FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2011145349> ?p ?o ?g. }

• W2011145349 endingPage "896" @default.
• W2011145349 startingPage "883" @default.
• W2011145349 abstract "•A complex of antimicrobials drives neutrophil elastase to the nucleus during NETosis•The “azurosome” complex mediates protein release across intact membranes•Myeloperoxidase is required for neutrophil elastase release•Neutrophil elastase degrades F-actin and arrests actin dynamics Neutrophils contain granules loaded with antimicrobial proteins and are regarded as impermeable organelles that deliver cargo via membrane fusion. However, during the formation of neutrophil extracellular traps (NETs), neutrophil elastase (NE) translocates from the granules to the nucleus via an unknown mechanism that does not involve membrane fusion and requires reactive oxygen species (ROS). Here, we show that the ROS triggers the dissociation of NE from a membrane-associated complex into the cytosol and activates its proteolytic activity in a myeloperoxidase (MPO)-dependent manner. In the cytosol, NE first binds and degrades F-actin to arrest actin dynamics and subsequently translocates to the nucleus. The complex is an example of an oxidative signaling scaffold that enables ROS and antimicrobial proteins to regulate neutrophil responses. Furthermore, granules contain protein machinery that transports and delivers cargo across membranes independently of membrane fusion. Neutrophils contain granules loaded with antimicrobial proteins and are regarded as impermeable organelles that deliver cargo via membrane fusion. However, during the formation of neutrophil extracellular traps (NETs), neutrophil elastase (NE) translocates from the granules to the nucleus via an unknown mechanism that does not involve membrane fusion and requires reactive oxygen species (ROS). Here, we show that the ROS triggers the dissociation of NE from a membrane-associated complex into the cytosol and activates its proteolytic activity in a myeloperoxidase (MPO)-dependent manner. In the cytosol, NE first binds and degrades F-actin to arrest actin dynamics and subsequently translocates to the nucleus. The complex is an example of an oxidative signaling scaffold that enables ROS and antimicrobial proteins to regulate neutrophil responses. Furthermore, granules contain protein machinery that transports and delivers cargo across membranes independently of membrane fusion. Neutrophils are the foot soldiers of the innate immune system as they are plentiful, short-lived, and armed with antimicrobial effector strategies. They are the first immune cells to arrive at a site of infection and are ready to respond, carrying presynthesized antimicrobial effectors and the enzymes needed to mount an intense burst of reactive oxygen species (ROS) (Amulic et al., 2012Amulic B. Cazalet C. Hayes G.L. Metzler K.D. Zychlinsky A. Neutrophil function: from mechanisms to disease.Annu. Rev. Immunol. 2012; 30: 459-489Crossref PubMed Scopus (1100) Google Scholar). Antimicrobial effectors are synthesized during neutrophil development and are stored in specialized membrane-bound vesicles called granules. Granules contain different cargo depending on when they were synthesized. This results in a continuum of granule contents that are classified into four categories: secretory vesicles and azurophilic, specific, and gelatinase granules (Borregaard, 2010Borregaard N. Neutrophils, from marrow to microbes.Immunity. 2010; 33: 657-670Abstract Full Text Full Text PDF PubMed Scopus (972) Google Scholar). Granule membranes are regarded as impermeable barriers that allow for delivery of their cargo through membrane fusion. Neutrophils ingest and kill microbes intracellularly through phagocytosis. During this process, microbes are enclosed in a membrane compartment known as the phagosome, where exposure to ROS and antimicrobial effectors eliminates pathogens. The antimicrobial load of granules is delivered to the phagosome by fusion of the granule and phagosomal membranes. In addition, granules can fuse with the plasma membrane to release granule cargo extracellularly through degranulation. In contrast to this classical view, an antimicrobial strategy that involves some unconventional cell biology was recently uncovered. Neutrophils were shown to release web-like structures known as neutrophil extracellular traps (NETs) that ensnare and kill a variety of microbes. NETs are composed of decondensed chromatin and a subset of granule and cytoplasmic proteins (Brinkmann et al., 2004Brinkmann V. Reichard U. Goosmann C. Fauler B. Uhlemann Y. Weiss D.S. Weinrauch Y. Zychlinsky A. Neutrophil extracellular traps kill bacteria.Science. 