs) are important components of the human innate immune system and are rapidly recruited at the site of bacterial infection. Despite the effective phagocytic activity of PMNs, Neisseria gonorrhoeae infections are characterized by high survival within PMNs. We reveal a novel type IV pilus-mediated adherence of pathogenic Neisseria to the uropod (the rear) of polarized PMNs. The direct pilus-uropod interaction was visualized by scanning electron microscopy and total internal reflection fluorescence (TIRF) microscopy. We showed that N. meningitidis adhesion to the PMN uropod depended on both pilus-associated proteins PilC1 and PilC2, while N. gonorrhoeae adhesion did not. Bacterial adhesion elicited accumulation of the complement regulator CD46, but not I-domain-containing integrins, beneath the adherent bacterial microcolony. Electrographs and live-cell imaging of PMNs suggested that bacterial adherence to the uropod is followed by internalization into PMNs via the uropod. We also present data showing that pathogenic Neisseria can hitchhike on PMNs to hide from their phagocytic activity as well as to facilitate the spread of the pathogen through the epithelial cell layer.

  17. Differential involvement of NF-kappaB and MAP kinase pathways in the generation of inflammatory cytokines by human neutrophils.

    PubMed

    Cloutier, Alexandre; Ear, Thornin; Blais-Charron, Emilie; Dubois, Claire M; McDonald, Patrick P

    2007-02-01

    The ability of human neutrophils to express a variety of genes encoding inflammatory mediators is well documented, and mounting evidence suggests that neutrophil-derived cytokines and chemokines contribute to the recruitment of discrete leukocyte populations at inflammatory sites. Despite this, our understanding of the signaling intermediates governing the generation of inflammatory cytokines by neutrophils remains fragmentary. Here, we report that inhibitors of the p38 MAPK and MEK pathways substantially diminish the release of (and in the case of p38 inhibitors, the gene expression of) several inflammatory cytokines in neutrophils stimulated with LPS or TNF. In addition, various NF-kappaB inhibitors were found to profoundly impede the inducible gene expression and release of inflammatory cytokines in these cells. The MAPK inhibitors did not affect NF-kappaB activation; instead, the transcriptional effects of the p38 MAPK inhibitor appear to involve transcriptional factor IID. Conversely, the NF-kappaB inhibitors failed to affect the activation of MAPKs. Finally, the MAPK inhibitors were found to prevent the activation a key component of the translational machinery, S6 ribosomal protein, in keeping with their post-transcriptional impact on cytokine generation. To our knowledge, this constitutes the first demonstration that in neutrophils, the inducible expression of proinflammatory cytokines by physiological stimuli largely reflects the ability of the latter to activate NF-kappaB and selected MAPK pathways. Our data also raise the possibility that NF-kappaB or MAPK inhibitors could be useful in the treatment of inflammatory disorders in which neutrophils predominate.

  18. Targeting Rac1 signaling inhibits streptococcal M1 protein-induced CXC chemokine formation, neutrophil infiltration and lung injury.

    PubMed

    Zhang, Songen; Rahman, Milladur; Zhang, Su; Song, Lei; Herwald, Heiko; Thorlacius, Henrik

    2013-01-01

    Infections with Streptococcus pyogenes exhibit a wide spectrum of infections ranging from mild pharyngitis to severe Streptococcal toxic shock syndrome (STSS). The M1 serotype of Streptococcus pyogenes is most commonly associated with STSS. In the present study, we hypothesized that Rac1 signaling might regulate M1 protein-induced lung injury. We studied the effect of a Rac1 inhibitor (NSC23766) on M1 protein-provoked pulmonary injury. Male C57BL/6 mice received NSC23766 prior to M1 protein challenge. Bronchoalveolar fluid and lung tissue were harvested for quantification of neutrophil recruitment, edema and CXC chemokine formation. Neutrophil expression of Mac-1 was quantified by use of flow cytometry. Quantitative RT-PCR was used to determine gene expression of CXC chemokines in alveolar macrophages. Treatment with NSC23766 decreased M1 protein-induced neutrophil infiltration, edema formation and tissue injury in the lung. M1 protein challenge markedly enhanced Mac-1 expression on neutrophils and CXC chemokine levels in the lung. Inhibition of Rac1 activity had no effect on M1 protein-induced expression of Mac-1 on neutrophils. However, Rac1 inhibition markedly decreased M1 protein-evoked formation of CXC chemokines in the lung. Moreover, NSC23766 completely inhibited M1 protein-provoked gene expression of CXC chemokines in alveolar macrophages. We conclude that these novel results suggest that Rac1 signaling is a significant regulator of neutrophil infiltration and CXC chemokine production in the lung. Thus, targeting Rac1 activity might be a potent strategy to attenuate streptococcal M1 protein-triggered acute lung damage.

  19. Network crosstalk dynamically changes during neutrophil polarization.

    PubMed

    Ku, Chin-Jen; Wang, Yanqin; Weiner, Orion D; Altschuler, Steven J; Wu, Lani F

    2012-05-25

    How complex signaling networks shape highly coordinated, multistep cellular responses is poorly understood. Here, we made use of a network-perturbation approach to investigate causal influences, or "crosstalk," among signaling modules involved in the cytoskeletal response of neutrophils to chemoattractant. We quantified the intensity and polarity of cytoskeletal marker proteins over time to characterize stereotyped cellular responses. Analyzing the effects of network disruptions revealed that, not only does crosstalk evolve rapidly during polarization, but also that intensity and polarity responses are influenced by different patterns of crosstalk. Interestingly, persistent crosstalk is arranged in a surprisingly simple circuit: a linear cascade from front to back to microtubules influences intensities, and a feed-forward network in the reverse direction influences polarity. Our approach provided a rational strategy for decomposing a complex, dynamically evolving signaling system and revealed evolving paths of causal influence that shape the neutrophil polarization response.

  20. [Inhibition of neutrophil adhesion by pectic galacturonans].

    PubMed

    Popov, S V; Ovodova, R G; Popova, G Iu; Nikitina, I R; Ovodov, Iu S

    2007-01-01

    The inhibition of the adhesion of neutrophils to fibronectin by the fragments of the main galacturonan chain of the following pectins was demonstrated: comaruman from the marsh cinquefoil Comarum polustre, bergenan from the Siberian tea Bergenia crassifolia, lemnan from the duckweed Lemna minor, zosteran from the seagrass Zostera marina, and citrus pectin. The parent pectins, except for comaruman, did not affect the cell adhesion. Galacturonans prepared from the starting pectins by acidic hydrolysis were shown to reduce the neutrophil adhesion stimulated by phorbol 12-myristate 13-acetate (1.625 microM) and dithiothreitol (0.5 mM) at a concentration of 50-200 microg/ml. The presence of carbohydrate chains with molecular masses higher than 300, from 100 to 300, and from 50 to 100 kDa in the galacturonan fractions was proved by membrane ultrafiltration. PMID:17375675

  1. Neutrophil myeloperoxidase destruction by ultraviolet irradiation

    SciTech Connect

    Hanker, J.; Giammara, B.; Strauss, G.

    1988-01-01

    The peroxidase activity of enriched leukocyte preparations on coverslips was determined cytochemically with a newly developed method. The techniques utilizes diaminobenzidine medium and cupric nitrate intensification and is suitable for analysis with light microscopy, SEM, and TEM. Blood specimens from control individuals were studied with and without in vitro UV irradiation and compared with those from psoriasis patients exposed therapeutically to various types of UV in phototherapy. All UV irradiated samples showed diminished neutrophil myeloperoxidase (MP) activity although that of the principal eosinophil peroxidase was unaffected. The SEMs supported the contention that decreased neutrophil MP activity might be related to UV induced degranulation. It is believed to be possible, eventually, to equate the observed MP degranulation effect after UV irradiation with diminished ability to fight bacterial infections.

  2. Prognostic value of neutrophil distribution in cholangiocarcinoma

    PubMed Central

    Mao, Zhi-Yuan; Zhu, Guang-Qing; Xiong, Mei; Ren, Li; Bai, Li

    2015-01-01

    AIM: To explore the relationship of clinicopathological features and the distribution of neutrophils in the tumor microenvironment with the prognosis of cholangiocarcinoma. METHODS: Two hundred and fifty-four formalin-fixed and paraffin embedded tissue blocks were analyzed, including tissues from cholangiocarcinoma (n = 254), and tumor adjacent tissues (n = 238). Tissue sections were stained for CD15 using immunohistochemical staining. CD15 expression was detected to identify the distribution of neutrophils in the local tumor microenvironment. The neutrophil density of the tumor tissues and the adjacent tumor tissues was detected to reflect their inflammatory status. Clinical data and follow-up information of cholangiocarcinoma patients who underwent surgery from January 2004 to December 2010 were analyzed retrospectively. The relationship between clinicopathological features and the distribution of neutrophils with prognosis of the patients were analyzed. RESULTS: The positive expression level of CD15 was only significantly related to the TNM stage. CD15 expression was higher in tumor tissues than in adjacent tissues (73.6% vs 54.6%), with significant differences. Patients with high expression of CD15 had significantly shorter overall survival (OS) than those with low expression of CD15 (median overall survival time 39.77 mo vs 16.87 mo, P = 0.008). Patients with high CD15 expression had significantly shorter disease free survival time (DFS) than those with low expression of CD15 (median DFS 38.27 mo vs 16.83 mo, P = 0.029). COX multivariate analysis indicated that high CD15 expression in tumor tissues was an independent risk factor for predicting OS for patients with cholangiocarcinoma [P = 0.012, relative risk (RR) = 1.601], but it was not an independent risk factor for predicting DFS (P = 0.073, RR = 1.462). CONCLUSION: Patients with high CD15 expression in cancer tissues had shorter DFS and OS. High expression of CD15 is an independent risk factor for OS. PMID

  3. Characterization of prostanoid receptors on rat neutrophils.

    PubMed Central

    Wise, H; Jones, R L

    1994-01-01

    1. The effects of various prostanoid agonists have been compared on the increase in intracellular free calcium ([Ca2+]i) and the aggregation reaction of rat peritoneal neutrophils induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP). 2. Prostaglandin E2 (PGE2) and the specific IP-receptor agonist, cicaprost, both inhibited the FMLP-induced increase in [Ca2+]i (IC50 33 nM and 18 nM respectively) and the FMLP-induced aggregation reaction (IC50 5.6 nM and 7.9 nM respectively). PGD2, PGF2 alpha, and the TP-receptor agonist, U 46619, were inactive at the highest concentration tested (1 microM). 3. The EP1-receptor agonist, 17-phenyl-omega-trinor PGE2, and the EP3-receptor agonists, GR 63799X and sulprostone, had no inhibitory effect on FMLP-stimulated rat neutrophils. 4. PGE1 (EP/IP-receptor agonist) and iloprost (IP-receptor agonist) inhibited the FMLP-induced increase in [Ca2+]i with IC50 values of 34 nM and 38 nM respectively. The EP2-receptor agonists, butaprost and misoprostol (1 microM), inhibited both FMLP-stimulated [Ca2+]i and aggregation. However another EP2-receptor agonist, AH 13205, was inactive in both assays. 5. Prostanoid receptors present on rat neutrophils were further characterized by measuring [3H]-adenosine 3':5'-cyclic monophosphate ([3H]-cyclic AMP) accumulation. Only those agonists capable of stimulating [3H]-cyclic AMP accumulation were able to inhibit both FMLP-stimulated [Ca2+]i and aggregation. 6. These results indicate that rat neutrophils possess inhibitory IP and EP-receptors; the relative potencies of PGE2, misoprostol and butaprost are those expected for the EP2-receptor subtype. No evidence for DP, FP, TP or EP1 and EP3-receptors was obtained. PMID:7834211

  4. Capsular polysaccharides from Cryptococcus neoformans modulate production of neutrophil extracellular traps (NETs) by human neutrophils.

    PubMed

    Rocha, Juliana D B; Nascimento, Michelle T C; Decote-Ricardo, Debora; Côrte-Real, Suzana; Morrot, Alexandre; Heise, Norton; Nunes, Marise P; Previato, José Osvaldo; Mendonça-Previato, Lucia; DosReis, George A; Saraiva, Elvira M; Freire-de-Lima, Célio G

    2015-01-01

    In the present study, we characterized the in vitro modulation of NETs (neutrophil extracellular traps) induced in human neutrophils by the opportunistic fungus Cryptococcus neoformans, evaluating the participation of capsular polysaccharides glucuronoxylomanan (GXM) and glucuronoxylomannogalactan (GXMGal) in this phenomenon. The mutant acapsular strain CAP67 and the capsular polysaccharide GXMGal induced NET production. In contrast, the wild-type strain and the major polysaccharide GXM did not induce NET release. In addition, C. neoformans and the capsular polysaccharide GXM inhibited PMA-induced NET release. Additionally, we observed that the NET-enriched supernatants induced through CAP67 yeasts showed fungicidal activity on the capsular strain, and neutrophil elastase, myeloperoxidase, collagenase and histones were the key components for the induction of NET fungicidal activity. The signaling pathways associated with NET induction through the CAP67 strain were dependent on reactive oxygen species (ROS) and peptidylarginine deiminase-4 (PAD-4). Neither polysaccharide induced ROS production however both molecules blocked the production of ROS through PMA-activated neutrophils. Taken together, the results demonstrate that C. neoformans and the capsular component GXM inhibit the production of NETs in human neutrophils. This mechanism indicates a potentially new and important modulation factor for this fungal pathogen. PMID:25620354

  5. Delayed human neutrophil apoptosis by Trichomonas vaginalis lysate.

    PubMed

    Song, Hyun-Ouk; Lim, Young-Su; Moon, Sun-Joo; Ahn, Myoung-Hee; Ryu, Jae-Sook

    2010-03-01

    Neutrophils play an important role in the human immune system for protection against such microorganisms as a protozoan parasite, Trichomonas vaginalis; however, the precise role of neutrophils in the pathogenesis of trichomoniasis is still unknown. Moreover, it is thought that trichomonal lysates and excretory-secretory products (ESP), as well as live T. vaginalis, could possibly interact with neutrophils in local tissues, including areas of inflammation induced by T. vaginalis in humans. The aim of this study was to investigate the influence of T. vaginalis lysate on the fate of neutrophils. We found that T. vaginalis lysate inhibits apoptosis of human neutrophils as revealed by Giemsa stain. Less altered mitochondrial membrane potential (MMP) and surface CD16 receptor expression also supported the idea that neutrophil apoptosis is delayed after T. vaginalis lysate stimulation. In contrast, ESP stimulated-neutrophils were similar in apoptotic features of untreated neutrophils. Maintained caspase-3 and myeloid cell leukemia-1 (Mcl-1) in neutrophils co-cultured with trichomonad lysate suggest that an intrinsic mitochondrial pathway of apoptosis was involved in T. vaginalis lysate-induced delayed neutrophil apoptosis; this phenomenon may contribute to local inflammation in trichomoniasis. PMID:20333279

  6. Phagocytosis and killing of Staphylococcus aureus by human neutrophils.

    PubMed

    Lu, Thea; Porter, Adeline R; Kennedy, Adam D; Kobayashi, Scott D; DeLeo, Frank R

    2014-01-01

    Neutrophils are essential for host defense against Staphylococcus aureus infections. Although significant progress has been made, our understanding of neutrophil interactions with S. aureus remains incomplete. To provide a more comprehensive view of this process, we investigated phagocytosis and killing of S. aureus by human neutrophils using varied assay conditions in vitro. A greater percentage of bacteria were internalized by adherent neutrophils compared to those in suspension, and, unexpectedly, uptake of S. aureus by adherent neutrophils occurred efficiently in the absence of opsonins. An antibody specific for S. aureus promoted uptake of unopsonized bacteria in suspension, but had little or no capacity to enhance phagocytosis of S. aureus opsonized with normal human serum or by adherent neutrophils. Collectively, these results indicate that assay conditions can have a significant influence on the phagocytosis and killing of S. aureus by neutrophils. More importantly, the results suggest a vaccine approach directed to enhance opsonophagocytosis alone is not sufficient to promote increased killing of S. aureus by human neutrophils. With the emergence and reemergence of antibiotic-resistant microorganisms, establishing parameters that are optimal for studying neutrophil-S. aureus interactions will pave the way towards developing immune-directed strategies for anti-staphylococcal therapies.

  7. Acetaminophen prevents oxidative burst and delays apoptosis in human neutrophils.

    PubMed

    Freitas, Marisa; Costa, Vera M; Ribeiro, Daniela; Couto, Diana; Porto, Graça; Carvalho, Félix; Fernandes, Eduarda

    2013-05-23

    Acetaminophen is a frequently prescribed over-the-counter drug to reduce fever and pain in the event of inflammatory process. As neutrophils are relevant cells in inflammatory processes, the putative interaction of acetaminophen with these cells, if present, would be of paramount importance. The present study was undertaken to evaluate the effect of acetaminophen in human neutrophils' oxidative burst and lifespan in vitro. The obtained results demonstrate that acetaminophen efficiently modulates neutrophils' oxidative burst in phorbol myristate acetate-activated neutrophils, in a concentration-dependent manner, at in vivo relevant concentrations. It was clearly demonstrated that acetaminophen is a strong scavenger of HOCl and H2O2, which probably contributed to the effect observed in neutrophils. Acetaminophen also induced the depletion of glutathione in stimulated neutrophils, suggesting its transformation into a reactive intermediate. Obtained results further revealed that acetaminophen affects programmed cell death of human neutrophils, resulting in a delay of previously stimulated neutrophils-mediated apoptosis. Overall, our data suggested that acetaminophen has considerable potential to be included in anti-inflammatory therapeutic strategies, by preventing biological damage induced by an excessive production of reactive species generated in activated neutrophils and by extending the lifespan of neutrophils, favoring the elimination of pathogens, thus contributing to tissue healing and resolution of inflammation. PMID:23518321

  8. Prevention of vascular inflammation by nanoparticle targeting of adherent neutrophils

    NASA Astrophysics Data System (ADS)

    Wang, Zhenjia; Li, Jing; Cho, Jaehyung; Malik, Asrar B.

