Azurophilic Granule

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Hans Tapper - One of the best experts on this subject based on the ideXlab platform.

  • Fusion of Azurophilic Granules with phagosomes containing S. pyogenes bacteria.
    2013
    Co-Authors: Pontus Nordenfelt, Susanne Bauer, Per Lonnbro, Hans Tapper
    Abstract:

    A. Western blot of isolated phagosomes and cell lysate. Differentiated HL-60 cells were allowed to phagocytose IgG-opsonized, heat-killed and magnetically labeled S. pyogenes bacteria for 20 min at a bacteria/cell ratio of 5∶1. Following washes, the cells were lysed by nitrogen cavitation and phagosomes were retrieved by magnetic selection. Equal amounts of phagosomes were loaded and probed with antibodies against cathepsin D (Azurophilic Granule content marker) and GM130 (Golgi marker) with anti-S. pyogenes as loading control. Increasing amounts of cell lysate (relative protein content.05×, 0.5× and 1.0×) was used as a control. B. Azurophilic Granule–phagosome fusion. Differentiated HL-60 cells were allowed to phagocytose opsonized Oregon Green-labeled S. pyogenes bacteria, either the BMJ71 (i-iii) or the AP1 (iv–v) strains, at a bacteria/cell ratio of 10∶1. After a synchronized presentation, the samples were incubated at 37°C for 5 min, fixed and incubated with antibodies directed against CD63, subsequently detected using Alexa 594 F(ab')2 fragments. Single deconvolved focal planes from serial z-stacks were taken from the mid-part of the HL-60 cells. Oregon Green staining shows the localization of bacteria (ii, v). Magenta staining shows the localization of the Azurophilic Granule membrane marker CD63 (i, iv). The images are also presented as merged (iii, vi). Size bar: 5 µm.

  • different requirements for early and late phases of Azurophilic Granule phagosome fusion
    Traffic, 2009
    Co-Authors: Pontus Nordenfelt, Martin E Winberg, Per Lonnbro, Birgitta Rasmusson, Hans Tapper
    Abstract:

    Phagocytosis and killing of microorganisms are complex processes that involve tightly regulated membrane traffic events. Because many signaling molecules associate with membrane rafts and because these structures can be found on Azurophilic Granules, we decided to investigate raft recruitment and the signaling requirements for Azurophilic Granule secretion during phagosome maturation. At the site of phagocytosis of immunoglobulin G-opsonized prey in human neutrophils, we found that early secretion of Azurophilic Granules was both raft- and calcium-dependent. Subsequently, rafts at the phagocytic site were internalized with the prey. At the fully formed phagosome, the fusion of Azurophilic Granules was no longer dependent on rafts or calcium. These findings were found to be true also when using Streptococcus pyogenes bacteria as prey, and depletion of calcium affected the kinetics of bacterial intracellular survival. These findings suggest that the mechanisms for delivery of Azurophilic content to nascent and sealed phagosomes, respectively, differ in their dependence on calcium and membrane rafts.

  • Phagocytosis of Streptococcus pyogenes by all-trans retinoic acid-differentiated HL-60 cells: roles of Azurophilic Granules and NADPH oxidase.
    Public Library of Science (PLoS), 2009
    Co-Authors: Pontus Nordenfelt, Susanne Bauer, Per Lonnbro, Hans Tapper
    Abstract:

    BACKGROUND:New experimental approaches to the study of the neutrophil phagosome and bacterial killing prompted a reassessment of the usefulness of all-trans retinoic acid (ATRA)-differentiated HL-60 cells as a neutrophil model. HL-60 cells are special in that they possess Azurophilic Granules while lacking the specific Granules with their associated oxidase components. The resulting inability to mount an effective intracellular respiratory burst makes these cells more dependent on other mechanisms when killing internalized bacteria. METHODOLOGY/PRINCIPAL FINDINGS:In this work phagocytosis and phagosome-related responses of ATRA-differentiated HL-60 cells were compared to those earlier described in human neutrophils. We show that intracellular survival of wild-type S. pyogenes bacteria in HL-60 cells is accompanied by inhibition of Azurophilic Granule-phagosome fusion. A mutant S. pyogenes bacterium, deficient in M-protein expression, is, on the other hand, rapidly killed in phagosomes that avidly fuse with Azurophilic Granules. CONCLUSIONS/SIGNIFICANCE:The current data extend our previous findings by showing that a system lacking in oxidase involvement also indicates a link between inhibition of Azurophilic Granule fusion and the intraphagosomal fate of S. pyogenes bacteria. We propose that differentiated HL-60 cells can be a useful tool to study certain aspects of neutrophil phagosome maturation, such as Azurophilic Granule fusion