    2014-03-01

    Inflammatory diseases such as acute lung injury and ischaemic tissue injury are caused by the adhesion of a type of white blood cell--polymorphonuclear neutrophils--to the lining of the circulatory system or vascular endothelium and unchecked neutrophil transmigration. Nanoparticle-mediated targeting of activated neutrophils on vascular endothelial cells at the site of injury may be a useful means of directly inactivating neutrophil transmigration and hence mitigating vascular inflammation. Here, we report a method employing drug-loaded albumin nanoparticles, which efficiently deliver drugs into neutrophils adherent to the surface of the inflamed endothelium. Using intravital microscopy of tumour necrosis factor-α-challenged mouse cremaster post-capillary venules, we demonstrate that fluorescently tagged albumin nanoparticles are largely internalized by neutrophils adherent to the activated endothelium via cell surface Fcɣ receptors. Administration of albumin nanoparticles loaded with the spleen tyrosine kinase inhibitor, piceatannol, which blocks `outside-in' β2 integrin signalling in leukocytes, detached the adherent neutrophils and elicited their release into the circulation. Thus, internalization of drug-loaded albumin nanoparticles into neutrophils inactivates the pro-inflammatory function of activated neutrophils, thereby offering a promising approach for treating inflammatory diseases resulting from inappropriate neutrophil sequestration and activation.

  9. Anoxia and glucose supplementation preserve neutrophil viability and function.

    PubMed

    Monceaux, Valérie; Chiche-Lapierre, Clarisse; Chaput, Catherine; Witko-Sarsat, Véronique; Prevost, Marie-Christine; Taylor, Cormac T; Ungeheuer, Marie-Noelle; Sansonetti, Philippe J; Marteyn, Benoit S

    2016-08-18

    Functional studies of human neutrophils and their transfusion for clinical purposes have been hampered by their short life span after isolation. Here, we demonstrate that neutrophil viability is maintained for 20 hours in culture media at 37°C under anoxic conditions with 3 mM glucose and 32 μg/mL dimethyloxalylglycine supplementation, as evidenced by stabilization of Mcl-1, proliferating cell nuclear antigen (PCNA), and pro-caspase-3. Notably, neutrophil morphology (nucleus shape and cell-surface markers) and functions (phagocytosis, degranulation, calcium release, chemotaxis, and reactive oxygen species production) were comparable to blood circulating neutrophils. The observed extension in neutrophil viability was reversed upon exposure to oxygen. Extending neutrophil life span allowed efficient transfection of plasmids (40% transfection efficiency) and short interfering RNA (interleukin-8, PCNA, and Bax), as a validation of effective and functional genetic manipulation of neutrophils both in vitro and in vivo. In vivo, transfusion of conditioned neutrophils in a neutropenic guinea pig model increased bacterial clearance of Shigella flexneri upon colonic infection, strongly suggesting that these conditioned neutrophils might be suitable for transfusion purposes. In summary, such conditioning of neutrophils in vitro should facilitate their study and offer new opportunities for genetic manipulation and therapeutic use. PMID:27402974

  10. Prevention of vascular inflammation by nanoparticle targeting of adherent neutrophils.

    PubMed

    Wang, Zhenjia; Li, Jing; Cho, Jaehyung; Malik, Asrar B

    2014-03-01

    Inflammatory diseases such as acute lung injury and ischaemic tissue injury are caused by the adhesion of a type of white blood cell--polymorphonuclear neutrophils--to the lining of the circulatory system or vascular endothelium and unchecked neutrophil transmigration. Nanoparticle-mediated targeting of activated neutrophils on vascular endothelial cells at the site of injury may be a useful means of directly inactivating neutrophil transmigration and hence mitigating vascular inflammation. Here, we report a method employing drug-loaded albumin nanoparticles, which efficiently deliver drugs into neutrophils adherent to the surface of the inflamed endothelium. Using intravital microscopy of tumour necrosis factor-α-challenged mouse cremaster post-capillary venules, we demonstrate that fluorescently tagged albumin nanoparticles are largely internalized by neutrophils adherent to the activated endothelium via cell surface Fcɣ receptors. Administration of albumin nanoparticles loaded with the spleen tyrosine kinase inhibitor, piceatannol, which blocks 'outside-in' β2 integrin signalling in leukocytes, detached the adherent neutrophils and elicited their release into the circulation. Thus, internalization of drug-loaded albumin nanoparticles into neutrophils inactivates the pro-inflammatory function of activated neutrophils, thereby offering a promising approach for treating inflammatory diseases resulting from inappropriate neutrophil sequestration and activation.

  11. DAMPs-activated neutrophil extracellular trap exacerbates sterile inflammatory liver injury

    PubMed Central

    Huang, Hai; Tohme, Samer; Al-Khafaji, Ahmed B; Tai, Sheng; Loughran, Patricia; Chen, Li; Wang, Shu; Kim, Jiyun; Billiar, Timothy; Wang, Yanming; Tsung, Allan

    2015-01-01

    Innate immunity plays a crucial role in the response to sterile inflammation such as liver ischemia/reperfusion (I/R) injury. The initiation of liver I/R injury results in the release of damage associated molecular patterns (DAMPs), which trigger innate immune and inflammatory cascade via pattern recognition receptors. Neutrophils are recruited to the liver after I/R and contribute to the organ damage, innate immune and inflammatory responses. Formation of neutrophil extracellular trap (NET) has been recently found in response to various stimuli. However, the role of NETs during liver I/R injury remains unknown. We show that NETs form in the sinusoids of ischemic liver lobes in vivo. This was associated with increased NET markers, serum level of myeloperoxidase (MPO)-DNA complexes and tissue level of citrullinated-histone H3 compared to control mice. Treatment with peptidyl-arginine-deiminase (PAD) 4 inhibitor or DNase I significantly protected hepatocytes and reduced inflammation after liver I/R as evidenced by inhibition of NET formation, indicating the pathophysiological role of NETs in liver I/R injury. In vitro, NETs increase hepatocyte death and induce Kupffer cells to release proinflammatory cytokines. DAMPs, such as HMGB1 and histones, released by injured hepatocytes stimulate NET formation through Toll-like receptor (TLR4)- and TLR9-MyD88 signaling pathways. After neutrophil depletion in mice, the adoptive transfer of TLR4 knockout (KO) or TLR9 KO neutrophils confers significant protection from liver I/R injury with significant decrease in NET formation. In addition, we found inhibition of NET formation by PAD4 inhibitor or DNase I reduces HMGB1 and histone-mediated liver I/R injury. Conclusion DAMPs released during liver I/R promotes NET formation through TLRs signaling pathway. Development of NETs subsequently exacerbates organ damage and initiates inflammatory responses during liver I/R. PMID:25855125

  12. Neutrophil activator of matrix metalloproteinase-2 (NAM).

    PubMed

    Rollo, Ellen E; Hymowitz, Michelle; Schmidt, Cathleen E; Montana, Steve; Foda, Hussein; Zucker, Stanley

    2006-01-01

    We have isolated a novel soluble factor(s), neutrophil activator of matrix metalloproteinases (NAM), secreted by unstimulated normal human peripheral blood neutrophils that causes the activation of cell secreted promatrix metalloproteinase-2 (proMMP-2). Partially purified preparations of NAM have been isolated from the conditioned media of neutrophils employing gelatin-Sepharose chromatography and differential membrane filter centrifugation. NAM activity, as assessed by exposing primary human umbilical vein endothelial cells (HUVEC) or HT1080 cells to NAM followed by gelatin zymography, was seen within one hour. Tissue inhibitor of metalloproteinase-2 (TIMP-2) and hydroxamic acid derived inhibitors of MMPs (CT1746 and BB94) abrogated the activation of proMMP-2 by NAM, while inhibitors of serine and cysteine proteases showed no effect. NAM also produced an increase in TIMP-2 binding to HUVEC and HT1080 cell surfaces that was inhibited by TIMP-2, CT1746, and BB94. Time-dependent increases in MT1-MMP protein and mRNA were seen following the addition of NAM to cells. These data support a role for NAM in cancer dissemination.

  13. Differentiating neutrophils using the optical coulter counter

    NASA Astrophysics Data System (ADS)

    Schonbrun, E.; Di Caprio, G.

    2015-03-01

    We present an opto-fluidic measurement system that quantifies cell volume, dry mass and nuclear morphology of neutrophils in high-throughput. While current clinical hematology analyzers can differentiate neutrophils from a blood sample, they do not give other quantitative information beyond their count. In order to better understand the distribution of neutrophil phenotypes in a blood sample, we perform two distinct multivariate measurements. In both measurements, white blood cells are driven through a microfluidic channel and imaged while in flow onto a color camera using a single exposure. In the first measurement, we quantify cell volume, scattering strength, and cell dry mass by combining quantitative phase imaging with dye exclusion cell volumetric imaging. In the second measurement, we quantify cell volume and nuclear morphology using a nucleic acid fluorescent stain. In this way, we can correlate cell volume to other cellular characteristics, which would not be possible using an electrical coulter counter. Unlike phase imaging or cell scattering analysis, the optical coulter counter is capable of quantifying cell volume virtually independent of the cell's refractive index and unlike optical tomography, measurements are possible on quickly flowing cells, enabling high-throughput.

  14. Differentiating neutrophils using the optical coulter counter

    NASA Astrophysics Data System (ADS)

    Schonbrun, Ethan; Di Caprio, Giuseppe

    2015-11-01

    We present an optofluidic measurement system that quantifies cell volume, dry mass, and nuclear morphology of neutrophils in high-throughput. While current clinical hematology analyzers can differentiate neutrophils from a blood sample, they do not give other quantitative information beyond their count. In order to better understand the distribution of neutrophil phenotypes in a blood sample, we perform two distinct multivariate measurements. In both measurements, white blood cells are driven through a microfluidic channel and imaged while in flow onto a color camera using a single exposure. In the first measurement, we quantify cell volume, scattering strength, and cell dry mass by combining quantitative phase imaging with dye exclusion cell volumetric imaging. In the second measurement, we quantify cell volume and nuclear morphology using a nucleic acid fluorescent stain. In this way, we can correlate cell volume to other cellular characteristics, which would not be possible using an electrical coulter counter. Unlike phase imaging or cell scattering analysis, the optical coulter counter is capable of quantifying cell volume virtually independent of the cell's refractive index and unlike optical tomography, measurements are possible on quickly flowing cells, enabling high-throughput.

  15. Key role for neutrophils in radiation-induced antitumor immune responses: Potentiation with G-CSF

    PubMed Central

    Takeshima, Tsuguhide; Pop, Laurentiu M.; Laine, Aaron; Iyengar, Puneeth; Vitetta, Ellen S.; Hannan, Raquibul

    2016-01-01

    Radiation therapy (RT), a major modality for treating localized tumors, can induce tumor regression outside the radiation field through an abscopal effect that is thought to involve the immune system. Our studies were designed to understand the early immunological effects of RT in the tumor microenvironment using several syngeneic mouse tumor models. We observed that RT induced sterile inflammation with a rapid and transient infiltration of CD11b+Gr-1high+ neutrophils into the tumors. RT-recruited tumor-associated neutrophils (RT-Ns) exhibited an increased production of reactive oxygen species and induced apoptosis of tumor cells. Tumor infiltration of RT-Ns resulted in sterile inflammation and, eventually, the activation of tumor-specific cytotoxic T cells, their recruitment into the tumor site, and tumor regression. Finally, the concurrent administration of granulocyte colony-stimulating factor (G-CSF) enhanced RT-mediated antitumor activity by activating RT-Ns. Our results suggest that the combination of RT and G-CSF should be further evaluated in preclinical and clinical settings. PMID:27651484

  16. Neutrophil Elastase-Generated Fragment of Vascular Endothelial Growth Factor-A Stimulates Macrophage and Endothelial Progenitor Cell Migration

    PubMed Central

    Kurtagic, Elma; Rich, Celeste B.; Buczek-Thomas, Jo Ann; Nugent, Matthew A.

    2015-01-01

    Elastase released from neutrophils as part of the innate immune system has been implicated in chronic diseases such as emphysema and cardiovascular disease. We have previously shown that neutrophil elastase targets vascular endothelial growth factor-A (VEGF) for partial degradation to generate a fragment of VEGF (VEGFf) that has distinct activities. Namely, VEGFf binds to VEGF receptor 1 but not to VEGF receptor 2 and shows altered signaling compared to intact VEGF. In the present study we investigated the chemotactic function of VEGF and VEGFf released from cells by neutrophil elastase. We found that endothelial cells migrated in response to intact VEGF but not VEGFf whereas RAW 264.7 macrophages/monocytes and embryonic endothelial progenitor cells were stimulated to migrate by either VEGF or VEGFf. To investigate the role of elastase-mediated release of VEGF from cells/extracellular matrices, a co-culture system was established. High or low VEGF producing cells were co-cultured with macrophages, endothelial or endothelial progenitor cells and treated with neutrophil elastase. Elastase treatment stimulated macrophage and endothelial progenitor cell migration with the response being greater with the high VEGF expressing cells. However, elastase treatment led to decreased endothelial cell migration due to VEGF cleavage to VEGF fragment. These findings suggest that the tissue response to NE-mediated injury might involve the generation of diffusible VEGF fragments that stimulate inflammatory cell recruitment. PMID:26672607

  17. Slit2 regulates attractive eosinophil and repulsive neutrophil chemotaxis through differential srGAP1 expression during lung inflammation.

    PubMed

    Ye, Bu-Qing; Geng, Zhen H; Ma, Li; Geng, Jian-Guo

    2010-11-15

    Directional migration of leukocytes is an essential step in leukocyte trafficking during inflammatory responses. However, the molecular mechanisms governing directional chemotaxis of leukocytes remain poorly understood. The Slit family of guidance cues has been implicated for inhibition of leuocyte migration. We report that Clara cells in the bronchial epithelium secreted Slit2, whereas eosinophils and neutrophils expressed its cell-surface receptor, Robo1. Compared to neutrophils, eosinophils exhibited a significantly lower level of Slit-Robo GTPase-activating protein 1 (srGAP1), leading to activation of Cdc42, recruitment of PI3K to Robo1, enhancment of eotaxin-induced eosinophil chemotaxis, and exaggeration of allergic airway inflammation. Notably, OVA sensitization elicited a Slit2 gradient at so-called bronchus-alveoli axis, with a higher level of Slit2 in the bronchial epithelium and a lower level in the alveolar tissue. Aerosol administration of rSlit2 accelerated eosinophil infiltration, whereas i.v. administered Slit2 reduced eosinophil deposition. In contrast, Slit2 inactivated Cdc42 and suppressed stromal cell-derived factor-1α-induced chemotaxis of neutrophils for inhibiting endotoxin-induced lung inflammation, which were reversed by blockade of srGAP1 binding to Robo1. These results indicate that the newly identified Slit2 gradient at the bronchus-alveoli axis induces attractive PI3K signaling in eosinophils and repulsive srGAP1 signaling in neutrophils through differential srGAP1 expression during lung inflammation.

  18. Human neutrophils and oral microbiota: a constant tug-of-war between a harmonious and a discordant coexistence.

    PubMed

    Uriarte, Silvia M; Edmisson, Jacob S; Jimenez-Flores, Emeri

    2016-09-01

    Neutrophils are a major component of the innate host response, and the outcome of the interaction between the oral microbiota and neutrophils is a key determinant of oral health status. The composition of the oral microbiome is very complex and different in health and disease. Neutrophils are constantly recruited to the oral cavity, and their protective role is highlighted in cases where their number or functional responses are impeded, resulting in different forms of periodontal disease. Periodontitis, one of the more severe and irreversible forms of periodontal disease, is a microbial-induced chronic inflammatory disease that affects the gingival tissues supporting the tooth. This chronic inflammatory disease is the result of a shift of the oral bacterial symbiotic community to a dysbiotic more complex community. Chronic inflammatory infectious diseases such as periodontitis can occur because the pathogens are able to evade or disable the innate immune system. In this review, we discuss how human neutrophils interact with both the symbiotic and the dysbiotic oral community; an understanding of which is essential to increase our knowledge of the periodontal disease process. PMID:27558341

  19. Pulmonary vascular sequestration of neutrophils in endotoxemia is initiated by an effect of endotoxin on the neutrophil in the rabbit

    SciTech Connect

    Haslett, C.; Worthen, G.S.; Giclas, P.C.; Morrison, D.C.; Henson, J.E.; Henson, P.M.

    1987-07-01

    Endotoxemia causes neutrophil sequestration in the pulmonary vascular bed. Such sequestration may be a critical initiating event in the generation of microvascular injury, although the mechanisms that lead to this localization are not understood. To investigate these phenomena, the following study employed intravenous pulses of /sup 111/Indium-tropolonate-labeled neutrophils (/sup 111/In-neutrophils), which circulated in the rabbit with normal kinetics and responded in a manner indistinguishable from unlabeled, circulating neutrophils in response to an intravenous injection of purified endotoxic lipopolysaccharide (LPS) or epinephrine. Pulmonary sequestration of /sup 111/In-neutrophils was assessed by quantitative external gamma camera scintigraphy of a lung suprahilar region of interest. Noninvasive assessment of radioactivity by this method accurately reflected total lung radioactivity, which was shown by autoradiography to be confined to the injected /sup 111/In-neutrophils. Intravenously administered LPS caused a marked, dose-dependent sequestration of /sup 111/In-neutrophils in the pulmonary vasculature, and exhaustive ultrastructural autoradiography showed discretely radiolabeled neutrophils located within pulmonary capillaries. A distinct effect was seen with an intravenous injection of as little as 100 ng per rabbit (i.e., 500 pg/ml blood). A 5-min ex vivo pretreatment of /sup 111/In-neutrophils with 10 ng to 10 micrograms/ml LPS in heat-inactivated plasma also caused dose-dependent pulmonary sequestration of the pretreated /sup 111/In-neutrophils but did not cause generalized neutropenia in recipient rabbits.

  20. Human neutrophil kinetics: modeling of stable isotope labeling data supports short blood neutrophil half-lives.

    PubMed

    Lahoz-Beneytez, Julio; Elemans, Marjet; Zhang, Yan; Ahmed, Raya; Salam, Arafa; Block, Michael; Niederalt, Christoph; Asquith, Becca; Macallan, Derek

    2016-06-30

    Human neutrophils have traditionally been thought to have a short half-life in blood; estimates vary from 4 to 18 hours. This dogma was recently challenged by stable isotope labeling studies with heavy water, which yielded estimates in excess of 3 days. To investigate this disparity, we generated new stable isotope labeling data in healthy adult subjects using both heavy water (n = 4) and deuterium-labeled glucose (n = 9), a compound with more rapid labeling kinetics. To interpret results, we developed a novel mechanistic model and applied it to previously published (n = 5) and newly generated data. We initially constrained the ratio of the blood neutrophil pool to the marrow precursor pool (ratio = 0.26; from published values). Analysis of heavy water data sets yielded turnover rates consistent with a short blood half-life, but parameters, particularly marrow transit time, were poorly defined. Analysis of glucose-labeling data yielded more precise estimates of half-life (0.79 ± 0.25 days; 19 hours) and marrow transit time (5.80 ± 0.42 days). Substitution of this marrow transit time in the heavy water analysis gave a better-defined blood half-life of 0.77 ± 0.14 days (18.5 hours), close to glucose-derived values. Allowing the ratio of blood neutrophils to mitotic neutrophil precursors (R) to vary yielded a best-fit value of 0.19. Reanalysis of the previously published model and data also revealed the origin of their long estimates for neutrophil half-life: an implicit assumption that R is very large, which is physiologically untenable. We conclude that stable isotope labeling in healthy humans is consistent with a blood neutrophil half-life of less than 1 day. PMID:27136946

  1. Human neutrophil kinetics: modeling of stable isotope labeling data supports short blood neutrophil half-lives

    PubMed Central

    Lahoz-Beneytez, Julio; Elemans, Marjet; Zhang, Yan; Ahmed, Raya; Salam, Arafa; Block, Michael; Niederalt, Christoph; Macallan, Derek

    2016-01-01

    Human neutrophils have traditionally been thought to have a short half-life in blood; estimates vary from 4 to 18 hours. This dogma was recently challenged by stable isotope labeling studies with heavy water, which yielded estimates in excess of 3 days. To investigate this disparity, we generated new stable isotope labeling data in healthy adult subjects using both heavy water (n = 4) and deuterium-labeled glucose (n = 9), a compound with more rapid labeling kinetics. To interpret results, we developed a novel mechanistic model and applied it to previously published (n = 5) and newly generated data. We initially constrained the ratio of the blood neutrophil pool to the marrow precursor pool (ratio = 0.26; from published values). Analysis of heavy water data sets yielded turnover rates consistent with a short blood half-life, but parameters, particularly marrow transit time, were poorly defined. Analysis of glucose-labeling data yielded more precise estimates of half-life (0.79 ± 0.25 days; 19 hours) and marrow transit time (5.80 ± 0.42 days). Substitution of this marrow transit time in the heavy water analysis gave a better-defined blood half-life of 0.77 ± 0.14 days (18.5 hours), close to glucose-derived values. Allowing the ratio of blood neutrophils to mitotic neutrophil precursors (R) to vary yielded a best-fit value of 0.19. Reanalysis of the previously published model and data also revealed the origin of their long estimates for neutrophil half-life: an implicit assumption that R is very large, which is physiologically untenable. We conclude that stable isotope labeling in healthy humans is consistent with a blood neutrophil half-life of less than 1 day. PMID:27136946

  2. 6 CFR 17.310 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 6 Domestic Security 1 2011-01-01 2011-01-01 false Recruitment. 17.310 Section 17.310 Domestic... in Admission and Recruitment Prohibited § 17.310 Recruitment. (a) Nondiscriminatory recruitment. A... recruitment and admission of students. A recipient may be required to undertake additional recruitment...

  3. 41 CFR 101-4.310 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 41 Public Contracts and Property Management 2 2011-07-01 2007-07-01 true Recruitment. 101-4.310... Admission and Recruitment Prohibited § 101-4.310 Recruitment. (a) Nondiscriminatory recruitment. A recipient... recruitment and admission of students. A recipient may be required to undertake additional recruitment...

  4. 41 CFR 101-4.310 - Recruitment.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 41 Public Contracts and Property Management 2 2010-07-01 2010-07-01 true Recruitment. 101-4.310... Admission and Recruitment Prohibited § 101-4.310 Recruitment. (a) Nondiscriminatory recruitment. A recipient... recruitment and admission of students. A recipient may be required to undertake additional recruitment...

  5. 40 CFR 5.310 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 1 2011-07-01 2011-07-01 false Recruitment. 5.310 Section 5.310... in Admission and Recruitment Prohibited § 5.310 Recruitment. (a) Nondiscriminatory recruitment. A... recruitment and admission of students. A recipient may be required to undertake additional recruitment...

  6. 6 CFR 17.310 - Recruitment.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 6 Domestic Security 1 2010-01-01 2010-01-01 false Recruitment. 17.310 Section 17.310 Domestic... in Admission and Recruitment Prohibited § 17.310 Recruitment. (a) Nondiscriminatory recruitment. A... recruitment and admission of students. A recipient may be required to undertake additional recruitment...

  7. 15 CFR 8a.310 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 15 Commerce and Foreign Trade 1 2011-01-01 2011-01-01 false Recruitment. 8a.310 Section 8a.310... in Admission and Recruitment Prohibited § 8a.310 Recruitment. (a) Nondiscriminatory recruitment. A... recruitment and admission of students. A recipient may be required to undertake additional recruitment...

  8. 43 CFR 41.310 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 43 Public Lands: Interior 1 2011-10-01 2011-10-01 false Recruitment. 41.310 Section 41.310 Public... in Admission and Recruitment Prohibited § 41.310 Recruitment. (a) Nondiscriminatory recruitment. A... recruitment and admission of students. A recipient may be required to undertake additional recruitment...

  9. 28 CFR 54.310 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 28 Judicial Administration 2 2011-07-01 2011-07-01 false Recruitment. 54.310 Section 54.310... in Admission and Recruitment Prohibited § 54.310 Recruitment. (a) Nondiscriminatory recruitment. A... recruitment and admission of students. A recipient may be required to undertake additional recruitment...

  10. Genetically determined resistance to listeriosis is associated with increased accumulation of inflammatory neutrophils and macrophages which have enhanced listericidal activity.

    PubMed Central

    Czuprynski, C J; Canono, B P; Henson, P M; Campbell, P A

    1985-01-01

    The C57BL/6 and A/J inbred strains of mice differ markedly in their resistance to the facultative intracellular bacterium Listeria monocytogenes. One possible explanation for this genetically determined resistance is that phagocytes from Listeria-resistant strains of mice can kill L. monocytogenes more effectively than phagocytes from Listeria-susceptible strains of mice. We report here that inflammatory neutrophils and macrophages from Listeria-resistant mice (C57BL/6) exhibit a slight but significantly enhanced ability to kill L. monocytogenes in vitro as compared to inflammatory phagocytes from Listeria-susceptible mice (A/J). More importantly, however, Listeria-resistant mice recruited more inflammatory neutrophils and macrophages to the peritoneal cavity in response to i.p. injection of heat-killed Listeria than did Listeria-susceptible mice. These data suggest that genetically determined resistance to listeriosis is dependent on the enhanced inflammatory responsiveness of Listeria-resistant mice. Further support for this hypothesis was provided by experiments in which the passive transfer to A/J mice (C5-deficient) of plasma from C57BL/6 mice (C5-sufficient) enhanced the ability of the recipient A/J mice both to recruit inflammatory neutrophils to the peritoneal cavity in response to i.p. injection of heat-killed Listeria, and to clear L. monocytogenes from the spleen after a sublethal challenge of viable Listeria. PMID:4018836

  11. iNKT Cell Emigration out of the Lung Vasculature Requires Neutrophils and Monocyte-Derived Dendritic Cells in Inflammation.

    PubMed

    Thanabalasuriar, Ajitha; Neupane, Arpan S; Wang, Jing; Krummel, Matthew F; Kubes, Paul

    2016-09-20

    iNKT cells are a subset of innate T cells that recognize glycolipids presented on CD1d molecules and protect against bacterial infections, including S. pneumoniae. Using lung intravital imaging, we examined the behavior and mechanism of pulmonary iNKT cell activation in response to the specific iNKT cell ligand α-galactosylceramide or S. pneumoniae infection. In untreated mice, the major fraction of iNKT cells resided in the vasculature, but a small critical population resided in the extravascular space in proximity to monocyte-derived DCs. Administration of either α-GalCer or S. pneumoniae induced CD1d-dependent rapid recruitment of neutrophils out of the vasculature. The neutrophils guided iNKT cells from the lung vasculature via CCL17. Depletion of monocyte-derived DCs abrogated both the neutrophil and subsequent iNKT cell extravasation. Moreover, impairing iNKT cell recruitment by blocking CCL17 increased susceptibility to S. pneumoniae infection, suggesting a critical role for the influx of iNKT cells in host defense.

  12. iNKT Cell Emigration out of the Lung Vasculature Requires Neutrophils and Monocyte-Derived Dendritic Cells in Inflammation.

    PubMed

    Thanabalasuriar, Ajitha; Neupane, Arpan S; Wang, Jing; Krummel, Matthew F; Kubes, Paul

    2016-09-20

    iNKT cells are a subset of innate T cells that recognize glycolipids presented on CD1d molecules and protect against bacterial infections, including S. pneumoniae. Using lung intravital imaging, we examined the behavior and mechanism of pulmonary iNKT cell activation in response to the specific iNKT cell ligand α-galactosylceramide or S. pneumoniae infection. In untreated mice, the major fraction of iNKT cells resided in the vasculature, but a small critical population resided in the extravascular space in proximity to monocyte-derived DCs. Administration of either α-GalCer or S. pneumoniae induced CD1d-dependent rapid recruitment of neutrophils out of the vasculature. The neutrophils guided iNKT cells from the lung vasculature via CCL17. Depletion of monocyte-derived DCs abrogated both the neutrophil and subsequent iNKT cell extravasation. Moreover, impairing iNKT cell recruitment by blocking CCL17 increased susceptibility to S. pneumoniae infection, suggesting a critical role for the influx of iNKT cells in host defense. PMID:27653688

  13. Neutrophils in host defense: new insights from zebrafish

    PubMed Central

    Harvie, Elizabeth A.; Huttenlocher, Anna

    2015-01-01

    Neutrophils are highly motile phagocytic cells that play a critical role in the immune response to infection. Zebrafish (Danio rerio) are increasingly used to study neutrophil function and host-pathogen interactions. The generation of transgenic zebrafish lines with fluorescently labeled leukocytes has made it possible to visualize the neutrophil response to infection in real time by use of optically transparent zebrafish larvae. In addition, the genetic tractability of zebrafish has allowed for the generation of models of inherited neutrophil disorders. In this review, we discuss several zebrafish models of infectious disease, both in the context of immunocompetent, as well as neutrophil-deficient hosts and how these models have shed light on neutrophil behavior during infection. PMID:25717145

  14. Neutrophil depletion delays wound repair in aged mice

    PubMed Central

    Nishio, Naomi; Okawa, Yayoi; Sakurai, Hidetoshi

    2008-01-01

    One of the most important clinical problems in caring for elderly patients is treatment of pressure ulcers. One component of normal wound healing is the generation of an inflammatory reaction, which is characterized by the sequential infiltration of neutrophils, macrophages and lymphocytes. Neutrophils migrate early in the wound healing process. In aged C57BL/6 mice, wound healing is relatively inefficient. We examined the effects of neutrophil numbers on wound healing in both young and aged mice. We found that the depletion of neutrophils by anti-Gr-1 antibody dramatically delayed wound healing in aged mice. The depletion of neutrophils in young mice had less effect on the kinetics of wound healing. Intravenous G-CSF injection increased the migration of neutrophils to the wound site. While the rate of wound repair did not change significantly in young mice following G-CSF injection, it increased significantly in old mice. PMID:19424869

  15. Neutrophil elastase inhibitor reduces ventilation-induced lung injury via nuclear factor-κB and NF-κB repressing factor in mice.

    PubMed

    Li, Li-Fu; Lai, Yi-Ting; Chang, Chih-Hao; Lin, Meng-Chih; Liu, Yung-Yang; Kao, Kuo-Chin; Tsai, Ying-Huang

    2014-04-11

    Mechanical ventilation used in patients with acute lung injury can damage pulmonary epithelial cells through production of inflammatory cytokines, oxygen radicals, and neutrophil infiltration, termed ventilator-induced lung injury. Neutrophil elastase, nuclear factor-κB (NF-κB), and NF-κB repressing factor (NRF) have previously been shown to participate in the regulation of macrophage inflammatory protein-2 (MIP-2) during airway inflammation. However, the mechanisms regulating interactions among mechanical ventilation, neutrophil influx, and NF-κB/NRF remain unclear. Thus, we hypothesized that neutrophil elastase inhibitor attenuated ventilation-induced neutrophil recruitment and MIP-2 production through inhibition of the NF-κB/NRF pathway. Male C57BL/6 mice were exposed to low-tidal-volume (6 mL/kg) or high-tidal-volume (30 mL/kg) mechanical ventilation using room air with or without 2 µg/g NF-κB inhibitor SN50 or 6 µg/g NRF short interfering RNA or 100 µg/g neutrophil elastase inhibitor administration. Nonventilated mice served as a control group. Evan blue dye, lung wet-to-dry weight ratio, free radicals, myeloperoxidase, histopathologic grading of lung tissue, inflammatory cytokines, Western blot of NF-κB and NRF, and gene expression of NRF were measured to establish the extent of lung injury. Neutrophil elastase inhibitor ameliorated high-tidal-volume ventilation-induced lung injury, neutrophil influx, production of MIP-2 and malondialdehyde, activation of NF-κB and NRF, apoptotic epithelial cell death, and disruption of bronchial microstructure in mice. Mechanical stretch-augmented acute lung injury was also attenuated through pharmacological inhibition of NF-κB activity by SN50 and NRF expression by NRF short interfering RNA. Our data suggest that neutrophil elastase inhibitor attenuates high-tidal-volume mechanical ventilation-induced neutrophil influx, oxidative stress, and production of MIP-2, at least partly, through inhibition of

  16. Netting neutrophils in autoimmune small-vessel vasculitis.

    PubMed

    Kessenbrock, Kai; Krumbholz, Markus; Schönermarck, Ulf; Back, Walter; Gross, Wolfgang L; Werb, Zena; Gröne, Hermann-Josef; Brinkmann, Volker; Jenne, Dieter E

    2009-06-01

    Small-vessel vasculitis (SVV) is a chronic autoinflammatory condition linked to antineutrophil cytoplasm autoantibodies (ANCAs). Here we show that chromatin fibers, so-called neutrophil extracellular traps (NETs), are released by ANCA-stimulated neutrophils and contain the targeted autoantigens proteinase-3 (PR3) and myeloperoxidase (MPO). Deposition of NETs in inflamed kidneys and circulating MPO-DNA complexes suggest that NET formation triggers vasculitis and promotes the autoimmune response against neutrophil components in individuals with SVV.

  17. Distinct Functions of Neutrophil in Cancer and Its Regulation

    PubMed Central

    Granot, Zvi; Jablonska, Jadwiga

    2015-01-01

    Neutrophils are the most abundant of all white blood cells in the human circulation and are usually associated with inflammation and with fighting infections. In recent years the role immune cells play in cancer has been a matter of increasing interest. In this context the function of neutrophils is controversial as neutrophils were shown to possess both tumor promoting and tumor limiting properties. Here we provide an up-to-date review of the pro- and antitumor properties neutrophils possess as well as the environmental cues that regulate these distinct functions. PMID:26648665

  18. Neutrophilic Skin Lesions in Autoimmune Connective Tissue Diseases

    PubMed Central

    Hau, Estelle; Vignon Pennamen, Marie-Dominique; Battistella, Maxime; Saussine, Anne; Bergis, Maud; Cavelier-Balloy, Benedicte; Janier, Michel; Cordoliani, Florence; Bagot, Martine; Rybojad, Michel; Bouaziz, Jean-David

    2014-01-01

    Abstract The pathophysiology of neutrophilic dermatoses (NDs) and autoimmune connective tissue diseases (AICTDs) is incompletely understood. The association between NDs and AICTDs is rare; recently, however, a distinctive subset of cutaneous lupus erythematosus (LE, the prototypical AICTD) with neutrophilic histological features has been proposed to be included in the spectrum of lupus. The aim of our study was to test the validity of such a classification. We conducted a monocentric retrospective study of 7028 AICTDs patients. Among these 7028 patients, a skin biopsy was performed in 932 cases with mainly neutrophilic infiltrate on histology in 9 cases. Combining our 9 cases and an exhaustive literature review, pyoderma gangrenosum, Sweet syndrome (n = 49), Sweet-like ND (n = 13), neutrophilic urticarial dermatosis (n = 6), palisaded neutrophilic granulomatous dermatitis (n = 12), and histiocytoid neutrophilic dermatitis (n = 2) were likely to occur both in AICTDs and autoinflammatory diseases. Other NDs were specifically encountered in AICTDs: bullous LE (n = 71), amicrobial pustulosis of the folds (n = 28), autoimmunity-related ND (n = 24), ND resembling erythema gyratum repens (n = 1), and neutrophilic annular erythema (n = 1). The improvement of AICTDS neutrophilic lesions under neutrophil targeting therapy suggests possible common physiopathological pathways between NDs and AICTDs. PMID:25546688

  19. Interference with glycosaminoglycan-chemokine interactions with a probe to alter leukocyte recruitment and inflammation in vivo.

    PubMed

    Li, Sandra; Pettersson, Ulrika S; Hoorelbeke, Bart; Kolaczkowska, Elzbieta; Schelfhout, Katrien; Martens, Erik; Kubes, Paul; Van Damme, Jo; Phillipson, Mia; Opdenakker, Ghislain

    2014-01-01

    In vivo leukocyte recruitment is not fully understood and may result from interactions of chemokines with glycosaminoglycans/GAGs. We previously showed that chlorite-oxidized oxyamylose/COAM binds the neutrophil chemokine GCP-2/CXCL6. Here, mouse chemokine binding by COAM was studied systematically and binding affinities of chemokines to COAM versus GAGs were compared. COAM and heparan sulphate bound the mouse CXC chemokines KC/CXCL1, MIP-2/CXCL2, IP-10/CXCL10 and I-TAC/CXCL11 and the CC chemokine RANTES/CCL5 with affinities in the nanomolar range, whereas no binding interactions were observed for mouse MCP-1/CCL2, MIP-1α/CCL3 and MIP-1β/CCL4. The affinities of COAM-interacting chemokines were similar to or higher than those observed for heparan sulphate. Although COAM did not display chemotactic activity by itself, its co-administration with mouse GCP-2/CXCL6 and MIP-2/CXCL2 or its binding of endogenous chemokines resulted in fast and cooperative peritoneal neutrophil recruitment and in extravasation into the cremaster muscle in vivo. These local GAG mimetic features by COAM within tissues superseded systemic effects and were sufficient and applicable to reduce LPS-induced liver-specific neutrophil recruitment and activation. COAM mimics glycosaminoglycans and is a nontoxic probe for the study of leukocyte recruitment and inflammation in vivo. PMID:25093679

  20. Diversity employment and recruitment sources

    SciTech Connect

    Not Available

    1994-08-01

    Effective human resources management has been identified as one of four critical success factors in the Department of Energy Strategic Plan. The Plan states relative to this factor: ``The Department seeks greater alignment of resources with agency priorities and increased diversification of the workforce, including gender, ethnicity, age, and skills. This diversification will bring new thinking and perspectives that heretofore have not had a voice in departmental decision-making.`` This Guide has been developed as a key tool to assist Department of Energy management and administrative staff in achieving Goal 2 of this critical success factor, which is to ``Ensure a diverse and talented workforce.`` There are numerous sources from which to recruit minorities, women and persons with disabilities. Applying creativity and proactive effort, using traditional and non-traditional approaches, and reaching out to various professional, academic and social communities will increase the reservoir of qualified candidates from which to make selections. In addition, outreach initiatives will undoubtedly yield further benefits such as a richer cultural understanding and diversity awareness. The resource listings presented in this Guide are offered to encourage active participation in the diversity recruitment process. This Guide contains resource listings by state for organizations in the following categories: (1) African American Recruitment Sources; (2) Asian American/Pacific Islander Recruitment Sources; (3) Hispanic Recruitment Sources; (4) Native American/Alaskan Native Recruitment Sources; (5) Persons with Disabilities Recruitment Sources; and (6) Women Recruitment Sources.