  • streptococcus pyogenes bacteria modulate membrane traffic in human neutrophils and selectively inhibit Azurophilic Granule fusion with phagosomes
    Cellular Microbiology, 2006
    Co-Authors: Leila Staali, Matthias Morgelin, Susanne Bauer, Lars Bjorck, Hans Tapper
    Abstract:

    Summary We recently reported that the human pathogen Strep- tococcus pyogenes of the M1 serotype survives and replicates intracellularly after being phagocytosed by human neutrophils. These data raised the possibility that the generation of reactive oxygen metabolites by neutrophils, and the release of microbicidal mole- cules from their Azurophilic and specific Granules into phagosomes, can be modulated by S. pyogenes bac- teria expressing surface-associated M and/or M-like proteins. We now demonstrate, using flow cytometry, immunofluorescence microscopy and transmission electron microscopy, that live wild-type S. pyogenes , after internalization by human neutrophils, inhibits the fusion of Azurophilic Granules with phagosomes. In contrast, Azurophilic Granule-content is efficiently delivered to phagosomes containing bacteria not expressing M and/or M-like proteins. Also, when heat- killed wild-type bacteria are used as the phagocytic prey, fusion of Azurophilic Granules with phagosomes is observed. The inhibition caused by live wild-type S. pyogenes is specific for Azurophilic Granule-pha- gosome fusion, because the mobilization of specific Granules and the production of reactive oxygen spe- cies are induced to a similar extent by all strains tested. In conclusion, our results demonstrate that viable S. pyogenes bacteria expressing M and M-like proteins selectively prevent the fusion of Azurophilic Granules with phagosomes.

  • secretion of heparin binding protein from human neutrophils is determined by its localization in Azurophilic Granules and secretory vesicles
    Blood, 2002
    Co-Authors: Hans Tapper, Hans Flodgaard, Anna Karlsson, Matthias Morgelin, Heiko Herwald
    Abstract:

    Human neutrophils have an important role in host defense against microbial infection. At different stages of an infectious process, neutrophils progressively up-regulate receptors and release various effector molecules. These are stored in several distinct types of Granules with varying propensity to be secreted. Heparin-binding protein (HBP), also known as CAP37 or azurocidin, is a multifunctional, inactive serine-protease homologue. The present work shows that HBP is released from neutrophils on stimulation with secretagogues that do not trigger the secretion of Azurophilic Granule content. Therefore, the subcellular localization of HBP was investigated in more detail. Immunofluorescence microscopy revealed that HBP was localized close to the plasma membrane. Further analysis by fractionation of postnuclear supernatants from cavitated neutrophils showed that HBP is stored in Azurophilic Granules and secretory vesicles but that it is also detected to a minor extent in the plasma membrane. These findings were confirmed by immunoelectron microscopy showing that HBP colocalized with marker proteins of Azurophilic Granules and secretory vesicles. The presence of HBP in secretory vesicles possibly depends on the stage of cell differentiation, since the promyelocytic cell line HL-60 contains less HBP than mature neutrophils, stored exclusively in the less easily mobilized Azurophilic Granules. Our findings suggest that HBP can be synthesized or targeted to easily mobilized compartments at a late stage of neutrophil maturation. The ability of neutrophils to secrete HBP from secretory vesicles may be important for proinflammatory functions of this protein, such as the alteration of vascular permeability. (Blood. 2002;99:1785-1793)

Sergio D. Catz - One of the best experts on this subject based on the ideXlab platform.

  • the trafficking protein jfc1 regulates rac1 gtp localization at the uropod controlling neutrophil chemotaxis and in vivo migration
    Journal of Leukocyte Biology, 2019
    Co-Authors: Mahalakshmi Ramadass, Jennifer L. Johnson, Jinzhong Zhang, Alex Marki, Dennis Wolf, William B Kiosses, Kersi Pestonjamasp, Klaus Ley, Sergio D. Catz
    Abstract:

    Neutrophil chemotaxis is essential in responses to infection and underlies inflammation. In neutrophils, the small GTPase Rac1 has discrete functions at both the leading edge and in the retraction of the trailing structure at the cell's rear (uropod), but how Rac1 is regulated at the uropod is unknown. Here, we identified a mechanism mediated by the trafficking protein synaptotagmin-like 1 (SYTL1 or JFC1) that controls Rac1-GTP recycling from the uropod and promotes directional migration of neutrophils. JFC1-null neutrophils displayed defective polarization and impaired directional migration to N-formyl-methionine-leucyl-phenylalanine in vitro, but chemoattractant-induced actin remodeling, calcium signaling and Erk activation were normal in these cells. Defective chemotaxis was not explained by impaired Azurophilic Granule exocytosis associated with JFC1 deficiency. Mechanistically, we show that active Rac1 localizes at dynamic vesicles where endogenous JFC1 colocalizes with Rac1-GTP. Super-resolution microscopy (STORM) analysis shows adjacent distribution of JFC1 and Rac1-GTP, which increases upon activation. JFC1 interacts with Rac1-GTP in a Rab27a-independent manner to regulate Rac1-GTP trafficking. JFC1-null cells exhibited Rac1-GTP accumulation at the uropod and increased tail length, and Rac1-GTP uropod accumulation was recapitulated by inhibition of ROCK or by interference with microtubule remodeling. In vivo, neutrophil dynamic studies in mixed bone marrow chimeric mice show that JFC1-/- neutrophils are unable to move directionally toward the source of the chemoattractant, supporting the notion that JFC1 deficiency results in defective neutrophil migration. Our results suggest that defective Rac1-GTP recycling from the uropod affects directionality and highlight JFC1-mediated Rac1 trafficking as a potential target to regulate chemotaxis in inflammation and immunity.

  • Increased Neutrophil Secretion Induced by NLRP3 Mutation Links the Inflammasome to Azurophilic Granule Exocytosis
    Frontiers Media S.A., 2017
    Co-Authors: Jennifer L. Johnson, Mahalakshmi Ramadass, Ariela Haimovich, Matthew D. Mcgeough, Jinzhong Zhang, Hal M. Hoffman, Sergio D. Catz
    Abstract:

    Heterozygous mutations in the NLRP3 gene in patients with cryopyrin associated periodic syndrome (CAPS) lead to hyper-responsive inflammasome function. CAPS is a systemic auto-inflammatory syndrome characterized by the activation of the innate immune system induced by elevated pro-inflammatory cytokines, but the involvement of selective innate immune cells in this process is not fully understood. Neutrophil secretion and the toxic components of their Granules are mediators of inflammation associated with several human diseases and inflammatory conditions. Here, using the Nlrp3A350V inducible mouse model (MWS CreT) that recapitulates human patients with the A352V mutation in NLRP3 observed in the Muckle-Wells sub-phenotype of CAPS, we studied the relationship between hyper-activation of the inflammasome and neutrophil exocytosis. Using a flow cytometry approach, we show that Nlrp3A350V (MWS) neutrophils express normal basal levels of CD11b at the plasma membrane and that the upregulation of CD11b from secretory vesicles in response to several plasma membrane or endocytic agonist including the bacterial-derived mimetic peptide formyl-Leu-Met-Phe (fMLF) and the unmethylated oligonucleotide CpG is normal in MWS neutrophils. Significant but modest CD11b upregulation in MWS neutrophils compared to wild type was only observed in response to GM-CSF and CpG. The same pattern was observed for the secretion of matrix metalloproteinase-9 (MMP-9) from gelatinase Granules in that MMP-9 secretion in MWS neutrophils was not different from that observed in wild-type neutrophils except when stimulated with GM-CSF and CpG. In contrast, Azurophilic Granule secretion, whose cargoes constitute the most toxic secretory and pro-inflammatory factors of the neutrophil, was markedly dysregulated in MWS neutrophils under both basal and stimulated conditions. This could not be attributed to paracrine effects of secretory cytokines because IL-1β secretion by neutrophils was undetectable under these experimental conditions. The increased Azurophilic Granule exocytosis in MWS neutrophils was attenuated by treatment with the neutrophil exocytosis inhibitor Nexinhib20. In agreement with a possible neutrophil contribution to systemic inflammation in CAPS, the levels of neutrophil secretory proteins were significantly elevated in the plasma from Nlrp3A350V mice. Altogether, our data indicates an Azurophilic Granule-selective dysregulation of neutrophil exocytosis in CAPS

  • increased neutrophil secretion induced by nlrp3 mutation links the inflammasome to Azurophilic Granule exocytosis
    Frontiers in Cellular and Infection Microbiology, 2017
    Co-Authors: Jennifer L. Johnson, Mahalakshmi Ramadass, Ariela Haimovich, Matthew D. Mcgeough, Jinzhong Zhang, Hal M. Hoffman, Sergio D. Catz
    Abstract:

    Heterozygous mutations in the NLRP3 gene in patients with cryopyrin associated periodic syndrome (CAPS) lead to hyper-responsive inflammasome function. CAPS is a systemic auto-inflammatory syndrome characterized by the activation of the innate immune system induced by elevated pro-inflammatory cytokines, but the involvement of selective innate immune cells in this process is not fully understood. Neutrophil secretion and the toxic components of their Granules are mediators of inflammation associated with several human diseases and inflammatory conditions. Here, using the Nlrp3A350V inducible mouse model (MWS CreT) that recapitulates human patients with the A352V mutation in NLRP3 observed in the Muckle-Wells sub-phenotype of CAPS, we studied the relationship between hyper-activation of the inflammasome and neutrophil exocytosis. Using a flow cytometry approach, we show that Nlrp3A350V (MWS) neutrophils express normal basal levels of CD11b at the plasma membrane and that the upregulation of CD11b from secretory vesicles in response to several plasma membrane or endocytic agonist including the bacterial-derived mimetic peptide formyl-Leu-Met-Phe (fMLF) and the unmethylated oligonucleotide CpG is normal in MWS neutrophils. Significant but modest CD11b upregulation in MWS neutrophils was only observed in response to GM-CSF and CpG. The same pattern was observed for the secretion of matrix metalloproteinase-9 (MMP-9) from gelatinase Granules in that MMP-9 secretion in MWS neutrophils was not different from that observed in wild-type neutrophils except when stimulated with GM-CSF and CpG. In contrast, Azurophilic Granule secretion, whose cargoes constitute the most toxic secretory and pro-inflammatory factors of the neutrophil, was markedly dysregulated in MWS neutrophils under both basal and stimulated conditions. This could not be attributed to paracrine effects of secretory cytokines because IL-1 secretion by neutrophils was undetectable under these experimental conditions. The increased Azurophilic Granule exocytosis in MWS neutrophils was attenuated by treatment with the neutrophil exocytosis inhibitor Nexinhib20. In agreement with a possible neutrophil contribution to systemic inflammation in CAPS, the levels of neutrophil secretory proteins were significantly elevated in the plasma from Nlrp3A350V mice. Altogether, our data indicates an Azurophilic Granule-selective dysregulation of neutrophil exocytosis in CAPS.

  • increased survival and reduced neutrophil infiltration of the liver in rab27a but not munc13 4 deficient mice in lipopolysaccharide induced systemic inflammation
    Infection and Immunity, 2011
    Co-Authors: Jennifer L. Johnson, Hong Hong, Jlenia Monfregola, Sergio D. Catz
    Abstract:

    Genetic defects in the Rab27a or Munc13-4 gene lead to immunodeficiencies in humans, characterized by frequent viral and bacterial infections. However, the role of Rab27a and Munc13-4 in the regulation of systemic inflammation initiated by Gram-negative bacterium-derived pathogenic molecules is currently unknown. Using a model of lipopolysaccharide-induced systemic inflammation, we show that Rab27a-deficient (Rab27aash/ash) mice are resistant to lipopolysaccharide (LPS)-induced death, while Munc13-4-deficient (Munc13-4jinx/jinx) mice show only moderate protection. Rab27aash/ash but not Munc13-4jinx/jinx mice showed significantly decreased tumor necrosis factor alpha (TNF-α) plasma levels after LPS administration. Neutrophil sequestration in lungs from Rab27aash/ash and Munc13-4jinx/jinx LPS-treated mice was similar to that observed for wild-type mice. In contrast, Rab27a- but not Munc13-4-deficient mice showed decreased neutrophil infiltration in liver and failed to undergo LPS-induced neutropenia. Decreased liver infiltration in Rab27aash/ash mice was accompanied by lower CD44 but normal CD11a and CD11b expression in neutrophils. Both Rab27a- and Munc13-4-deficient mice showed decreased Azurophilic Granule secretion in vivo, suggesting that impaired liver infiltration and improved survival in Rab27aash/ash mice is not fully explained by deficient exocytosis of this Granule subset. Altogether, our data indicate that Rab27a but not Munc13-4 plays an important role in neutrophil recruitment to liver and LPS-induced death during endotoxemia, thus highlighting a previously unrecognized role for Rab27a in LPS-mediated systemic inflammation.

  • Rab27a and Rab27b regulate neutrophil Azurophilic Granule exocytosis and NADPH oxidase activity by independent mechanisms.
    Traffic (Copenhagen Denmark), 2009
    Co-Authors: Jennifer L. Johnson, Miguel C. Seabra, Agnieszka A. Brzezinska, Tanya Tolmachova, Daniela B. Munafo, Beverly A. Ellis, Hong Hong, Sergio D. Catz
    Abstract:

    Neutrophils rely on exocytosis to mobilize receptors and adhesion molecules and to release microbicidal factors. This process should be strictly regulated because uncontrolled release of toxic proteins would be injurious to the host. In vivo studies showed that the small GTPase Rab27a regulates Azurophilic Granule exocytosis. Using mouse neutrophils deficient in Rab27a (Rab27aash/ash), Rab27b [Rab27b knockout (KO)] or both [Rab27a/b double KO (DoKo)], we investigated the role of the Rab27 isoforms in neutrophils. We found that both Rab27a and Rab27b deficiencies impaired Azurophilic Granule exocytosis. Rab27aash/ash neutrophils showed upregulation of Rab27b expression which did not compensate for the secretory defects observed in Rab27a-deficient cells, suggesting that Rab27 isoforms play independent roles in neutrophil exocytosis. Total internal reflection fluorescence microscopy analysis showed that Rab27aash/ash and Rab27b KO neutrophils have a decreased number of Azurophilic Granules near the plasma membrane. The effect was exacerbated in Rab27a/b DoKo neutrophils. Rab27-deficient neutrophils showed impaired activation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase at the plasma membrane although intraphagosomal reactive oxygen species (ROS) production was not affected. Exocytosis of secretory vesicles in Rab27-deficient neutrophils was functional, suggesting that Rab27 GTPases selectively control the exocytosis of neutrophil Granules.

Nicolle H Packer - One of the best experts on this subject based on the ideXlab platform.

  • human neutrophils secrete bioactive paucimannosidic proteins from Azurophilic Granules into pathogen infected sputum
    Journal of Biological Chemistry, 2015
    Co-Authors: Morten Thaysenandersen, Vignesh Venkatakrishnan, Ian Loke, Christine Laurini, Simone Diestel, Benjamin L Parker, Nicolle H Packer
    Abstract:

    Abstract Unlike plants and invertebrates, mammals reportedly lack proteins displaying asparagine (N)-linked paucimannosylation (mannose1–3fucose0–1N-acetylglucosamine2Asn). Enabled by technology advancements in system-wide biomolecular characterization, we document that protein paucimannosylation is a significant host-derived molecular signature of neutrophil-rich sputum from pathogen-infected human lungs and is negligible in pathogen-free sputum. Five types of paucimannosidic N-glycans were carried by compartment-specific and inflammation-associated proteins of the Azurophilic Granules of human neutrophils including myeloperoxidase (MPO), azurocidin, and neutrophil elastase. The timely expressed human Azurophilic Granule-resident β-hexosaminidase A displayed the capacity to generate paucimannosidic N-glycans by trimming hybrid/complex type N-glycan intermediates with relative broad substrate specificity. Paucimannosidic N-glycoepitopes showed significant co-localization with β-hexosaminidase A and the Azurophilic marker MPO in human neutrophils using immunocytochemistry. Furthermore, promyelocyte stage-specific expression of genes coding for paucimannosidic proteins and biosynthetic enzymes indicated a novel spatio-temporal biosynthetic route in early neutrophil maturation. The absence of bacterial exoglycosidase activities and paucimannosidic N-glycans excluded exogenous origins of paucimannosylation. Paucimannosidic proteins from isolated and sputum neutrophils were preferentially secreted upon inoculation with virulent Pseudomonas aeruginosa. Finally, paucimannosidic proteins displayed affinities to mannose-binding lectin, suggesting immune-related functions of paucimannosylation in activated human neutrophils. In conclusion, we are the first to document that human neutrophils produce, store and, upon activation, selectively secrete bioactive paucimannosidic proteins into sputum of lungs undergoing pathogen-based inflammation.

Jennifer L. Johnson - One of the best experts on this subject based on the ideXlab platform.

  • the trafficking protein jfc1 regulates rac1 gtp localization at the uropod controlling neutrophil chemotaxis and in vivo migration
    Journal of Leukocyte Biology, 2019
    Co-Authors: Mahalakshmi Ramadass, Jennifer L. Johnson, Jinzhong Zhang, Alex Marki, Dennis Wolf, William B Kiosses, Kersi Pestonjamasp, Klaus Ley, Sergio D. Catz
    Abstract:

    Neutrophil chemotaxis is essential in responses to infection and underlies inflammation. In neutrophils, the small GTPase Rac1 has discrete functions at both the leading edge and in the retraction of the trailing structure at the cell's rear (uropod), but how Rac1 is regulated at the uropod is unknown. Here, we identified a mechanism mediated by the trafficking protein synaptotagmin-like 1 (SYTL1 or JFC1) that controls Rac1-GTP recycling from the uropod and promotes directional migration of neutrophils. JFC1-null neutrophils displayed defective polarization and impaired directional migration to N-formyl-methionine-leucyl-phenylalanine in vitro, but chemoattractant-induced actin remodeling, calcium signaling and Erk activation were normal in these cells. Defective chemotaxis was not explained by impaired Azurophilic Granule exocytosis associated with JFC1 deficiency. Mechanistically, we show that active Rac1 localizes at dynamic vesicles where endogenous JFC1 colocalizes with Rac1-GTP. Super-resolution microscopy (STORM) analysis shows adjacent distribution of JFC1 and Rac1-GTP, which increases upon activation. JFC1 interacts with Rac1-GTP in a Rab27a-independent manner to regulate Rac1-GTP trafficking. JFC1-null cells exhibited Rac1-GTP accumulation at the uropod and increased tail length, and Rac1-GTP uropod accumulation was recapitulated by inhibition of ROCK or by interference with microtubule remodeling. In vivo, neutrophil dynamic studies in mixed bone marrow chimeric mice show that JFC1-/- neutrophils are unable to move directionally toward the source of the chemoattractant, supporting the notion that JFC1 deficiency results in defective neutrophil migration. Our results suggest that defective Rac1-GTP recycling from the uropod affects directionality and highlight JFC1-mediated Rac1 trafficking as a potential target to regulate chemotaxis in inflammation and immunity.