  1. Three Keys to Better Recruiting

    ERIC Educational Resources Information Center

    Brazington, Alicia

    2012-01-01

    Recruitment is an expensive business: In 2010-2011, the median cost to recruit an undergraduate was $2,185 among private colleges and universities, according to Noel-Levitz, an enrollment management consultancy. In these tough fiscal times, admissions departments are under pressure to keep those costs down even as they pursue higher enrollment and…

  2. Teachers, Recruitment and the Law

    ERIC Educational Resources Information Center

    Degazon-Johnson, Roli

    2007-01-01

    This article offers a critical review and evaluation of the statutory environment in which recruitment agencies and businesses ply their trade in the United Kingdom (UK), with specific reference to the employment of overseas teachers. It focuses especially on teachers recruited from the Commonwealth over the period 1999-2005, a significant time…

  3. Recruiting Mexican American Adoptive Parents.

    ERIC Educational Resources Information Center

    Bausch, Robert S.; Serpe, Richard T.

    1999-01-01

    Interviews were conducted with 591 Mexican Americans to determine adoption interest and create recruiting practices for prospective parents. Approximately one-third of sample reported an interest in adoption, but many perceived both structural and cultural obstacles to adoption. Based on findings, recommendations for increasing recruitment of…

  4. Recruiting Migrant Students Reference Supplement.

    ERIC Educational Resources Information Center

    Johnson, Leon, Ed.

    This document reports on a study evaluating the practices of identification and recruitment of migrant students. Guided by a 14-state advisory council, data were collected by ethnographic research of practice in migrant recruitment and focus-group studies of migrant families. The document consists of five sections each written by a different…

  5. Recruit and ADVANCE

    NASA Astrophysics Data System (ADS)

    Rosser, Sue V.

    2007-04-01

    Beginning in 2001, the National Science Foundation launched the ADVANCE Initiative, which has now awarded more than 70 million to some thirty institutions for transformations to advance women. Results of studies on how to attract and retain women students and faculty underpinned our ADVANCE Institutional Transformation grant funded by the NSF for 3.7 million for five years, beginning in 2001. As co-principal investigator on this grant, I insured that this research informed the five major threads of the grant: 1) Four termed ADVANCE professors to mentor junior women faculty in each college; 2) Collection of MIT-Report-like data indicators to assess whether advancement of women really occurs during and after the institutional transformation undertaken through ADVANCE; 3) Family-friendly policies and practices to stop the tenure clock and provide active service, modified duties, lactation stations and day care; 4) Mini-retreats to facilitate access for tenure-track women faculty to male decision-makers and administrators for informal conversations and discussion on topics important to women faculty; 5) Removal of subtle gender, racial, and other biases in promotion and tenure. The dynamic changes resulting from the grant in quality of mentoring, new understanding of promotion and tenure, numbers of women retained and given endowed chairs, and emergence of new family friendly policies gave me hope for genuine diversification of leadership in science and technology. As the grant funding ends, the absence of NSF prestige and monitoring, coupled with a change in academic leadership at the top, provide new challenges for institutionalization, recruitment, and advancement of women into leadership positions in science and engineering.

  6. Imaging G Protein-coupled Receptor-mediated Chemotaxis and its Signaling Events in Neutrophil-like HL60 Cells.

    PubMed

    Wen, Xi; Jin, Tian; Xu, Xuehua

    2016-01-01

    Eukaryotic cells sense and move towards a chemoattractant gradient, a cellular process referred as chemotaxis. Chemotaxis plays critical roles in many physiological processes, such as embryogenesis, neuron patterning, metastasis of cancer cells, recruitment of neutrophils to sites of inflammation, and the development of the model organism Dictyostelium discoideum. Eukaryotic cells sense chemo-attractants using G protein-coupled receptors. Visual chemotaxis assays are essential for a better understanding of how eukaryotic cells control chemoattractant-mediated directional cell migration. Here, we describe detailed methods for: 1) real-time, high-resolution monitoring of multiple chemotaxis assays, and 2) simultaneously visualizing the chemoattractant gradient and the spatiotemporal dynamics of signaling events in neutrophil-like HL60 cells. PMID:27684322

  7. Differential Use of Human Neutrophil Fcγ Receptors for Inducing Neutrophil Extracellular Trap Formation

    PubMed Central

    Alemán, Omar Rafael; Mora, Nancy; Cortes-Vieyra, Ricarda; Uribe-Querol, Eileen; Rosales, Carlos

    2016-01-01

    Neutrophils (PMN) are the most abundant leukocytes in the blood. PMN migrate from the circulation to sites of infection, where they are responsible for antimicrobial functions. PMN use phagocytosis, degranulation, and formation of neutrophil extracellular traps (NETs) to kill microbes. NETs are fibers composed of chromatin and neutrophil-granule proteins. Several pathogens, including bacteria, fungi, and parasites, and also some pharmacological stimuli such as phorbol 12-myristate 13-acetate (PMA) are efficient inducers of NETs. Antigen-antibody complexes are also capable of inducing NET formation. However the particular Fcγ receptor involved in triggering this function is a matter of controversy. In order to provide some insight into what Fcγ receptor is responsible for NET formation, each of the two human Fcγ receptors was stimulated individually by specific monoclonal antibodies and NET formation was evaluated. FcγRIIa cross-linking did not promote NET formation. Cross-linking other receptors such as integrins also did not promote NET formation. In contrast FcγRIIIb cross-linking induced NET formation similarly to PMA stimulation. NET formation was dependent on NADPH-oxidase, PKC, and ERK activation. These data show that cross-linking FcγRIIIb is responsible for NET formation by the human neutrophil. PMID:27034964

  8. Francisella tularensis alters human neutrophil gene expression: insights into the molecular basis of delayed neutrophil apoptosis.

    PubMed

    Schwartz, Justin T; Bandyopadhyay, Sarmistha; Kobayashi, Scott D; McCracken, Jenna; Whitney, Adeline R; Deleo, Frank R; Allen, Lee-Ann H

    2013-01-01

    We demonstrated recently that Francisella tularensis profoundly impairs human neutrophil apoptosis, but how this is achieved is largely unknown. Herein we used human oligonucleotide microarrays to test the hypothesis that changes in neutrophil gene expression contribute to this phenotype, and now demonstrate that F. tularensis live vaccine strain (LVS) caused significant changes in neutrophil gene expression over a 24-hour time period relative to the uninfected controls. Of approximately 47,000 genes analyzed, 3,435 were significantly up- or downregulated by LVS, including 365 unique genes associated with apoptosis and cell survival. Specific targets in this category included genes asso-ciated with the intrinsic and extrinsic apoptotic pathways (CFLAR, TNFAIP3, TNFRSF10D, SOD2, BCL2A1, BIRC4, PIM2, TNFSF10, TNFRSF10C, CASP2 and CASP8) and genes that act via the NFĸB pathway and other mechanisms to prolong cell viability (NFKB1, NFKB2 and RELA, IL1B, CAST, CDK2,GADD45B, BCL3, BIRC3, CDK2, IL1A, PBEF1, IL6, CXCL1, CCL4 and VEGF). The microarray data were confirmed by qPCR and pathway analysis. Moreover, we demonstrate that the X-linked inhibitor of apoptosis protein remained abundant in polymorphonuclear leukocytes over 48 h of LVS infection, whereas BAX mRNA and protein were progressively downregulated. These data strongly suggest that antiapoptotic and prosurvival mechanisms collaborate to sustain the viability of F. tularensis--infected neutrophils. PMID:22986450

  9. Characterization of C1 inhibitor binding to neutrophils.

    PubMed Central

    Chang, N S; Boackle, R J; Leu, R W

    1991-01-01

    In a previous study we have isolated neutrophil membrane proteins that non-covalently bind to native C1-INH (105,000 MW) and a non-functional, degraded C1-INH (88,000 MW; C1-INH-88). To further characterize the binding nature, we have designed a novel kinetic C1 titration assay which enables not only a quantification of the removal of fluid-phase C1-INH by neutrophils, but also a concomitant measure of residual C1-INH function. Native C1-INH, when adsorbed to EDTA-pretreated neutrophils, lost its function in the inhibition of fluid-phase C1. The non-functional C1-INH-88, which is probably devoid of a reactive centre, was found to block the binding of native C1-INH to neutrophils. Pretreatment of neutrophils with serine esterase inhibitors did not abrogate binding capacity of the cells for C1-INH, whereas the binding affinity for C1-INH was lost when the cells were pretreated with trypsin. An array of human peripheral blood leucocytes and several lymphoid cell lines has surface binding sites for C1-INH, but not on human erythrocytes and U937 cells. Binding was further confirmed using (i) C1-INH-microsphere beads to neutrophils, in which the binding was blocked when pretreating neutrophils with excess C1-INH or with trypsin, and (ii) radiolabelled C1-INH to neutrophils, which was competitively blocked by unlabelled non-functional C1-INH-88. Desialylation of C1-INH significantly reduced its binding affinity for neutrophils, indicating that the membrane receptor sites on neutrophils could be specific for the binding of sialic acid residues on C1-INH. Overall, our studies indicate that neutrophils or other leucocytes possess specific surface binding sites for the sialic acid-containing portion of C1-INH. PMID:2045131

  10. Exogenous melatonin inhibits neutrophil migration through suppression of ERK activation.

    PubMed

    Ren, Da-Long; Sun, Ai-Ai; Li, Ya-Juan; Chen, Min; Ge, Shu-Chao; Hu, Bing

    2015-10-01

    Neutrophil migration to inflammatory sites is the fundamental process of innate immunity among organisms against pathogen invasion. As a major sleep adjusting hormone, melatonin has also been proved to be involved in various inflammatory events. This study aimed to evaluate the impact of exogenous melatonin on neutrophil migration to the injury site in live zebrafish and further investigate whether ERK signaling is involved in this process. Using the tail fin transection model, the fluorescently labeled neutrophil was in vivo visualized in transgenic Tg(lyz:EGFP), Tg(lyz:DsRed) zebrafish. We found that exogenous melatonin administration dramatically inhibited the injury-induced neutrophil migration in a dose-dependent and time-dependent manner. The inhibited effect of melatonin on neutrophil migration could be attenuated by melatonin receptor 1, 2, and 3 antagonists. The ERK phosphorylation level was significantly decreased post injury when treated with melatonin. The blocking of ERK activation with inhibitor PD0325901 suppressed the number of migrated neutrophils in response to injury. However, the activation of ERK with the epidermal growth factor could impair the inhibited effect of melatonin on neutrophil migration. We also detected that PD0325901 significantly suppressed the in vivo neutrophils transmigrating over the vessel endothelial cell using the transgenic Tg(flk:EGFP);(lyz:DsRed) line labeled as both vessel and neutrophil. Taking all of these data together, the results indicated that exogenous melatonin had an anti-migratory effect on neutrophils by blocking the ERK phosphorylation signal, and it led to the subsequent adhesion molecule expression. Thus, the crossing of the vessel endothelial cells of neutrophils became difficult.

  11. Role of Granulocyte-Macrophage Colony-Stimulating Factor Signaling in Regulating Neutrophil Antifungal Activity and the Oxidative Burst During Respiratory Fungal Challenge.

    PubMed

    Kasahara, Shinji; Jhingran, Anupam; Dhingra, Sourabh; Salem, Anand; Cramer, Robert A; Hohl, Tobias M

    2016-04-15

    Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a pleiotropic cytokine that plays a critical role in regulating myeloid cell host defense. In this study, we demonstrated that GM-CSF signaling plays an essential role in antifungal defense against Aspergillus fumigatus. Mice that lack the GM-CSF receptor β chain (GM-CSFRβ) developed invasive hyphal growth and exhibited impaired survival after pulmonary challenge with A. fumigatus conidia. GM-CSFRβ signaling regulated the recruitment of inflammatory monocytes to infected lungs, but not the recruitment of effector neutrophils. Cell-intrinsic GM-CSFRβ signaling mediated neutrophil and inflammatory monocyte antifungal activity, because lung GM-CSFRβ(-/-) leukocytes exhibited impaired conidial killing compared with GM-CSFRβ(+/+) counterparts in mixed bone marrow chimeric mice. GM-CSFRβ(-/-) neutrophils exhibited reduced (hydrogenated) nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity in vivo. Conversely, administration of recombinant GM-CSF enhanced neutrophil NADPH oxidase function, conidiacidal activity, and lung fungal clearance in A. fumigatus-challenged mice. Thus, our study illustrates the functional role of GM-CSFRβ signaling on lung myeloid cell responses against inhaled A. fumigatus conidia and demonstrates a benefit for systemic GM-CSF administration. PMID:26908736

  12. Bromelain decreases neutrophil interactions with P-selectin, but not E-selectin, in vitro by proteolytic cleavage of P-selectin glycoprotein ligand-1.

    PubMed

    Banks, Jessica M; Herman, Christine T; Bailey, Ryan C

    2013-01-01

    Stem bromelain, a cysteine protease isolated from pineapples, is a natural anti-inflammatory treatment, yet its mechanism of action remains unclear. Curious as to whether bromelain might affect selectin-mediated leukocyte rolling, we studied the ability of bromelain-treated human neutrophils to tether to substrates presenting immobilized P-selectin or E-selectin under shear stress. Bromelain treatment attenuated P-selectin-mediated tethering but had no effect on neutrophil recruitment on E-selectin substrates. Flow cytometric analysis of human neutrophils, using two antibodies against distinct epitopes within the P-selectin glycoprotein ligand-1 (PSGL-1) active site, revealed that bromelain cleaves PSGL-1 to remove one of two sites required for P-selectin binding, while leaving the region required for E-selectin binding intact. These findings suggest one molecular mechanism by which bromelain may exert its anti-inflammatory effects is via selective cleavage of PSGL-1 to reduce P-selectin-mediated neutrophil recruitment.

  13. Study of neutrophils isolated from peripheral blood of patients suffering from aggressive periodontitis at the cellular level: Receptors and cytoskeletal reorganization

    PubMed Central

    Mukherjee, Saswati; Kundu, Debabrata

    2012-01-01

    Background: Aggressive periodontitis (AgP) has been associated with polymorphonuclear leukocyte's (PMNL) dysfunction and periodontal pathogens possess variety of virulence factors that can impair PMNL's function. This study investigated the possible association between defective neutrophil adhesion and β2 -integrin expression and defective neutrophil migration and actin polymerization level in the peripheral blood of neutrophils from the patients with AgP. Materials and Methods: A total of 30 individuals both male and female, age ranges between 13 – 48 years, were included in the study. Healthy controls (group I, n=10), chronic periodontitis (ChP) (group II, n=10), and AgP (group III, n=10), all without any systemic diseases and non-smokers, were recruited. Peripheral blood samples were taken and β2 -integrin expression and actin polymerization levels were estimated by using fluorescence activated cell sorter analysis. Results: In AgP cases, both average values (β2 -integrin and actin level) were significantly less than that of normal subjects (<0.001). But for ChP cases, only the average value of actin level is significantly lower than that of normal subjects (<0.025). Conclusion: Lower β2 -integrin expression in the AgP cases signifies lower neutrophil adhesion in AgP cases than normal, and the lower average value of actin polymerization for the AgP cases suggest lower migration capacity of neutrophils in AgP cases than normal. PMID:22628965

  14. How Neutrophil Extracellular Traps Become Visible

    PubMed Central

    2016-01-01

    Neutrophil extracellular traps (NETs) have been identified as a fundamental innate immune defense mechanism against different pathogens. NETs are characterized as released nuclear DNA associated with histones and granule proteins, which form an extracellular web-like structure that is able to entrap and occasionally kill certain microbes. Furthermore, NETs have been shown to contribute to several noninfectious disease conditions when released by activated neutrophils during inflammation. The identification of NETs has mainly been succeeded by various microscopy techniques, for example, immunofluorescence microscopy, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). Since the last years the development and improvement of new immunofluorescence-based techniques enabled optimized visualization and quantification of NETs. On the one hand in vitro live-cell imaging led to profound new ideas about the mechanisms involved in the formation and functionality of NETs. On the other hand different intravital, in vivo, and in situ microscopy techniques led to deeper insights into the role of NET formation during health and disease. This paper presents an overview of the main used microscopy techniques to visualize NETs and describes their advantages as well as disadvantages. PMID:27294157

  15. Pondering neutrophil extracellular traps with healthy skepticism.

    PubMed

    Nauseef, William M; Kubes, Paul

    2016-10-01

    The authors engage in a dialogue that evaluates critically the state of the study of neutrophil extracellular traps (NETs), a phenomenon currently the object of considerable interest, with the goal of identifying those aspects that merit clarification in order to assign the process its proper place in our current understanding of cell biology. Since the seminal observations in the Zychlinsky laboratory that described the extrusion of filaments of nuclear DNA associated with histones and granule proteins from neutrophils stimulated in vitro, many investigators have examined the phenomenon of NET formation in numerous and diverse settings. However, an overview of work in this rapidly growing field prompts several fundamental questions about NETs, including their precise composition, the mechanisms by which they arise, their clinical relevance, and the interrelationship of those observed in vitro and in vivo. In this discussion, the authors challenge interpretation of data from some experimental settings and provide recommendations for specific studies that would address the concerns raised, improve understanding of the biological relevance of NETs, and strengthen the field. PMID:27470975

  16. Pondering neutrophil extracellular traps with healthy skepticism.

    PubMed

    Nauseef, William M; Kubes, Paul

    2016-10-01

    The authors engage in a dialogue that evaluates critically the state of the study of neutrophil extracellular traps (NETs), a phenomenon currently the object of considerable interest, with the goal of identifying those aspects that merit clarification in order to assign the process its proper place in our current understanding of cell biology. Since the seminal observations in the Zychlinsky laboratory that described the extrusion of filaments of nuclear DNA associated with histones and granule proteins from neutrophils stimulated in vitro, many investigators have examined the phenomenon of NET formation in numerous and diverse settings. However, an overview of work in this rapidly growing field prompts several fundamental questions about NETs, including their precise composition, the mechanisms by which they arise, their clinical relevance, and the interrelationship of those observed in vitro and in vivo. In this discussion, the authors challenge interpretation of data from some experimental settings and provide recommendations for specific studies that would address the concerns raised, improve understanding of the biological relevance of NETs, and strengthen the field.

  17. Fluorescent Ly6G antibodies determine macrophage phagocytosis of neutrophils and alter the retrieval of neutrophils in mice.

    PubMed

    Bucher, Kirsten; Schmitt, Fee; Autenrieth, Stella E; Dillmann, Inken; Nürnberg, Bernd; Schenke-Layland, Katja; Beer-Hammer, Sandra

    2015-09-01

    Fluorescently labeled Ly6G antibodies enable the tracking of neutrophils in mice, whereas purified anti-Ly6G rapidly depletes neutrophils from the circulation. The mechanisms underlying neutrophil depletion are still under debate. Here, we examined how identical Ly6G antibodies coupled to different fluorochromes affect neutrophil fate in vivo. BM cells stained with Ly6G antibodies were injected into mice. The number of retrieved anti-Ly6G-FITC(+) cells was reduced significantly in comparison with anti-Ly6G-APC(+) or anti-Ly6G-PE(+) cells. Flow cytometry and multispectral imaging flow cytometry analyses revealed that anti-Ly6G-FITC(+) neutrophils were preferentially phagocytosed by BMMs in vitro and by splenic, hepatic, and BM macrophages in vivo. Direct antibody injection of anti-Ly6G-FITC but not anti-Ly6G-PE depleted neutrophils to the same degree as purified anti-Ly6G, indicating that the FITC-coupled antibody eliminates neutrophils by a similar mechanism as the uncoupled antibody. With the use of a protein G-binding assay, we demonstrated that APC and PE but not FITC coupling inhibited access to interaction sites on the anti-Ly6G antibody. We conclude the following: 1) that neutrophil phagocytosis by macrophages is a central mechanism in anti-Ly6G-induced neutrophil depletion and 2) that fluorochrome-coupling can affect functional properties of anti-Ly6G antibodies, thereby modifying macrophage uptake of Ly6G-labeled neutrophils and neutrophil retrieval following adoptive cell transfer or injection of fluorescent anti-Ly6G.

  18. Fluorescent Ly6G antibodies determine macrophage phagocytosis of neutrophils and alter the retrieval of neutrophils in mice.

    PubMed

    Bucher, Kirsten; Schmitt, Fee; Autenrieth, Stella E; Dillmann, Inken; Nürnberg, Bernd; Schenke-Layland, Katja; Beer-Hammer, Sandra

    2015-09-01

    Fluorescently labeled Ly6G antibodies enable the tracking of neutrophils in mice, whereas purified anti-Ly6G rapidly depletes neutrophils from the circulation. The mechanisms underlying neutrophil depletion are still under debate. Here, we examined how identical Ly6G antibodies coupled to different fluorochromes affect neutrophil fate in vivo. BM cells stained with Ly6G antibodies were injected into mice. The number of retrieved anti-Ly6G-FITC(+) cells was reduced significantly in comparison with anti-Ly6G-APC(+) or anti-Ly6G-PE(+) cells. Flow cytometry and multispectral imaging flow cytometry analyses revealed that anti-Ly6G-FITC(+) neutrophils were preferentially phagocytosed by BMMs in vitro and by splenic, hepatic, and BM macrophages in vivo. Direct antibody injection of anti-Ly6G-FITC but not anti-Ly6G-PE depleted neutrophils to the same degree as purified anti-Ly6G, indicating that the FITC-coupled antibody eliminates neutrophils by a similar mechanism as the uncoupled antibody. With the use of a protein G-binding assay, we demonstrated that APC and PE but not FITC coupling inhibited access to interaction sites on the anti-Ly6G antibody. We conclude the following: 1) that neutrophil phagocytosis by macrophages is a central mechanism in anti-Ly6G-induced neutrophil depletion and 2) that fluorochrome-coupling can affect functional properties of anti-Ly6G antibodies, thereby modifying macrophage uptake of Ly6G-labeled neutrophils and neutrophil retrieval following adoptive cell transfer or injection of fluorescent anti-Ly6G. PMID:26019296

  19. Diverse novel functions of neutrophils in immunity, inflammation, and beyond

    PubMed Central

    Mócsai, Attila

    2013-01-01

    Neutrophils have long been considered simple suicide killers at the bottom of the hierarchy of the immune response. That view began to change 10–20 yr ago, when the sophisticated mechanisms behind how neutrophils locate and eliminate pathogens and regulate immunity and inflammation were discovered. The last few years witnessed a new wave of discoveries about additional novel and unexpected functions of these cells. Neutrophils have been proposed to participate in protection against intracellular pathogens such as viruses and mycobacteria. They have been shown to intimately shape the adaptive immune response at various levels, including marginal zone B cells, plasmacytoid dendritic cells and T cell populations, and even to control NK cell homeostasis. Neutrophils have been shown to mediate an alternative pathway of systemic anaphylaxis and to participate in allergic skin reactions. Finally, neutrophils were found to be involved in physiological and pathological processes beyond the immune system, such as diabetes, atherosclerosis, and thrombus formation. Many of those functions appear to be related to their unique ability to release neutrophil extracellular traps even in the absence of pathogens. This review summarizes those novel findings on versatile functions of neutrophils and how they change our view of neutrophil biology in health and disease. PMID:23825232

  20. Human neutrophil leukocyte elastase activity is inhibited by Phenol Red

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Neutrophil elastase (NE) activity in urine, sputum and nasal mucous is used as an indicator of inflammation due to viral or bacterial infection. However, bovine nasal mucous neutrophils collected, lysed and stored in Dulbecco's minimal medium containing Phenol Red, showed no NE activity with methox...

  1. Intracellular localization of VAMP-1 protein in human neutrophils.

    PubMed

    Nabokina, S M

    2001-02-01

    We studied the intracellular localization of vesicle-associated membrane protein VAMP-1 in human neutrophils. VAMP-1 was associated with membranes of gelatinase and specific secretory granules rapidly mobilized during exocytosis. VAMP-1 probably acts as a component of the SNARE complex during exocytosis of gelatinase and specific granules in human neutrophils.

  2. 49 CFR 25.310 - Recruitment.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 1 2010-10-01 2010-10-01 false Recruitment. 25.310 Section 25.310 Transportation... Recruitment Prohibited § 25.310 Recruitment. (a) Nondiscriminatory recruitment. A recipient to which §§ 25.300 through 25.310 apply shall not discriminate on the basis of sex in the recruitment and admission...

  3. 22 CFR 146.310 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 22 Foreign Relations 1 2011-04-01 2011-04-01 false Recruitment. 146.310 Section 146.310 Foreign... Recruitment Prohibited § 146.310 Recruitment. (a) Nondiscriminatory recruitment. A recipient to which §§ 146.300 through 146.310 apply shall not discriminate on the basis of sex in the recruitment and...

  4. 22 CFR 229.310 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 22 Foreign Relations 1 2011-04-01 2011-04-01 false Recruitment. 229.310 Section 229.310 Foreign... and Recruitment Prohibited § 229.310 Recruitment. (a) Nondiscriminatory recruitment. A recipient to which §§ 229.300 through 229.310 apply shall not discriminate on the basis of sex in the recruitment...

  5. 10 CFR 1042.310 - Recruitment.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 4 2010-01-01 2010-01-01 false Recruitment. 1042.310 Section 1042.310 Energy DEPARTMENT... Recruitment Prohibited § 1042.310 Recruitment. (a) Nondiscriminatory recruitment. A recipient to which §§ 1042.300 through 1042.310 apply shall not discriminate on the basis of sex in the recruitment and...

  6. 49 CFR 25.310 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 1 2011-10-01 2011-10-01 false Recruitment. 25.310 Section 25.310 Transportation... Recruitment Prohibited § 25.310 Recruitment. (a) Nondiscriminatory recruitment. A recipient to which §§ 25.300 through 25.310 apply shall not discriminate on the basis of sex in the recruitment and admission...

  7. Swell activated chloride channel function in human neutrophils

    SciTech Connect

    Salmon, Michael D.; Ahluwalia, Jatinder

    2009-04-17

    Non-excitable cells such as neutrophil granulocytes are the archetypal inflammatory immune cell involved in critical functions of the innate immune system. The electron current generated (I{sub e}) by the neutrophil NADPH oxidase is electrogenic and rapidly depolarises the membrane potential. For continuous function of the NADPH oxidase, I{sub e} has to be balanced to preserve electroneutrality, if not; sufficient depolarisation would prevent electrons from leaving the cell and neutrophil function would be abrogated. Subsequently, the depolarisation generated by the neutrophil NADPH oxidase I{sub e} must be counteracted by ion transport. The finding that depolarisation required counter-ions to compensate electron transport was followed by the observation that chloride channels activated by swell can counteract the NADPH oxidase membrane depolarisation. In this mini review, we discuss the research findings that revealed the essential role of swell activated chloride channels in human neutrophil function.

  8. Effects of pentoxifylline on equine neutrophil function and flow properties.

    PubMed Central

    Weiss, D J; Geor, R J; Burris, S M; Smith, C M

    1992-01-01

    Pentoxifylline has been reported to improve peripheral vascular circulation by altering the flow properties of blood. To determine if the hemorrheological effects of pentoxifylline were mediated by alterations in neutrophil function and/or flow properties, we evaluated the drug's effects on equine neutrophils in vitro. Pentoxifylline, at a concentration of 1 x 10(-1) M, but not at concentrations of 1 x 10(-6) M to 1 x 10(-2) M, markedly suppressed neutrophil superoxide production, zymosan phagocytosis and adherence to nylon wool. Pentoxifylline failed to improve neutrophil filterability through 3 mu polycarbonate filters at any concentration tested. We conclude that equine neutrophil function and flow properties are unlikely to be affected by pentoxifylline concentrations achievable in vivo. PMID:1335832

  9. Paradoxical Roles of the Neutrophil in Sepsis: Protective and Deleterious

    PubMed Central

    Sônego, Fabiane; Castanheira, Fernanda Vargas e Silva; Ferreira, Raphael Gomes; Kanashiro, Alexandre; Leite, Caio Abner Vitorino Gonçalves; Nascimento, Daniele Carvalho; Colón, David Fernando; Borges, Vanessa de Fátima; Alves-Filho, José Carlos; Cunha, Fernando Queiróz

    2016-01-01

    Sepsis, an overwhelming inflammatory response syndrome secondary to infection, is one of the costliest and deadliest medical conditions worldwide. Neutrophils are classically considered to be essential players in the host defense against invading pathogens. However, several investigations have shown that impairment of neutrophil migration to the site of infection, also referred to as neutrophil paralysis, occurs during severe sepsis, resulting in an inability of the host to contain and eliminate the infection. On the other hand, the neutrophil antibacterial arsenal contributes to tissue damage and the development of organ dysfunction during sepsis. In this review, we provide an overview of the main events in which neutrophils play a beneficial or deleterious role in the outcome of sepsis. PMID:27199981

  10. Spatial control of actin polymerization during neutrophil chemotaxis

    PubMed Central

    Weiner, Orion D.; Servant, Guy; Welch, Matthew D.; Mitchison, Timothy J.; Sedat, John W.; Bourne, Henry R.

    2010-01-01

    Neutrophils respond to chemotactic stimuli by increasing the nucleation and polymerization of actin filaments, but the location and regulation of these processes are not well understood. Here, using a permeabilized-cell assay, we show that chemotactic stimuli cause neutrophils to organize many discrete sites of actin polymerization, the distribution of which is biased by external chemotactic gradients. Furthermore, the Arp2/3 complex, which can nucleate actin polymerization, dynamically redistributes to the region of living neutrophils that receives maximal chemotactic stimulation, and the least-extractable pool of the Arp2/3 complex co-localizes with sites of actin polymerization. Our observations indicate that chemoattractant-stimulated neutrophils may establish discrete foci of actin polymerization that are similar to those generated at the posterior surface of the intracellular bacterium Listeria monocytogenes. We propose that asymmetrical establishment and/or maintenance of sites of actin polymerization produces directional migration of neutrophils in response to chemotactic gradients. PMID:10559877

  11. The role of neutrophils in the immune system: an overview.

    PubMed

    Malech, Harry L; Deleo, Frank R; Quinn, Mark T

    2014-01-01

    Neutrophils, also known as polymorphonuclear leukocytes (PMNs), have long been considered as the short-lived, nonspecific white cells that form pus-and also happen to kill invading microbes. Indeed, neutrophils were often neglected (and largely not considered) as immune cells. This historic view of neutrophils has changed considerably over the past several decades, and we know now that, in addition to playing the predominant role in the clearance of bacteria and fungi, they play a major role in shaping the host response to infection and immune system homeostasis. The change in our view of the role of neutrophils in the immune system has been due in large part to the study of these cells in vitro. Such work has been made possible by new and/or improved methods and approaches used to investigate neutrophils. These methods are the focus of this volume.

  12. Clearance of apoptotic neutrophils and resolution of inflammation.

    PubMed

    Greenlee-Wacker, Mallary C

    2016-09-01

    The engulfment of apoptotic cells by phagocytes, a process referred to as efferocytosis, is essential for maintenance of normal tissue homeostasis and a prerequisite for the resolution of inflammation. Neutrophils are the predominant circulating white blood cell in humans, and contain an arsenal of toxic substances that kill and degrade microbes. Neutrophils are short-lived and spontaneously die by apoptosis. This review will highlight how the engulfment of apoptotic neutrophils by human phagocytes occurs, how heterogeneity of phagocyte populations influences efferocytosis signaling, and downstream consequences of efferocytosis. The efferocytosis of apoptotic neutrophils by macrophages promotes anti-inflammatory signaling, prevents neutrophil lysis, and dampens immune responses. Given the immunomodulatory properties of efferocytosis, understanding pathways that regulate and enhance efferocytosis could be harnessed to combat infection and chronic inflammatory conditions. PMID:27558346

  13. Student Recruitment: The Hard Sell?

    ERIC Educational Resources Information Center

    McAdams, Tony

    1975-01-01

    Presents the pros and cons concerning advertising for recruitment using three modes of analyses - economics, ethics, and law. The author concludes that advertising is an invaluable technique for information dispersal in higher education. (Author/PG)

  14. Heparan sulphate as a regulator of leukocyte recruitment in inflammation.

    PubMed

    Kumar, Archana V; Katakam, Sampath K; Urbanowitz, Ann-Kathrin; Gotte, Martin

    2015-01-01

    A key event in inflammatory disease is the transendothelial recruitment of leukocytes from the circulation to the site of inflammation. Intense research in the past decades indicates that the polyanionic carbohydrate heparan sulphate (HS) modulates multiple steps in the leukocyte recruitment cascade. Leukocyte recruitment is initiated by endothelial cell activation and presentation of chemokines to rolling leukocytes, which, via integrin activation, results in adhesion and diapedesis through the vessel wall. Heparan sulfate proteoglycans (HSPGs) immobilize the chemokines on the luminal endothelial cells, rendering them more robust against mechanical or hydrodynamic perturbations. During inflammation, endothelial HSPGs serve as ligands to L-selectin on leukocytes, transport chemokines in a basolateral to apical direction across the endothelium, and present chemokines at the luminal surface of the endothelium to circulating cells. HSPGs also promote chemokine oligomerization, which influences chemokine receptor signaling. Furthermore, proteoglycans of the syndecan family are involved in modulating integrin-mediated tight adhesion of leukocytes to the endothelium. Creation of a chemokine gradient by a localized chemokine release influences the speed of leukocyte recruitment from the blood to the tissue by attracting crawling neutrophils to optimal sites for transmigration. The directionality of intraluminal crawling is thought to be influenced by both mechanotactic and haptotactic signals, which are modulated by HS-dependent signaling processes. Finally, diapedesis is influenced by HS regarding transendothelial chemokine gradient formation and integrin- CAM interactions, and further enhanced by heparanase-mediated degradation of the endothelial basement membrane. Overall, the multifunctional role of HS in inflammation marks it as a potential target of glycan-centered therapeutic approaches.

  15. Human plasma kallikrein releases neutrophil elastase during blood coagulation.

    PubMed Central

    Wachtfogel, Y T; Kucich, U; James, H L; Scott, C F; Schapira, M; Zimmerman, M; Cohen, A B; Colman, R W

    1983-01-01

    Elastase is released from human neutrophils during the early events of blood coagulation. Human plasma kallikrein has been shown to stimulate neutrophil chemotaxis, aggregation, and oxygen consumption. Therefore, the ability of kallikrein to release neutrophil elastase was investigated. Neutrophils were isolated by dextran sedimentation, and elastase release was measured by both an enzyme-linked immunosorbent assay, and an enzymatic assay using t-butoxy-carbonyl-Ala-Ala-Pro-Val-amino methyl coumarin as the substrate. Kallikrein, 0.1-1.0 U/ml, (0.045-0.45 microM), was incubated with neutrophils that were preincubated with cytochalasin B (5 micrograms/ml). The release of elastase was found to be proportional to the kallikrein concentration. Kallikrein released a maximum of 34% of the total elastase content, as measured by solubilizing the neutrophils in the nonionic detergent Triton X-100. A series of experiments was carried out to determine if kallikrein was a major enzyme involved in neutrophil elastase release during blood coagulation. When 10 million neutrophils were incubated in 1 ml of normal plasma in the presence of 30 mM CaCl2 for 90 min, 2.75 micrograms of elastase was released. In contrast, neutrophils incubated in prekallikrein-deficient or Factor XII-deficient plasma released less than half of the elastase, as compared with normal plasma. The addition of purified prekallikrein to prekallikrein-deficient plasma restored neutrophil elastase release to normal levels. Moreover, release of elastase was enhanced in plasma deficient in C1-inhibitor, the major plasma inhibitor of kallikrein. This release was not dependent upon further steps in the coagulation pathway, or on C5a, since levels of elastase, released in Factor XI- or C5-deficient plasma, were similar to that in normal plasma, and an antibody to C5 failed to inhibit elastase release. These data suggest that kallikrein may be a major enzyme responsible for the release of elastase during blood

  16. Human resistin promotes neutrophil proinflammatory activation and neutrophil extracellular trap formation and increases severity of acute lung injury.