  • Increased Neutrophil Secretion Induced by NLRP3 Mutation Links the Inflammasome to Azurophilic Granule Exocytosis
    Frontiers Media S.A., 2017
    Co-Authors: Jennifer L. Johnson, Mahalakshmi Ramadass, Ariela Haimovich, Matthew D. Mcgeough, Jinzhong Zhang, Hal M. Hoffman, Sergio D. Catz
    Abstract:

    Heterozygous mutations in the NLRP3 gene in patients with cryopyrin associated periodic syndrome (CAPS) lead to hyper-responsive inflammasome function. CAPS is a systemic auto-inflammatory syndrome characterized by the activation of the innate immune system induced by elevated pro-inflammatory cytokines, but the involvement of selective innate immune cells in this process is not fully understood. Neutrophil secretion and the toxic components of their Granules are mediators of inflammation associated with several human diseases and inflammatory conditions. Here, using the Nlrp3A350V inducible mouse model (MWS CreT) that recapitulates human patients with the A352V mutation in NLRP3 observed in the Muckle-Wells sub-phenotype of CAPS, we studied the relationship between hyper-activation of the inflammasome and neutrophil exocytosis. Using a flow cytometry approach, we show that Nlrp3A350V (MWS) neutrophils express normal basal levels of CD11b at the plasma membrane and that the upregulation of CD11b from secretory vesicles in response to several plasma membrane or endocytic agonist including the bacterial-derived mimetic peptide formyl-Leu-Met-Phe (fMLF) and the unmethylated oligonucleotide CpG is normal in MWS neutrophils. Significant but modest CD11b upregulation in MWS neutrophils compared to wild type was only observed in response to GM-CSF and CpG. The same pattern was observed for the secretion of matrix metalloproteinase-9 (MMP-9) from gelatinase Granules in that MMP-9 secretion in MWS neutrophils was not different from that observed in wild-type neutrophils except when stimulated with GM-CSF and CpG. In contrast, Azurophilic Granule secretion, whose cargoes constitute the most toxic secretory and pro-inflammatory factors of the neutrophil, was markedly dysregulated in MWS neutrophils under both basal and stimulated conditions. This could not be attributed to paracrine effects of secretory cytokines because IL-1β secretion by neutrophils was undetectable under these experimental conditions. The increased Azurophilic Granule exocytosis in MWS neutrophils was attenuated by treatment with the neutrophil exocytosis inhibitor Nexinhib20. In agreement with a possible neutrophil contribution to systemic inflammation in CAPS, the levels of neutrophil secretory proteins were significantly elevated in the plasma from Nlrp3A350V mice. Altogether, our data indicates an Azurophilic Granule-selective dysregulation of neutrophil exocytosis in CAPS

  • increased neutrophil secretion induced by nlrp3 mutation links the inflammasome to Azurophilic Granule exocytosis
    Frontiers in Cellular and Infection Microbiology, 2017
    Co-Authors: Jennifer L. Johnson, Mahalakshmi Ramadass, Ariela Haimovich, Matthew D. Mcgeough, Jinzhong Zhang, Hal M. Hoffman, Sergio D. Catz
    Abstract:

    Heterozygous mutations in the NLRP3 gene in patients with cryopyrin associated periodic syndrome (CAPS) lead to hyper-responsive inflammasome function. CAPS is a systemic auto-inflammatory syndrome characterized by the activation of the innate immune system induced by elevated pro-inflammatory cytokines, but the involvement of selective innate immune cells in this process is not fully understood. Neutrophil secretion and the toxic components of their Granules are mediators of inflammation associated with several human diseases and inflammatory conditions. Here, using the Nlrp3A350V inducible mouse model (MWS CreT) that recapitulates human patients with the A352V mutation in NLRP3 observed in the Muckle-Wells sub-phenotype of CAPS, we studied the relationship between hyper-activation of the inflammasome and neutrophil exocytosis. Using a flow cytometry approach, we show that Nlrp3A350V (MWS) neutrophils express normal basal levels of CD11b at the plasma membrane and that the upregulation of CD11b from secretory vesicles in response to several plasma membrane or endocytic agonist including the bacterial-derived mimetic peptide formyl-Leu-Met-Phe (fMLF) and the unmethylated oligonucleotide CpG is normal in MWS neutrophils. Significant but modest CD11b upregulation in MWS neutrophils was only observed in response to GM-CSF and CpG. The same pattern was observed for the secretion of matrix metalloproteinase-9 (MMP-9) from gelatinase Granules in that MMP-9 secretion in MWS neutrophils was not different from that observed in wild-type neutrophils except when stimulated with GM-CSF and CpG. In contrast, Azurophilic Granule secretion, whose cargoes constitute the most toxic secretory and pro-inflammatory factors of the neutrophil, was markedly dysregulated in MWS neutrophils under both basal and stimulated conditions. This could not be attributed to paracrine effects of secretory cytokines because IL-1 secretion by neutrophils was undetectable under these experimental conditions. The increased Azurophilic Granule exocytosis in MWS neutrophils was attenuated by treatment with the neutrophil exocytosis inhibitor Nexinhib20. In agreement with a possible neutrophil contribution to systemic inflammation in CAPS, the levels of neutrophil secretory proteins were significantly elevated in the plasma from Nlrp3A350V mice. Altogether, our data indicates an Azurophilic Granule-selective dysregulation of neutrophil exocytosis in CAPS.

  • increased survival and reduced neutrophil infiltration of the liver in rab27a but not munc13 4 deficient mice in lipopolysaccharide induced systemic inflammation
    Infection and Immunity, 2011
    Co-Authors: Jennifer L. Johnson, Hong Hong, Jlenia Monfregola, Sergio D. Catz
    Abstract:

    Genetic defects in the Rab27a or Munc13-4 gene lead to immunodeficiencies in humans, characterized by frequent viral and bacterial infections. However, the role of Rab27a and Munc13-4 in the regulation of systemic inflammation initiated by Gram-negative bacterium-derived pathogenic molecules is currently unknown. Using a model of lipopolysaccharide-induced systemic inflammation, we show that Rab27a-deficient (Rab27aash/ash) mice are resistant to lipopolysaccharide (LPS)-induced death, while Munc13-4-deficient (Munc13-4jinx/jinx) mice show only moderate protection. Rab27aash/ash but not Munc13-4jinx/jinx mice showed significantly decreased tumor necrosis factor alpha (TNF-α) plasma levels after LPS administration. Neutrophil sequestration in lungs from Rab27aash/ash and Munc13-4jinx/jinx LPS-treated mice was similar to that observed for wild-type mice. In contrast, Rab27a- but not Munc13-4-deficient mice showed decreased neutrophil infiltration in liver and failed to undergo LPS-induced neutropenia. Decreased liver infiltration in Rab27aash/ash mice was accompanied by lower CD44 but normal CD11a and CD11b expression in neutrophils. Both Rab27a- and Munc13-4-deficient mice showed decreased Azurophilic Granule secretion in vivo, suggesting that impaired liver infiltration and improved survival in Rab27aash/ash mice is not fully explained by deficient exocytosis of this Granule subset. Altogether, our data indicate that Rab27a but not Munc13-4 plays an important role in neutrophil recruitment to liver and LPS-induced death during endotoxemia, thus highlighting a previously unrecognized role for Rab27a in LPS-mediated systemic inflammation.

  • Rab27a and Rab27b regulate neutrophil Azurophilic Granule exocytosis and NADPH oxidase activity by independent mechanisms.
    Traffic (Copenhagen Denmark), 2009
    Co-Authors: Jennifer L. Johnson, Miguel C. Seabra, Agnieszka A. Brzezinska, Tanya Tolmachova, Daniela B. Munafo, Beverly A. Ellis, Hong Hong, Sergio D. Catz
    Abstract:

    Neutrophils rely on exocytosis to mobilize receptors and adhesion molecules and to release microbicidal factors. This process should be strictly regulated because uncontrolled release of toxic proteins would be injurious to the host. In vivo studies showed that the small GTPase Rab27a regulates Azurophilic Granule exocytosis. Using mouse neutrophils deficient in Rab27a (Rab27aash/ash), Rab27b [Rab27b knockout (KO)] or both [Rab27a/b double KO (DoKo)], we investigated the role of the Rab27 isoforms in neutrophils. We found that both Rab27a and Rab27b deficiencies impaired Azurophilic Granule exocytosis. Rab27aash/ash neutrophils showed upregulation of Rab27b expression which did not compensate for the secretory defects observed in Rab27a-deficient cells, suggesting that Rab27 isoforms play independent roles in neutrophil exocytosis. Total internal reflection fluorescence microscopy analysis showed that Rab27aash/ash and Rab27b KO neutrophils have a decreased number of Azurophilic Granules near the plasma membrane. The effect was exacerbated in Rab27a/b DoKo neutrophils. Rab27-deficient neutrophils showed impaired activation of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase at the plasma membrane although intraphagosomal reactive oxygen species (ROS) production was not affected. Exocytosis of secretory vesicles in Rab27-deficient neutrophils was functional, suggesting that Rab27 GTPases selectively control the exocytosis of neutrophil Granules.

Frank R Deleo - One of the best experts on this subject based on the ideXlab platform.

  • neutrophil microbicides induce a pathogen survival response in community associated methicillin resistant staphylococcus aureus
    Journal of Immunology, 2008
    Co-Authors: Amy M Palazzoloballance, Michelle L Reniere, Kevin R Braughton, Daniel E Sturdevant, Michael Otto, Barry N Kreiswirth, Eric P Skaar, Frank R Deleo
    Abstract:

    In recent years, there has been a dramatic increase in the incidence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections. MW2 (pulsed-field type USA400), the prototype CA-MRSA strain, is highly virulent and has enhanced ability to evade killing by neutrophils. Although progress has been made, the molecular basis for enhanced virulence of CA-MRSA remains incompletely defined. To that end, we studied resistance of MW2 to key microbicides of human neutrophils. Hydrogen peroxide (H 2 O 2 ), hypochlorous acid, and Azurophilic Granule proteins had significant bacteriostatic but limited staphylocidal activity toward MW2 under the conditions tested. An MW2-specific microarray revealed common changes in S. aureus gene expression following exposure to each microbicide, such as up-regulation of transcripts involved in gene regulation (e.g., saeRS and kdpDE ) and stress response. Azurophilic Granule proteins elicited the greatest number of changes in MW2 transcripts, including up-regulation of mRNAs encoding multiple toxins and hemolysins (e.g., hlgA , hlgB , hlgC , hla , lukS-PV , lukF-PV , sec4 , and set17 –26). Notably, H 2 O 2 triggered up-regulation of transcripts related to heme/iron uptake (e.g., isdA , isdB , and isdCDEFsrtBisdG ), and an isogenic isdAB -negative strain of MW2 had increased susceptibility to H 2 O 2 ( p p S. aureus survival response wherein Iron-regulated surface determinant (Isd) proteins are important for resistance to innate host defense. Collectively, the data provide an enhanced view of the mechanisms used by S. aureus to circumvent destruction by the innate immune system.

  • neutrophil microbicides induce a pathogen survival response in community associated methicillin resistant staphylococcus aureus
    Journal of Immunology, 2008
    Co-Authors: Amy M Palazzoloballance, Michelle L Reniere, Kevin R Braughton, Daniel E Sturdevant, Michael Otto, Barry N Kreiswirth, Eric P Skaar, Frank R Deleo
    Abstract:

    In recent years, there has been a dramatic increase in the incidence of community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) infections. MW2 (pulsed-field type USA400), the prototype CA-MRSA strain, is highly virulent and has enhanced ability to evade killing by neutrophils. Although progress has been made, the molecular basis for enhanced virulence of CA-MRSA remains incompletely defined. To that end, we studied resistance of MW2 to key microbicides of human neutrophils. Hydrogen peroxide (H 2 O 2 ), hypochlorous acid, and Azurophilic Granule proteins had significant bacteriostatic but limited staphylocidal activity toward MW2 under the conditions tested. An MW2-specific microarray revealed common changes in S. aureus gene expression following exposure to each microbicide, such as up-regulation of transcripts involved in gene regulation (e.g., saeRS and kdpDE ) and stress response. Azurophilic Granule proteins elicited the greatest number of changes in MW2 transcripts, including up-regulation of mRNAs encoding multiple toxins and hemolysins (e.g., hlgA , hlgB , hlgC , hla , lukS-PV , lukF-PV , sec4 , and set17 –26). Notably, H 2 O 2 triggered up-regulation of transcripts related to heme/iron uptake (e.g., isdA , isdB , and isdCDEFsrtBisdG ), and an isogenic isdAB -negative strain of MW2 had increased susceptibility to H 2 O 2 ( p p S. aureus survival response wherein Iron-regulated surface determinant (Isd) proteins are important for resistance to innate host defense. Collectively, the data provide an enhanced view of the mechanisms used by S. aureus to circumvent destruction by the innate immune system.