    PubMed

    Jiang, Shaoning; Park, Dae Won; Tadie, Jean-Marc; Gregoire, Murielle; Deshane, Jessy; Pittet, Jean Francois; Abraham, Edward; Zmijewski, Jaroslaw W

    2014-05-15

    Although resistin was recently found to modulate insulin resistance in preclinical models of type II diabetes and obesity, recent studies also suggested that resistin has proinflammatory properties. We examined whether the human-specific variant of resistin affects neutrophil activation and the severity of LPS-induced acute lung injury. Because human and mouse resistin have distinct patterns of tissue distribution, experiments were performed using humanized resistin mice that exclusively express human resistin (hRTN(+/-)(/-)) but are deficient in mouse resistin. Enhanced production of TNF-α or MIP-2 was found in LPS-treated hRtn(+/-/-) neutrophils compared with control Rtn(-/-/-) neutrophils. Expression of human resistin inhibited the activation of AMP-activated protein kinase, a major sensor and regulator of cellular bioenergetics that also is implicated in inhibiting inflammatory activity of neutrophils and macrophages. In addition to the ability of resistin to sensitize neutrophils to LPS stimulation, human resistin enhanced neutrophil extracellular trap formation. In LPS-induced acute lung injury, humanized resistin mice demonstrated enhanced production of proinflammatory cytokines, more severe pulmonary edema, increased neutrophil extracellular trap formation, and elevated concentration of the alarmins HMGB1 and histone 3 in the lungs. Our results suggest that human resistin may play an important contributory role in enhancing TLR4-induced inflammatory responses, and it may be a target for future therapies aimed at reducing the severity of acute lung injury and other inflammatory situations in which neutrophils play a major role.

  17. Recruiting Students into Nursing: Prior Questions.

    ERIC Educational Resources Information Center

    Hipps, Opal S.

    1983-01-01

    Raises fundamental questions regarding student recruitment: (1) why recruit students into nursing? (2) what are the issues that determine whether a school should have a nursing program? and (3) what are students being recruited into? (JOW)

  18. Eosinophils versus Neutrophils in Host Defense

    PubMed Central

    Bass, David A.; Szejda, Pamela

    1979-01-01

    Eosinophil leukocytes have been reported to have a major role in host defense against invasive, migratory phases of helminth infestations, yet the relative larvicidal abilities of eosinophils and neutrophils have not been thoroughly examined. This study examined the killing of newborn (migratory phase) larvae of Trichinella spiralis during incubation by human granulocytes in vitro. The assay employed cultue of larvae with cells, sera, and reagents in microtiter wells with direct counting of surviving larvae after incubation. Killed larvae appeared to be lysed. Verification of the microplate assay was obtained by demonstrating complete loss of infectivity of larvae incubated with leukocytes and immune serum. In the presence of optimal immune serum concentrations, purified neutrophils or eosinophils achieved ≥95% killing of larvae at cell:larva ratios of 2,000:1 or greater. Fresh normal serum prompted slight (19%) killing by leukocytes at a cell:larva ratio of 9,000:1. Cells plus heat-inactivated normal serum and all sera preparations in the absence of leukocytes killed <8% of the larvae. The activity of immune serum was opsonic. Cells adhered to larvae that had been preincubated in immune serum, and immunofluorescent studies indicated that such preopsonized larvae were coated with immunoglobulin (Ig)G. However, preopsonized larvae lost opsonic activity and surface IgG during incubation for 3 h in medium lacking immune serum. The rate of killing was dependent on the cell:larva ratio; at high leukocyte concentrations (4,200:1), 99% were killed within 7 h; at lower cell:larva ratios, killing increased steadily during a 20-h incubation period. Killing was inhibited by 20 μg catalase, 5 μg/ml cytochalasin B, or 5μM colchicine, but was unchanged by superoxide dismutase and was enhanced by azide or cyanide. Leukocytes from a patient with chronic granulomatous disease, lacking ability to mount a normal oxidative response, demonstrated a markedly suppressed larvicidal

  19. Polymorphonuclear neutrophil function in systemic sclerosis.

    PubMed Central

    Czirják, L; Dankó, K; Sipka, S; Zeher, M; Szegedi, G

    1987-01-01

    In vitro functions of polymorphonuclear (PMN) neutrophils were studied in 20 patients with progressive systemic sclerosis (PSS). An increase in the basal chemiluminescence (CL) activity of peripheral blood PMNs was found, suggesting that these cells had been preactivated in vivo. Patients with more extensive skin disease or signs of disease progression tended to have higher basal CL values. Active oxygen products during the respiratory burst may increase the extent of inflammatory and fibrotic processes and could be involved in the endothelial injury in PSS. The stimulatory capacity of CL response was normal in our study. No alterations were found in the opsonised yeast phagocytic activity of granulocytes when compared with control values. The binding of erythrocyte-antibody particles was found also to be normal. A depressed chemotactic activity of PMN cells against zymosan activated serum was also shown. The cause of the decreased chemotaxis of PMNs remains to be elucidated. PMID:3592786

  20. Platelet-neutrophil interactions during hemodialysis: a proposed biocompatibility approach.

    PubMed

    Stuard, S; Bonomini, M; Settefrati, N; Albertazzi, A

    1998-02-01

    Platelet interaction with neutrophils may occur to a significant degree during hemodialysis (HD). We have recently shown that the enhanced neutrophil reactive oxygen species (ROS) production during the early phase of HD with cuprophan (CUP) is sustained by neutrophils which have bound platelets through P-selectin (CD62P). The evaluation of platelet-neutrophil interactions during dialysis offers the novel aspect of cell-cell interactions as a new parameter for studying the biocompatibility of dialyzer membranes. By the use of flow cytometry techniques, the present study was set up to analyze intradialytic platelet-neutrophil coaggregate formation and neutrophil ROS (hydrogen peroxide) production from 6 HD patients each dialysed with CUP, cellulose diacetate (CDA), polymethylmethacrylate (PMMA), and polyacrylonitrile (PAN) in a cross-over clinical trial. Platelet-neutrophil coaggregate formation (percentage of neutrophil cells positive for CD62P) and ROS production by neutrophils (total population; CD62P+ cells; CD62P- cells) were determined before HD and after 10', 20'and 40'. CD62P+ neutrophils significantly increased during HD with CUP (10', 20', 40'), PMMA (20') and CDA (20), while no change was observed with PAN. The difference between CUP and the other membranes was significant at 10', 20' and 40'; at 20', PMMA vs PAN p<0.005. ROS production by total neutrophil population significantly increased with CUP (10', 20), PMMA (20) and CDA (20'). The increase with CUP was higher at 10' when compared to CDA (p<0.020) or PAN (p<0.005), and at 20' versus the other three membranes; at 20' PMMA vs PAN p<0.005. Only neutrophils gated in neutrophil-platelet coaggregate areas (CD26P+ neutrophils) produced hydrogen peroxide. ROS production by CD62P+ neutrophils significantly increased with CUP (10', 20), PMMA (20') and CDA (20'). The increase with CUP was significantly (p<0.0002) higher than the other three membranes at 10' and 20'; at 20', PMMA vs PAN p<0.02. With each

  1. Human intravenous immunoglobulin (IVIG) preparations degranulate human neutrophils in vitro.

    PubMed

    Teeling, J L; De Groot, E R; Eerenberg, A J; Bleeker, W K; Van Mierlo, G; Aarden, L A; Hack, C E

    1998-11-01

    IVIG preparations have biological effects in vivo that are not fully understood. Possible effects include the property to stimulate Fc receptors on various cell types. To study whether IVIG may interact with neutrophils we developed an in vitro system, in which neutrophils, in whole blood or purified, were incubated with IVIG and assessed for degranulation by measuring the release of elastase and lactoferrin in culture medium. All commercially available IVIG preparations tested induced degranulation of neutrophils when incubated for 2 h at therapeutically relevant concentrations. In studies with blocking antibodies against Fc receptors (FcR), this degranulation was shown to be dependent on Fc gammaRII, whereas Fc gammaRIII had no effect. Experiments with purified neutrophils as well as binding experiments with labelled IVIG preparations indicated that neutrophil degranulation resulted from a direct interaction of IVIG with neutrophils. Using gel filtration fractions, it was found that polymeric and dimeric IgG present in IVIG was mainly responsible for the degranulation. We suggest that degranulation of neutrophils may contribute to the (side)effects of IVIG treatment in vivo.

  2. Quantitative assessment of neutrophil phagocytosis using flow cytometry.

    PubMed

    Nordenfelt, Pontus

    2014-01-01

    Neutrophils have an incredible ability to find and eradicate intruders such as bacteria and fungi. They do this largely through the process of phagocytosis, where the target is internalized into a phagosome, and eventually destroyed by the hostile phagosomal environment. It is important to study phagocytosis in order to understand how neutrophils interact with various pathogens and how they respond to different stimuli. Here, I describe a method to study neutrophil phagocytosis of bacteria using flow cytometry. The bacteria are fluorescently labeled before being introduced to neutrophils. After phagocytosis, both any remaining extracellular bacteria and neutrophils are labeled using one-step staining before three-color analysis. To assess phagocytosis, first the average time it takes for the neutrophils to internalize all bound bacteria is determined. Experiments are then performed using that time point while varying the bacteria-to-neutrophil ratio for full control of the analysis. Due to the ease with which multiple samples can be analyzed, and the quantitative nature of flow cytometry, this approach is both reproducible and sensitive.

  3. Constitutive apoptosis in equine peripheral blood neutrophils in vitro

    PubMed Central

    Brazil, Timothy J.; Dixon, Padraic M.; Haslett, Christopher; Murray, Joanna; McGorum, Bruce C.

    2014-01-01

    The aim of this study was to characterise constitutive apoptosis in equine peripheral blood neutrophils, including assessment of factors that potentially modulate neutrophil survival through alteration of the rate of constitutive apoptosis. Cells underwent spontaneous time-dependent constitutive apoptosis when aged in culture for up to 36 h, developing the structural and functional features of apoptosis observed in many cell types, including human neutrophils. Neutrophils undergoing apoptosis also had diminished zymosan activated serum (ZAS)-stimulated chemiluminescence, but maintained responsiveness to phorbol myristate acetate (PMA). The constitutive rate of equine neutrophil apoptosis was promoted by lipopolysaccharide (LPS), tumour necrosis factor α and phagocytosis of opsonised ovine erythrocytes, while it was inhibited by dexamethasone and ZAS (a source of C5a). Formyl-Met-Leu-Phe, leukotriene B4, platelet activating factor and PMA had no demonstrable effect on equine neutrophil apoptosis. There was a difference between equine and human neutrophil apoptosis in response to LPS and the time-dependence of the response to dexamethasone. PMID:25239298

  4. Neutrophil function in an experimental model of hemolytic uremic syndrome.

    PubMed

    Vedanarayanan, V V; Kaplan, B S; Fong, J S

    1987-03-01

    To understand the role of neutrophil leukocytosis in hemolytic uremic syndrome, we studied the changes in neutrophil function in the modified generalized Shwartzman reaction in rabbits. This model resembles hemolytic uremic syndrome associated with endotoxemia. At the end of an endotoxin infusion, we observed leukopenia, thrombocytopenia, and a decrease in hematocrit associated with schistocytosis. Plasma B-glucuronidase levels increased and this was associated with a decrease in neutrophil content of the enzyme. The chemotactic index and neutrophil aggregation to zymosan-activated serum were impaired compared to controls. The neutrophil procoagulant content increased after endotoxin infusion. The serum creatinine concentration and proteinuria increased in the endotoxin-treated animals. The changes returned to normal by 48 h. Renal cortical malondialdehyde, a reflection of lipid peroxidation, was higher in the endotoxin-treated animals than in the controls. We have shown enzyme release by neutrophils, impairment of chemotaxis and aggregation, increased procoagulant content in neutrophils, and evidence of lipid peroxidation in renal cortical tissue in this model. These observations raise the possibility that leukocytes may have a role in the pathogenesis of the hemolytic uremic syndrome. PMID:3550673

  5. Mycobacterium tuberculosis- induced neutrophil extracellular traps activate human macrophages.

    PubMed

    Braian, Clara; Hogea, Valentin; Stendahl, Olle

    2013-01-01

    Neutrophils activated by Mycobacterium tuberculosis (Mtb) form neutrophil extracellular traps (NETs), containing DNA and several biologically active cytosolic and granular proteins. These NETs may assist in the innate immune defense against different pathogens. We investigated whether the NET-forming neutrophils mediate an activating signal to macrophages during the early multicellular inflammatory reaction and granuloma formation. Mtb-induced NETs were found to be reactive oxygen species dependent and phagocytosis dependent. A neutrophil elastase inhibitor also delayed NET formation. However, NET formation occurred independently of Mtb-induced apoptosis. We observed close interactions between macrophages and Mtb-activated neutrophils, where macrophages bound and phagocytosed NETs. Significant secretion of the cytokines interleukin (IL)-6, tumor necrosis factor-α, IL-1β and IL-10 were detected from macrophages cocultured with NETs from Mtb-activated but not phorbol myristate acetate-activated neutrophils. NETs binding heat shock protein 72 (Hsp72) or recombinant Hsp72 were able to trigger cytokine release from macrophages. Only Mtb-induced NETs contained Hsp72, suggesting that these NETs can transfer this danger signal to adjacent macrophages. We propose that Hsp72 sequestered in NETs plays an important role in the interaction between neutrophils and macrophages during the early innate immune phase of an Mtb infection. The immunomodulatory role of NETs and proteins derived from them may influence not only chronic inflammation during tuberculosis but also immune regulation and autoimmunity.

  6. Granulocytes in Ocular HSV-1 Infection: Opposing Roles of Mast Cells and Neutrophils

    PubMed Central

    Royer, Derek J.; Zheng, Min; Conrady, Christopher D.; Carr, Daniel J. J.

    2015-01-01

    Purpose. The contributions of mast cells (MCs) to immunologic defense against pathogens in the eye are unknown. We have characterized pericorneal MCs as tissue-resident innate sentinels and determined their impact on the immune response to herpes simplex virus type-1 (HSV-1), a common ocular pathogen. Methods. The impact of mast cells on the immune response to HSV-1 infection was investigated using MC-deficient KitW-sh mice. Virus titers, inflammatory cytokine production, eicosanoid profiles, cellular immune responses, and ocular pathology were evaluated and compared with C57BL/6J mice during an acute corneal HSV-1 infection. Results. Corneas of KitW-sh mice have higher viral titers, increased edema, and greater leukocyte infiltration following HSV-1 infection. Following infection, cytokine profiles were slightly elevated overall in KitW-sh mice. Eicosanoid profiles were remarkably different only when comparing uninfected corneas from both groups. Neutrophils within infected corneas expressed HSV-1 antigen, lytic genes, and served as a disease-causing vector when adoptively transferred into immunocompromised animals. Myeloid-derived suppressor cells did not infiltrate into the cornea or suppress the expansion, recruitment, or cytokine production by CD8+ T cells following acute HSV-1 infection. Conclusions. Collectively, these findings provide new insight into host defense in the cornea and the pathogenesis of HSV-1 infection by identifying previously unacknowledged MCs as protective innate sentinels for infection of the ocular surface and reinforcing that neutrophils are detrimental to corneal infection. PMID:26066745

  7. Neutrophil Extracellular Trap-Related Extracellular Histones Cause Vascular Necrosis in Severe GN.

    PubMed

    Kumar, Santhosh V R; Kulkarni, Onkar P; Mulay, Shrikant R; Darisipudi, Murthy N; Romoli, Simone; Thomasova, Dana; Scherbaum, Christina R; Hohenstein, Bernd; Hugo, Christian; Müller, Susanna; Liapis, Helen; Anders, Hans-Joachim

    2015-10-01

    Severe GN involves local neutrophil extracellular trap (NET) formation. We hypothesized a local cytotoxic effect of NET-related histone release in necrotizing GN. In vitro, histones from calf thymus or histones released by neutrophils undergoing NETosis killed glomerular endothelial cells, podocytes, and parietal epithelial cells in a dose-dependent manner. Histone-neutralizing agents such as antihistone IgG, activated protein C, or heparin prevented this effect. Histone toxicity on glomeruli ex vivo was Toll-like receptor 2/4 dependent, and lack of TLR2/4 attenuated histone-induced renal thrombotic microangiopathy and glomerular necrosis in mice. Anti-glomerular basement membrane GN involved NET formation and vascular necrosis, whereas blocking NET formation by peptidylarginine inhibition or preemptive anti-histone IgG injection significantly reduced all aspects of GN (i.e., vascular necrosis, podocyte loss, albuminuria, cytokine induction, recruitment or activation of glomerular leukocytes, and glomerular crescent formation). To evaluate histones as a therapeutic target, mice with established GN were treated with three different histone-neutralizing agents. Anti-histone IgG, recombinant activated protein C, and heparin were equally effective in abrogating severe GN, whereas combination therapy had no additive effects. Together, these results indicate that NET-related histone release during GN elicits cytotoxic and immunostimulatory effects. Furthermore, neutralizing extracellular histones is still therapeutic when initiated in established GN.

  8. Neutrophil Extracellular Trap-Related Extracellular Histones Cause Vascular Necrosis in Severe GN

    PubMed Central

    Kumar, Santhosh V.R.; Kulkarni, Onkar P.; Mulay, Shrikant R.; Darisipudi, Murthy N.; Romoli, Simone; Thomasova, Dana; Scherbaum, Christina R.; Hohenstein, Bernd; Hugo, Christian; Müller, Susanna; Liapis, Helen

    2015-01-01

    Severe GN involves local neutrophil extracellular trap (NET) formation. We hypothesized a local cytotoxic effect of NET-related histone release in necrotizing GN. In vitro, histones from calf thymus or histones released by neutrophils undergoing NETosis killed glomerular endothelial cells, podocytes, and parietal epithelial cells in a dose-dependent manner. Histone-neutralizing agents such as antihistone IgG, activated protein C, or heparin prevented this effect. Histone toxicity on glomeruli ex vivo was Toll-like receptor 2/4 dependent, and lack of TLR2/4 attenuated histone-induced renal thrombotic microangiopathy and glomerular necrosis in mice. Anti–glomerular basement membrane GN involved NET formation and vascular necrosis, whereas blocking NET formation by peptidylarginine inhibition or preemptive anti-histone IgG injection significantly reduced all aspects of GN (i.e., vascular necrosis, podocyte loss, albuminuria, cytokine induction, recruitment or activation of glomerular leukocytes, and glomerular crescent formation). To evaluate histones as a therapeutic target, mice with established GN were treated with three different histone-neutralizing agents. Anti-histone IgG, recombinant activated protein C, and heparin were equally effective in abrogating severe GN, whereas combination therapy had no additive effects. Together, these results indicate that NET-related histone release during GN elicits cytotoxic and immunostimulatory effects. Furthermore, neutralizing extracellular histones is still therapeutic when initiated in established GN. PMID:25644111

  9. Spatial synchrony in cisco recruitment

    USGS Publications Warehouse

    Myers, Jared T.; Yule, Daniel L.; Jones, Michael L.; Ahrenstorff, Tyler D.; Hrabik, Thomas R.; Claramunt, Randall M.; Ebener, Mark P.; Berglund, Eric K.

    2015-01-01

    We examined the spatial scale of recruitment variability for disparate cisco (Coregonus artedi) populations in the Great Lakes (n = 8) and Minnesota inland lakes (n = 4). We found that the scale of synchrony was approximately 400 km when all available data were utilized; much greater than the 50-km scale suggested for freshwater fish populations in an earlier global analysis. The presence of recruitment synchrony between Great Lakes and inland lake cisco populations supports the hypothesis that synchronicity is driven by climate and not dispersal. We also found synchrony in larval densities among three Lake Superior populations separated by 25–275 km, which further supports the hypothesis that broad-scale climatic factors are the cause of spatial synchrony. Among several candidate climate variables measured during the period of larval cisco emergence, maximum wind speeds exhibited the most similar spatial scale of synchrony to that observed for cisco. Other factors, such as average water temperatures, exhibited synchrony on broader spatial scales, which suggests they could also be contributing to recruitment synchrony. Our results provide evidence that abiotic factors can induce synchronous patterns of recruitment for populations of cisco inhabiting waters across a broad geographic range, and show that broad-scale synchrony of recruitment can occur in freshwater fish populations as well as those from marine systems.

  10. Heterogeneity in Neutrophil Microparticles Reveals Distinct Proteome and Functional Properties*

    PubMed Central

    Dalli, Jesmond; Montero-Melendez, Trinidad; Norling, Lucy V; Yin, Xiaoke; Hinds, Charles; Haskard, Dorian; Mayr, Manuel; Perretti, Mauro

    2013-01-01

    Altered plasma neutrophil microparticle levels have recently been implicated in a number of vascular and inflammatory diseases, yet our understanding of their actions is very limited. Herein, we investigate the proteome of neutrophil microparticles in order to shed light on their biological actions. Stimulation of human neutrophils, either in suspension or adherent to an endothelial monolayer, led to the production of microparticles containing >400 distinct proteins with only 223 being shared by the two subsets. For instance, postadherent microparticles were enriched in alpha-2 macroglobulin and ceruloplasmin, whereas microparticles produced by neutrophils in suspension were abundant in heat shock 70 kDa protein 1. Annexin A1 and lactotransferrin were expressed in both microparticle subsets. We next determined relative abundance of these proteins in three types of human microparticle samples: healthy volunteer plasma, plasma of septic patients and skin blister exudates finding that these proteins were differentially expressed on neutrophil microparticles from these samples reflecting in part the expression profiles we found in vitro. Functional assessment of the neutrophil microparticles subsets demonstrated that in response to direct stimulation neutrophil microparticles produced reactive oxygen species and leukotriene B4 as well as locomoted toward a chemotactic gradient. Finally, we investigated the actions of the two neutrophil microparticles subsets described herein on target cell responses. Microarray analysis with human primary endothelial cells incubated with either microparticle subset revealed a discrete modulation of endothelial cell gene expression profile. These findings demonstrate that neutrophil microparticles are heterogenous and can deliver packaged information propagating the activation status of the parent cell, potentially exerting novel and fundamental roles both under homeostatic and disease conditions. PMID:23660474

  11. Neutrophil adhesion and chemotaxis depend on substrate mechanics

    NASA Astrophysics Data System (ADS)

    Jannat, Risat A.; Robbins, Gregory P.; Ricart, Brendon G.; Dembo, Micah; Hammer, Daniel A.

    2010-05-01

    Neutrophil adhesion to the vasculature and chemotaxis within tissues play critical roles in the inflammatory response to injury and pathogens. Unregulated neutrophil activity has been implicated in the progression of numerous chronic and acute diseases such as rheumatoid arthritis, asthma and sepsis. Cell migration of anchorage-dependent cells is known to depend on both chemical and mechanical interactions. Although neutrophil responses to chemical cues have been well characterized, little is known about the effect of underlying tissue mechanics on neutrophil adhesion and migration. To address this question, we quantified neutrophil migration and traction stresses on compliant hydrogel substrates with varying elasticity in a micromachined gradient chamber in which we could apply either a uniform concentration or a precise gradient of the bacterial chemoattractant fMLP. Neutrophils spread more extensively on substrates of greater stiffness. In addition, increasing the stiffness of the substrate leads to a significant increase in the chemotactic index for each fMLP gradient tested. As the substrate becomes stiffer, neutrophils generate higher traction forces without significant changes in cell speed. These forces are often displayed in pairs and focused in the uropod. Increases in the mean fMLP concentration beyond the KD of the receptor lead to a decrease in chemotactic index on all surfaces. Blocking with an antibody against β2-integrins leads to a significant reduction, but not an elimination, of directed motility on stiff materials, but no change in motility on soft materials, suggesting neutrophils can display both integrin-dependent and integrin-independent motility. These findings are critical for understanding how neutrophil migration may change in different mechanical environments in vivo and can be used to guide the design of migration inhibitors that more efficiently target inflammation.

  12. Neutrophil adhesion and chemotaxis depend on substrate mechanics

    PubMed Central

    Jannat, Risat A.; Dembo, Micah; Hammer, Daniel A.

    2009-01-01

    Neutrophil adhesion to the vasculature and chemotaxis within tissues play critical roles in the inflammatory response to injury and pathogens. Unregulated neutrophil activity has been implicated in the progression of numerous chronic and acute diseases such as rheumatoid arthritis, asthma, and sepsis. Cell migration of anchorage-dependent cells is known to depend on both chemical and mechanical interactions. Although neutrophil responses to chemical cues have been well characterized, little is known about the effect of underlying tissue mechanics on neutrophil adhesion and migration. To address this question, we quantified neutrophil migration and traction stresses on compliant hydrogel substrates with varying elasticity in a micro-machined gradient chamber in which we could apply either a uniform concentration or a precise gradient of the bacterial chemoattractant fMLP. Neutrophils spread more extensively on substrates of greater stiffness. In addition, increasing the stiffness of the substrate leads to a significant increase in the chemotactic index for each fMLP gradient tested. As the substrate becomes stiffer, neutrophils generate higher traction forces without significant changes in cell speed. These forces are often displayed in pairs and focused in the uropod. Increases in the mean fMLP concentration beyond the KD of the receptor lead to a decrease in chemotactic index on all surfaces. Blocking with an antibody against β2-integrins leads to a significant reduction but not an elimination of directed motility on stiff materials, but no change in motility on soft materials, suggesting neutrophils can display both integrin-dependent and integrin-independent motility. These findings are critical for understanding how neutrophil migration may change in different mechanical environments in vivo and can be used to guide the design of migration inhibitors that more efficiently target inflammation. PMID:20473350

  13. Neutrophil localization in acute and chronic experimental abscesses

    SciTech Connect

    Bamberger, D.M.; Bettin, K.M.; Gerding, D.N.

    1987-04-01

    Abdominal abscesses are associated with a high mortality, and usually require surgical drainage for cure. A potential mechanism explaining the inability of the host to clear this infection may be in part a result of the inability of the neutrophil to localize at the site of an established infection. To study this question, either acute (4 hours old) or chronic (2 weeks old) abscesses caused by Staphylococcus aureus were created in perforated capsules implanted in the peritoneal cavity of rabbits. Homologous neutrophils were obtained from donor rabbits 4 hours after peritoneal glycogen stimulation and labeled with indium 111 oxine. Only 0.71% of injected /sup 111/In-labeled neutrophils localized in the chronic abscesses, compared with 1.77% in acute abscesses (P less than or equal to 0.01). Animals with chronic infections had a lower intravascular recovery of injected neutrophils (P less than 0.002). Failure of neutrophil localization was not associated with less chemotactic activity within the abscess, as measured by a chemotaxis-under-agarose assay, or caused by a barrier surrounding the abscess as detected by radionuclide imaging. Only 0.07% of injected neutrophils localized into acute abdominal abscesses in animals with a concomitant chronic subcutaneous abscess. These chronically infected animals also demonstrated a low peak intravascular recovery of injected neutrophils when compared with animals with only an acute infection (P less than 0.002). These data reveal that neutrophils localize to abscesses poorly in animals with chronic infections. The mechanism is possibly related to a systemic factor(s) associated with a lower intravascular recovery of injected neutrophils in chronically infected animals.

  14. Neutrophil Integrins and Matrix Ligands and NET Release

    PubMed Central

    O’Brien, Xian M.; Reichner, Jonathan S.

    2016-01-01

    Neutrophils are motile and responsive to tissue injury and infection. As neutrophils emigrate from the bloodstream and migrate toward a site of affliction, they encounter the tissue extracellular matrix (ECM) and thereby engage integrins. Our laboratory studies the neutrophilic response to the fungal pathogen Candida albicans either in the filamentous state of the microbe or to the purified pathogen-associated molecular pattern, β-glucan. We have gained an appreciation for the role of integrins in regulating the neutrophil anti-Candida response and how the presence or absence of ECM can drive experimental outcome. The β2 integrin CR3 (complement receptor 3; αMβ2; Mac-1; CD11b/CD18) plays an important role in fungal recognition by its ability to bind β-glucan at a unique lectin-like domain. The presence of ECM differentially regulates essential neutrophil anti-fungal functions, including chemotaxis, respiratory burst, homotypic aggregation, and the release of neutrophil extracellular traps (NETs). We have shown that NET release to C. albicans hyphae or immobilized β-glucan occurs rapidly and without the requirement for respiratory burst on ECM. This is in contrast to the more frequently reported mechanisms of NETosis to other pathogens without the context of ECM, which occur after a prolonged lag period and require respiratory burst. As expected for an ECM-dependent phenotype, NETosis and other neutrophil functions are dependent on specific integrins. The focus of this review is the role of ECM ligation by neutrophil integrins as it pertains to host defense functions with an emphasis on lessons we have learned studying the anti-Candida response of human neutrophils.

  15. Neutrophil Integrins and Matrix Ligands and NET Release

    PubMed Central

    O’Brien, Xian M.; Reichner, Jonathan S.

    2016-01-01

    Neutrophils are motile and responsive to tissue injury and infection. As neutrophils emigrate from the bloodstream and migrate toward a site of affliction, they encounter the tissue extracellular matrix (ECM) and thereby engage integrins. Our laboratory studies the neutrophilic response to the fungal pathogen Candida albicans either in the filamentous state of the microbe or to the purified pathogen-associated molecular pattern, β-glucan. We have gained an appreciation for the role of integrins in regulating the neutrophil anti-Candida response and how the presence or absence of ECM can drive experimental outcome. The β2 integrin CR3 (complement receptor 3; αMβ2; Mac-1; CD11b/CD18) plays an important role in fungal recognition by its ability to bind β-glucan at a unique lectin-like domain. The presence of ECM differentially regulates essential neutrophil anti-fungal functions, including chemotaxis, respiratory burst, homotypic aggregation, and the release of neutrophil extracellular traps (NETs). We have shown that NET release to C. albicans hyphae or immobilized β-glucan occurs rapidly and without the requirement for respiratory burst on ECM. This is in contrast to the more frequently reported mechanisms of NETosis to other pathogens without the context of ECM, which occur after a prolonged lag period and require respiratory burst. As expected for an ECM-dependent phenotype, NETosis and other neutrophil functions are dependent on specific integrins. The focus of this review is the role of ECM ligation by neutrophil integrins as it pertains to host defense functions with an emphasis on lessons we have learned studying the anti-Candida response of human neutrophils. PMID:27698655

  16. Neutrophils Oppose Uterine Epithelial Carcinogenesis via Debridement of Hypoxic Tumor Cells.

    PubMed

    Blaisdell, Adam; Crequer, Amandine; Columbus, Devin; Daikoku, Takiko; Mittal, Khush; Dey, Sudhansu K; Erlebacher, Adrian

    2015-12-14

    Polymorphonuclear neutrophils (PMNs) are largely considered to foster cancer development despite wielding an arsenal of cytotoxic agents. Using a mouse model of PTEN-deficient uterine cancer, we describe a surprising inhibitory role for PMNs in epithelial carcinogenesis. By inducing tumor cell detachment from the basement membrane, PMNs impeded early-stage tumor growth and retarded malignant progression. Unexpectedly, PMN recruitment and tumor growth control occurred independently of lymphocytes and cellular senescence and instead ensued as part of the tumor's intrinsic inflammatory response to hypoxia. In humans, a PMN gene signature correlated with improved survival in several cancer subtypes, including PTEN-deficient uterine cancer. These findings provide insight into tumor-associated PMNs and reveal a context-specific capacity for PMNs to directly combat tumorigenesis.

  17. Cytokine-induced neutrophil-derived interleukin-8.

    PubMed Central

    Strieter, R. M.; Kasahara, K.; Allen, R. M.; Standiford, T. J.; Rolfe, M. W.; Becker, F. S.; Chensue, S. W.; Kunkel, S. L.

    1992-01-01

    During acute inflammation, the first line of cellular response for host defense is the neutrophil. In addition to the historic role of the neutrophil as a phagocyte, recent studies have identified this cell as an important source of a number of cytokines. In this study, we provide evidence that the neutrophil is a significant source of interleukin-8 (IL-8). Neutrophils freshly isolated from whole blood were not found to constitutively express IL-8 mRNA. In contrast, when these leukocytes were cultured on plastic they were activated, leading to the significant expression of de novo steady-state levels of IL-8 mRNA. In addition, when neutrophils were treated with cycloheximide, there was evidence for "superinduction" of steady-state levels of IL-8 mRNA and inhibition of antigenic IL-8 production. Neutrophils were subsequently stimulated with lipopolysaccharide (LPS), tumor necrosis factor-alpha, or interleukin-1-beta and were found to express IL-8 mRNA and antigen in both a time- and dose-dependent manner. Furthermore, neutrophils stimulated with traditional chemotactic/activating factors, such as the split product of the fifth component of complement (C5a), formylmethionyleucylphenylalanine (fMLP), and leukotriene B4 (LTB4) in a dose-dependent manner did not produce significant antigenic IL-8, as compared with unstimulated controls. In contrast, when neutrophils were exposed to either of these neutrophil agonists in the presence of LPS, the production of antigenic IL-8 was significantly elevated, as compared with either of the stimuli alone, suggesting a synergistic response. These data would suggest that the neutrophil can no longer be viewed as only a phagocyte or warehouse for proteolytic enzymes, but is a pivotal effector cell that is able to respond to mediators in its environment and generate cytokines. This latter neutrophil response may be important for either the elicitation of additional neutrophils or to orchestrate the conventional immune response at

  18. Activation of calpains mediates early lung neutrophilic inflammation in ventilator-induced lung injury

    PubMed Central

    Liu, Dejie; Yan, Zhibo; Minshall, Richard D.; Schwartz, David E.; Chen, Yuguo

    2012-01-01

    Lung inflammatory responses in the absence of infection are considered to be one of primary mechanisms of ventilator-induced lung injury. Here, we determined the role of calpain in the pathogenesis of lung inflammation attributable to mechanical ventilation. Male C57BL/6J mice were subjected to high (28 ml/kg) tidal volume ventilation for 2 h in the absence and presence of calpain inhibitor I (10 mg/kg). To address the isoform-specific functions of calpain 1 and calpain 2 during mechanical ventilation, we utilized a liposome-based delivery system to introduce small interfering RNAs targeting each isoform in pulmonary vasculature in vivo. Mechanical ventilation with high tidal volume induced rapid (within minutes) and persistent calpain activation and lung inflammation as evidenced by neutrophil recruitment, production of TNF-α and IL-6, pulmonary vascular hyperpermeability, and lung edema formation. Pharmaceutical calpain inhibition significantly attenuated these inflammatory responses caused by lung hyperinflation. Depletion of calpain 1 or calpain 2 had a protective effect against ventilator-induced lung inflammatory responses. Inhibition of calpain activity by means of siRNA silencing or pharmacological inhibition also reduced endothelial nitric oxide (NO) synthase (NOS-3)-mediated NO production and subsequent ICAM-1 phosphorylation following high tidal volume ventilation. These results suggest that calpain activation mediates early lung inflammation during ventilator-induced lung injury via NOS-3/NO-dependent ICAM-1 phosphorylation and neutrophil recruitment. Inhibition of calpain activation may therefore provide a novel and promising strategy for the prevention and treatment of ventilator-induced lung injury. PMID:22140070

  19. Anti-Inflammation of Natural Components from Medicinal Plants at Low Concentrations in Brain via Inhibiting Neutrophil Infiltration after Stroke

    PubMed Central

    Chen, Jiannan; Zhang, Cong; Chen, Rong; Jiao, Honglei; Li, Linlin; Zhang, Lan; Cui, Lili

    2016-01-01

    Inflammation after stroke consists of activation of microglia/astrocytes in situ and infiltration of blood-borne leukocytes, resulting in brain damage and neurological deficits. Mounting data demonstrated that most natural components from medicinal plants had anti-inflammatory effects after ischemic stroke through inhibiting activation of resident microglia/astrocytes within ischemic area. However, it is speculated that this classical activity cannot account for the anti-inflammatory function of these natural components in the cerebral parenchyma, where they are detected at very low concentrations due to their poor membrane permeability and slight leakage of BBB. Could these drugs exert anti-inflammatory effects peripherally without being delivered across the BBB? Factually, ameliorating blood-borne neutrophil recruitment in peripheral circulatory system has been proved to reduce ischemic damage and improve outcomes. Thus, it is concluded that if drugs could achieve effective concentrations in the cerebral parenchyma, they can function via crippling resident microglia/astrocytes activation and inhibiting neutrophil infiltration, whereas the latter will be dominating when these drugs localize in the brain at a low concentration. In this review, the availability of some natural components crossing the BBB in stroke will be discussed, and how these drugs lead to improvements in stroke through inhibition of neutrophil rolling, adhesion, and transmigration will be illustrated. PMID:27688603

  20. Anti-Inflammation of Natural Components from Medicinal Plants at Low Concentrations in Brain via Inhibiting Neutrophil Infiltration after Stroke

    PubMed Central

    Chen, Jiannan; Zhang, Cong; Chen, Rong; Jiao, Honglei; Li, Linlin; Zhang, Lan; Cui, Lili

    2016-01-01

    Inflammation after stroke consists of activation of microglia/astrocytes in situ and infiltration of blood-borne leukocytes, resulting in brain damage and neurological deficits. Mounting data demonstrated that most natural components from medicinal plants had anti-inflammatory effects after ischemic stroke through inhibiting activation of resident microglia/astrocytes within ischemic area. However, it is speculated that this classical activity cannot account for the anti-inflammatory function of these natural components in the cerebral parenchyma, where they are detected at very low concentrations due to their poor membrane permeability and slight leakage of BBB. Could these drugs exert anti-inflammatory effects peripherally without being delivered across the BBB? Factually, ameliorating blood-borne neutrophil recruitment in peripheral circulatory system has been proved to reduce ischemic damage and improve outcomes. Thus, it is concluded that if drugs could achieve effective concentrations in the cerebral parenchyma, they can function via crippling resident microglia/astrocytes activation and inhibiting neutrophil infiltration, whereas the latter will be dominating when these drugs localize in the brain at a low concentration. In this review, the availability of some natural components crossing the BBB in stroke will be discussed, and how these drugs lead to improvements in stroke through inhibition of neutrophil rolling, adhesion, and transmigration will be illustrated.

  1. Effects of Montelukast on free radical production in whole blood and isolated human polymorphonuclear neutrophils (PMNs) in asthmatic children

    PubMed Central

    Al Saadi, Muslim M.; Meo, Sultan Ayoub; Mustafa, Ali; Shafi, Ahmed; Tuwajri, Ali S. Al

    2011-01-01

    Montelukast is a highly selective leukotriene-receptor antagonist (LTRA). It is widely used in the treatment of bronchial asthma, primarily as an adjunct to corticosteroids. Reactive oxygen species (ROSs) play an important role in the pathogenesis of asthma and oxidative stress contributing to the initiation and worsening of inflammatory respiratory disorders, such as asthma. Antioxidant drugs may have a role in minimizing or preventing damage in asthmatic children. The aim of this study was to assess the antioxidant effect of montelukast on the production of free radicals in the whole blood and polymorphonuclear neutrophils (PMNs) in asthmatic children. A group of 48 (38 males and 10 females), apparently healthy asthmatic children were recruited with ages ranging between 6 and 14 years. In asthmatic children, base line (premedication) and post medication free radicals activity in the whole blood and polymorphonuclear neutrophils (PMNs) was determined by measuring chemiluminescence (CL) response through chemiluminescence luminometer. Free radical productions were significantly decreased in the whole blood, when stimulated with Phorbol Myristate Acetate (p < 0.04) and Opsonised Zymosan (p < 0.05). The free radicals were also significantly decreased in isolated polymorphonuclear neutrophils (PMNs) when stimulated with Opsonised Zymosan (p < 0.05) after the post medication treatment of montelukast in asthmatic children. Montelukast decreased the reactive oxygen species production, both in the whole blood as well as isolated PMNs in asthmatic children. PMID:23960762

  2. Direct observation of phagocytosis and NET-formation by neutrophils in infected lungs using 2-photon microscopy.

    PubMed

    Hasenberg, Mike; Köhler, Anja; Bonifatius, Susanne; Jeron, Andreas; Gunzer, Matthias

    2011-06-02

    After the gastrointestinal tract, the lung is the second largest surface for interaction between the vertebrate body and the environment. Here, an effective gas exchange must be maintained, while at the same time avoiding infection by the multiple pathogens that are inhaled during normal breathing. To achieve this, a superb set of defense strategies combining humoral and cellular immune mechanisms exists. One of the most effective measures for acute defense of the lung is the recruitment of neutrophils, which either phagocytose the inhaled pathogens or kill them by releasing cytotoxic chemicals. A recent addition to the arsenal of neutrophils is their explosive release of extracellular DNA-NETs by which bacteria or fungi can be caught or inactivated even after the NET releasing cells have died. We present here a method that allows one to directly observe neutrophils, migrating within a recently infected lung, phagocytosing fungal pathogens as well as visualize the extensive NETs that they have produced throughout the infected tissue. The method describes the preparation of thick viable lung slices 7 hours after intratracheal infection of mice with conidia of the mold Aspergillus fumigatus and their examination by multicolor time-lapse 2-photon microscopy. This approach allows one to directly investigate antifungal defense in native lung tissue and thus opens a new avenue for the detailed investigation of pulmonary immunity.

  3. 38 CFR 23.310 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2011-07-01 2011-07-01 false Recruitment. 23.310... Discrimination on the Basis of Sex in Admission and Recruitment Prohibited § 23.310 Recruitment. (a) Nondiscriminatory recruitment. A recipient to which §§ 23.300 through 23.310 apply shall not discriminate on...

  4. 29 CFR 36.510 - Recruitment.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 1 2010-07-01 2010-07-01 true Recruitment. 36.510 Section 36.510 Labor Office of the... Activities Prohibited § 36.510 Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and hiring of employees. Where a recipient...

  5. 18 CFR 1317.310 - Recruitment.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 2 2010-04-01 2010-04-01 false Recruitment. 1317.310... Discrimination on the Basis of Sex in Admission and Recruitment Prohibited § 1317.310 Recruitment. (a) Nondiscriminatory recruitment. A recipient to which §§ 1317.300 through 1317.310 apply shall not discriminate on...

  6. 7 CFR 15a.23 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 1 2011-01-01 2011-01-01 false Recruitment. 15a.23 Section 15a.23 Agriculture Office... FEDERAL FINANCIAL ASSISTANCE Discrimination on the Basis of Sex in Admission and Recruitment Prohibited § 15a.23 Recruitment. (a) Nondiscriminatory recruitment. A recipient to which this subpart applies...

  7. 18 CFR 1317.310 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 18 Conservation of Power and Water Resources 2 2011-04-01 2011-04-01 false Recruitment. 1317.310... Discrimination on the Basis of Sex in Admission and Recruitment Prohibited § 1317.310 Recruitment. (a) Nondiscriminatory recruitment. A recipient to which §§ 1317.300 through 1317.310 apply shall not discriminate on...

  8. 44 CFR 19.510 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 44 Emergency Management and Assistance 1 2011-10-01 2011-10-01 false Recruitment. 19.510 Section... Programs or Activities Prohibited § 19.510 Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and hiring of employees. Where...

  9. 7 CFR 15a.53 - Recruitment.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 7 Agriculture 1 2010-01-01 2010-01-01 false Recruitment. 15a.53 Section 15a.53 Agriculture Office... Activities Prohibited § 15a.53 Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and hiring of employees. Where a recipient...

  10. 34 CFR 106.53 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 34 Education 1 2011-07-01 2011-07-01 false Recruitment. 106.53 Section 106.53 Education... Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and hiring of employees. Where a recipient has been found to be...

  11. 45 CFR 2555.510 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 45 Public Welfare 4 2011-10-01 2011-10-01 false Recruitment. 2555.510 Section 2555.510 Public... Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and hiring of employees. Where a recipient has been found to be...

  12. 22 CFR 229.510 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 22 Foreign Relations 1 2011-04-01 2011-04-01 false Recruitment. 229.510 Section 229.510 Foreign... in Education Programs or Activities Prohibited § 229.510 Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and...

  13. 49 CFR 25.510 - Recruitment.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 49 Transportation 1 2010-10-01 2010-10-01 false Recruitment. 25.510 Section 25.510 Transportation... Education Programs or Activities Prohibited § 25.510 Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and hiring of...

  14. 34 CFR 106.53 - Recruitment.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 34 Education 1 2010-07-01 2010-07-01 false Recruitment. 106.53 Section 106.53 Education... Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and hiring of employees. Where a recipient has been found to be...

  15. 32 CFR 196.510 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 32 National Defense 2 2011-07-01 2011-07-01 false Recruitment. 196.510 Section 196.510 National... Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and hiring of employees. Where a recipient has been found to be...

  16. 41 CFR 101-4.510 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 41 Public Contracts and Property Management 2 2011-07-01 2007-07-01 true Recruitment. 101-4.510... Employment in Education Programs or Activities Prohibited § 101-4.510 Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and...

  17. 45 CFR 2555.510 - Recruitment.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 45 Public Welfare 4 2010-10-01 2010-10-01 false Recruitment. 2555.510 Section 2555.510 Public... Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and hiring of employees. Where a recipient has been found to be...

  18. 20 CFR 655.156 - Recruitment report.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 20 Employees' Benefits 3 2010-04-01 2010-04-01 false Recruitment report. 655.156 Section 655.156... the United States (H-2A Workers) Post-Acceptance Requirements § 655.156 Recruitment report. (a) Requirements of a recruitment report. The employer must prepare, sign, and date a written recruitment...

  19. 22 CFR 146.510 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 22 Foreign Relations 1 2011-04-01 2011-04-01 false Recruitment. 146.510 Section 146.510 Foreign... Education Programs or Activities Prohibited § 146.510 Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and hiring of...

  20. 45 CFR 2555.310 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 45 Public Welfare 4 2011-10-01 2011-10-01 false Recruitment. 2555.310 Section 2555.310 Public... Discrimination on the Basis of Sex in Admission and Recruitment Prohibited § 2555.310 Recruitment. (a) Nondiscriminatory recruitment. A recipient to which §§ 2555.300 through 2555.310 apply shall not discriminate on...

  1. 18 CFR 1317.510 - Recruitment.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 2 2010-04-01 2010-04-01 false Recruitment. 1317.510... Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and hiring of employees. Where a recipient has been found to be...

  2. 45 CFR 83.12 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 45 Public Welfare 1 2011-10-01 2011-10-01 false Recruitment. 83.12 Section 83.12 Public Welfare... § 83.12 Recruitment. (a) Comparable recruitment. A federally supported entity shall, with respect to... demonstrate that such action is part of a recruitment program which does not have the effect of...

  3. 10 CFR 5.310 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 1 2011-01-01 2011-01-01 false Recruitment. 5.310 Section 5.310 Energy NUCLEAR REGULATORY... FINANCIAL ASSISTANCE Discrimination on the Basis of Sex in Admission and Recruitment Prohibited § 5.310 Recruitment. (a) Nondiscriminatory recruitment. A recipient to which §§ 5.300 through 5.310 apply shall...

  4. 36 CFR 1211.510 - Recruitment.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 36 Parks, Forests, and Public Property 3 2010-07-01 2010-07-01 false Recruitment. 1211.510 Section... Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and hiring of employees. Where a recipient has been found to be...

  5. 45 CFR 618.310 - Recruitment.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 45 Public Welfare 3 2010-10-01 2010-10-01 false Recruitment. 618.310 Section 618.310 Public... Discrimination on the Basis of Sex in Admission and Recruitment Prohibited § 618.310 Recruitment. (a) Nondiscriminatory recruitment. A recipient to which §§ 618.300 through 618.310 apply shall not discriminate on...

  6. 34 CFR 106.23 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 34 Education 1 2011-07-01 2011-07-01 false Recruitment. 106.23 Section 106.23 Education... Discrimination on the Basis of Sex in Admission and Recruitment Prohibited § 106.23 Recruitment. (a) Nondiscriminatory recruitment. A recipient to which this subpart applies shall not discriminate on the basis of...

  7. 13 CFR 113.310 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 13 Business Credit and Assistance 1 2011-01-01 2011-01-01 false Recruitment. 113.310 Section 113... Discrimination on the Basis of Sex in Admission and Recruitment Prohibited § 113.310 Recruitment. (a) Nondiscriminatory recruitment. A recipient to which §§ 113.300 through 113.310 apply shall not discriminate on...

  8. 36 CFR 1211.310 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 36 Parks, Forests, and Public Property 3 2011-07-01 2011-07-01 false Recruitment. 1211.310 Section... Discrimination on the Basis of Sex in Admission and Recruitment Prohibited § 1211.310 Recruitment. (a) Nondiscriminatory recruitment. A recipient to which §§ 1211.300 through 1211.310 apply shall not discriminate on...

  9. 10 CFR 5.510 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 10 Energy 1 2011-01-01 2011-01-01 false Recruitment. 5.510 Section 5.510 Energy NUCLEAR REGULATORY... Prohibited § 5.510 Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and hiring of employees. Where a recipient has been...

  10. 32 CFR 196.310 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 32 National Defense 2 2011-07-01 2011-07-01 false Recruitment. 196.310 Section 196.310 National... Discrimination on the Basis of Sex in Admission and Recruitment Prohibited § 196.310 Recruitment. (a) Nondiscriminatory recruitment. A recipient to which §§ 196.300 through 196.310 apply shall not discriminate on...

  11. 20 CFR 656.21 - Supervised recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 20 Employees' Benefits 3 2011-04-01 2011-04-01 false Supervised recruitment. 656.21 Section 656.21... Supervised recruitment. (a) Supervised recruitment. Where the Certifying Officer determines it appropriate, post-filing supervised recruitment may be required of the employer for the pending application...

  12. 29 CFR 36.310 - Recruitment.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 29 Labor 1 2010-07-01 2010-07-01 true Recruitment. 36.310 Section 36.310 Labor Office of the... FEDERAL FINANCIAL ASSISTANCE Discrimination on the Basis of Sex in Admission and Recruitment Prohibited § 36.310 Recruitment. (a) Nondiscriminatory recruitment. A recipient to which §§ 36.300 through...

  13. 10 CFR 1042.510 - Recruitment.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 4 2010-01-01 2010-01-01 false Recruitment. 1042.510 Section 1042.510 Energy DEPARTMENT... Education Programs or Activities Prohibited § 1042.510 Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and hiring of...

  14. 13 CFR 113.510 - Recruitment.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 13 Business Credit and Assistance 1 2010-01-01 2010-01-01 false Recruitment. 113.510 Section 113... Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and hiring of employees. Where a recipient has been found to be...

  15. 49 CFR 25.510 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 49 Transportation 1 2011-10-01 2011-10-01 false Recruitment. 25.510 Section 25.510 Transportation... Education Programs or Activities Prohibited § 25.510 Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and hiring of...

  16. 7 CFR 15a.53 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 7 Agriculture 1 2011-01-01 2011-01-01 false Recruitment. 15a.53 Section 15a.53 Agriculture Office... Activities Prohibited § 15a.53 Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and hiring of employees. Where a recipient...

  17. 32 CFR 196.510 - Recruitment.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 32 National Defense 2 2010-07-01 2010-07-01 false Recruitment. 196.510 Section 196.510 National... Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and hiring of employees. Where a recipient has been found to be...

  18. 45 CFR 2555.310 - Recruitment.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 45 Public Welfare 4 2010-10-01 2010-10-01 false Recruitment. 2555.310 Section 2555.310 Public... Discrimination on the Basis of Sex in Admission and Recruitment Prohibited § 2555.310 Recruitment. (a) Nondiscriminatory recruitment. A recipient to which §§ 2555.300 through 2555.310 apply shall not discriminate on...

  19. 45 CFR 618.510 - Recruitment.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 45 Public Welfare 3 2010-10-01 2010-10-01 false Recruitment. 618.510 Section 618.510 Public... Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and hiring of employees. Where a recipient has been found to be...

  20. 20 CFR 655.156 - Recruitment report.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 20 Employees' Benefits 3 2011-04-01 2011-04-01 false Recruitment report. 655.156 Section 655.156... the United States (H-2A Workers) Post-Acceptance Requirements § 655.156 Recruitment report. (a) Requirements of a recruitment report. The employer must prepare, sign, and date a written recruitment...

  1. 38 CFR 23.510 - Recruitment.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2010-07-01 2010-07-01 false Recruitment. 23.510... Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and hiring of employees. Where a recipient has been found to be...

  2. 10 CFR 5.510 - Recruitment.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 1 2010-01-01 2010-01-01 false Recruitment. 5.510 Section 5.510 Energy NUCLEAR REGULATORY... Prohibited § 5.510 Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and hiring of employees. Where a recipient has been...

  3. 13 CFR 113.510 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... 13 Business Credit and Assistance 1 2011-01-01 2011-01-01 false Recruitment. 113.510 Section 113... Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and hiring of employees. Where a recipient has been found to be...

  4. 38 CFR 23.510 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 38 Pensions, Bonuses, and Veterans' Relief 2 2011-07-01 2011-07-01 false Recruitment. 23.510... Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and hiring of employees. Where a recipient has been found to be...

  5. 10 CFR 5.310 - Recruitment.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 10 Energy 1 2010-01-01 2010-01-01 false Recruitment. 5.310 Section 5.310 Energy NUCLEAR REGULATORY... FINANCIAL ASSISTANCE Discrimination on the Basis of Sex in Admission and Recruitment Prohibited § 5.310 Recruitment. (a) Nondiscriminatory recruitment. A recipient to which §§ 5.300 through 5.310 apply shall...

  6. 44 CFR 19.310 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 44 Emergency Management and Assistance 1 2011-10-01 2011-10-01 false Recruitment. 19.310 Section... RECEIVING FEDERAL FINANCIAL ASSISTANCE Discrimination on the Basis of Sex in Admission and Recruitment Prohibited § 19.310 Recruitment. (a) Nondiscriminatory recruitment. A recipient to which §§ 19.300 through...

  7. 36 CFR 1211.510 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 36 Parks, Forests, and Public Property 3 2011-07-01 2011-07-01 false Recruitment. 1211.510 Section... Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and hiring of employees. Where a recipient has been found to be...

  8. 18 CFR 1317.510 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 18 Conservation of Power and Water Resources 2 2011-04-01 2011-04-01 false Recruitment. 1317.510... Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and hiring of employees. Where a recipient has been found to be...

  9. 41 CFR 101-4.510 - Recruitment.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 41 Public Contracts and Property Management 2 2010-07-01 2010-07-01 true Recruitment. 101-4.510... Employment in Education Programs or Activities Prohibited § 101-4.510 Recruitment. (a) Nondiscriminatory recruitment and hiring. A recipient shall not discriminate on the basis of sex in the recruitment and...

  10. 31 CFR 28.310 - Recruitment.

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 31 Money and Finance: Treasury 1 2011-07-01 2011-07-01 false Recruitment. 28.310 Section 28.310... Basis of Sex in Admission and Recruitment Prohibited § 28.310 Recruitment. (a) Nondiscriminato