2004; 303: 1532-1535Crossref PubMed Scopus (6357) Google Scholar). Patients and animals carrying mutations in the genes required for NET formation are more susceptible to infections (Brinkmann and Zychlinsky, 2012Brinkmann V. Zychlinsky A. Neutrophil extracellular traps: is immunity the second function of chromatin?.J. Cell Biol. 2012; 198: 773-783Crossref PubMed Scopus (717) Google Scholar). On the other hand, unregulated NET release or lack of NET degradation is linked to several diseases, including cystic fibrosis, preeclampsia, autoimmunity, and vascular diseases (Garcia-Romo et al., 2011Garcia-Romo G.S. Caielli S. Vega B. Connolly J. Allantaz F. Xu Z. Punaro M. Baisch J. Guiducci C. Coffman R.L. et al.Netting neutrophils are major inducers of type I IFN production in pediatric systemic lupus erythematosus.Sci. Transl. Med. 2011; 3: 73ra20Crossref PubMed Scopus (977) Google Scholar, Hakkim et al., 2010Hakkim A. Fürnrohr B.G. Amann K. Laube B. Abed U.A. Brinkmann V. Herrmann M. Voll R.E. Zychlinsky A. Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis.Proc. Natl. Acad. Sci. USA. 2010; 107: 9813-9818Crossref PubMed Scopus (1033) Google Scholar, Kessenbrock et al., 2009Kessenbrock K. Krumbholz M. Schönermarck U. Back W. Gross W.L. Werb Z. Gröne H.J. Brinkmann V. Jenne D.E. Netting neutrophils in autoimmune small-vessel vasculitis.Nat. Med. 2009; 15: 623-625Crossref PubMed Scopus (1188) Google Scholar, Khandpur et al., 2013Khandpur R. Carmona-Rivera C. Vivekanandan-Giri A. Gizinski A. Yalavarthi S. Knight J.S. Friday S. Li S. Patel R.M. Subramanian V. et al.NETs are a source of citrullinated autoantigens and stimulate inflammatory responses in rheumatoid arthritis.Sci. Transl. Med. 2013; 5: 178ra140Crossref Scopus (822) Google Scholar, Lande et al., 2011Lande R. Ganguly D. Facchinetti V. Frasca L. Conrad C. Gregorio J. Meller S. Chamilos G. Sebasigari R. Riccieri V. et al.Neutrophils activate plasmacytoid dendritic cells by releasing self-DNA-peptide complexes in systemic lupus erythematosus.Sci. Transl. Med. 2011; 3: 73ra19Crossref PubMed Scopus (947) Google Scholar, Papayannopoulos et al., 2011Papayannopoulos V. Staab D. Zychlinsky A. Neutrophil elastase enhances sputum solubilization in cystic fibrosis patients receiving DNase therapy.PLoS ONE. 2011; 6: e28526Crossref PubMed Scopus (174) Google Scholar, Villanueva et al., 2011Villanueva E. Yalavarthi S. Berthier C.C. Hodgin J.B. Khandpur R. Lin A.M. Rubin C.J. Zhao W. Olsen S.H. Klinker M. et al.Netting neutrophils induce endothelial damage, infiltrate tissues, and expose immunostimulatory molecules in systemic lupus erythematosus.J. Immunol. 2011; 187: 538-552Crossref PubMed Scopus (876) Google Scholar). Therefore, it is critical to understand the mechanisms that regulate NET formation. NETs form in response to specific stimuli through a unique form of cell death called “NETosis.” The nuclear material expands while chromatin decondenses and the nuclear envelope disintegrates. The cytoplasmic membrane ruptures, liberating the NETs (Fuchs et al., 2007Fuchs T.A. Abed U. Goosmann C. Hurwitz R. Schulze I. Wahn V. Weinrauch Y. Brinkmann V. Zychlinsky A. Novel cell death program leads to neutrophil extracellular traps.J. Cell Biol. 2007; 176: 231-241Crossref PubMed Scopus (2237) Google Scholar). A fraction of neutrophils have also been reported to release NETs without dying, leaving behind cytoplasts that continue to ingest microbes (Pilsczek et al., 2010Pilsczek F.H. Salina D. Poon K.K.H. Fahey C. Yipp B.G. Sibley C.D. Robbins S.M. Green F.H.Y. Surette M.G. Sugai M. et al.A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus.J. Immunol. 2010; 185: 7413-7425Crossref PubMed Scopus (746) Google Scholar, Yipp et al., 2012Yipp B.G. Petri B. Salina D. Jenne C.N. Scott B.N. Zbytnuik L.D. Pittman K. Asaduzzaman M. Wu K. Meijndert H.C. et al.Infection-induced NETosis is a dynamic process involving neutrophil multitasking in vivo.Nat. Med. 2012; 18: 1386-1393Crossref PubMed Scopus (744) Google Scholar). Α factor that is known to be critical for NET formation is neutrophil elastase (NE) (Papayannopoulos et al., 2010Papayannopoulos V. Metzler K.D. Hakkim A. Zychlinsky A. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps.J. Cell Biol. 2010; 191: 677-691Crossref PubMed Scopus (1270) Google Scholar). This serine protease is stored in azurophilic granules and contributes to antimicrobial activity in the phagosome. During NET formation, NE translocates from the granules to the nucleus and partially cleaves histones to promote chromatin decondensation (Papayannopoulos et al., 2010Papayannopoulos V. Metzler K.D. Hakkim A. Zychlinsky A. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps.J. Cell Biol. 2010; 191: 677-691Crossref PubMed Scopus (1270) Google Scholar). The mechanism of NE release from azurophilic granules remains unknown and does not involve membrane fusion. ROS are crucial for effective antimicrobial responses. Patients with chronic granulomatous disease (CGD), who are deficient in NADPH oxidase activity, and individuals who are completely deficient in myeloperoxidase (ΔMPO; Figure S1A) are susceptible to opportunistic infections, particularly to fungal pathogens (Nauseef, 2007Nauseef W.M. How human neutrophils kill and degrade microbes: an integrated view.Immunol. Rev. 2007; 219: 88-102Crossref PubMed Scopus (568) Google Scholar). Neutrophils from these patients fail to form NETs when stimulated with physiological NET stimuli such as fungi or the ROS agonist phorbol myristate acetate (PMA) (Fuchs et al., 2007Fuchs T.A. Abed U. Goosmann C. Hurwitz R. Schulze I. Wahn V. Weinrauch Y. Brinkmann V. Zychlinsky A. Novel cell death program leads to neutrophil extracellular traps.J. Cell Biol. 2007; 176: 231-241Crossref PubMed Scopus (2237) Google Scholar, Metzler et al., 2011Metzler K.D. Fuchs T.A. Nauseef W.M. Reumaux D. Roesler J. Schulze I. Wahn V. Papayannopoulos V. Zychlinsky A. Myeloperoxidase is required for neutrophil extracellular trap formation: implications for innate immunity.Blood. 2011; 117: 953-959Crossref PubMed Scopus (493) Google Scholar). Upon stimulation, neutrophils rapidly activate the NADPH oxidase to generate superoxide, a highly reactive molecule that dismutates to hydrogen peroxide (H2O2) (Winterbourn and Kettle, 2013Winterbourn C.C. Kettle A.J. Redox reactions and microbial killing in the neutrophil phagosome.Antioxid. Redox Signal. 2013; 18: 642-660Crossref PubMed Scopus (316) Google Scholar). H2O2 is consumed by MPO to produce hypochlorous acid (HOCl) and other oxidants. MPO is also required for NET formation, as shown in donors with complete MPO deficiency, but its role remains unclear (Metzler et al., 2011Metzler K.D. Fuchs T.A. Nauseef W.M. Reumaux D. Roesler J. Schulze I. Wahn V. Papayannopoulos V. Zychlinsky A. Myeloperoxidase is required for neutrophil extracellular trap formation: implications for innate immunity.Blood. 2011; 117: 953-959Crossref PubMed Scopus (493) Google Scholar). Although ROS are cytotoxic, they are also important signaling mediators that regulate protein function via the oxidation of specific amino acid residues (Nathan, 2003Nathan C. Specificity of a third kind: reactive oxygen and nitrogen intermediates in cell signaling.J. Clin. Invest. 2003; 111: 769-778Crossref PubMed Scopus (389) Google Scholar, Tonks, 2005Tonks N.K. Redox redux: revisiting PTPs and the control of cell signaling.Cell. 2005; 121: 667-670Abstract Full Text Full Text PDF PubMed Scopus (607) Google Scholar, Winterbourn, 2008Winterbourn C.C. Reconciling the chemistry and biology of reactive oxygen species.Nat. Chem. Biol. 2008; 4: 278-286Crossref PubMed Scopus (1789) Google Scholar). However, since ROS are highly reactive, short-lived molecules, it is unclear how they are able to produce specific cellular responses. In particular, during NET formation, it is not known whether and how ROS regulate the selective translocation of NE from the granules to the nucleus. Furthermore, as the nucleus begins to decondense during NET formation, neutrophil chemotaxis is arrested through an unknown mechanism. Using primary human neutrophils and proteins purified from healthy individuals and patient donors, we show that NE translocation involves a mechanism that does not require membrane fusion and regulates protease activation and actin dynamics. Since ROS production precedes NE translocation to the nucleus, we tested whether ROS and MPO are required for this process. In contrast to neutrophils from healthy “control” donors, in neutrophils derived from CGD and ΔMPO donors stimulated with Candida albicans (Figure 1A) or PMA (Figure S1B), NE failed to translocate to the nucleus and remained in granules. We examined whether NE is first released from the granules into the cytosol. We stimulated neutrophils with PMA, lysed them at different time points, and isolated cytoplasm containing the soluble cytosol and granules. We obtained cytosol, which contains only the released soluble proteins, by ultracentrifugation of cytoplasm to remove granules, and monitored the presence of NE in these subcellular fractions by ELISA. NE was detected transiently in the cytosol of control neutrophils 60 min after activation (Figure 1B). Consistent with previous observations (Papayannopoulos et al., 2010Papayannopoulos V. Metzler K.D. Hakkim A. Zychlinsky A. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps.J. Cell Biol. 2010; 191: 677-691Crossref PubMed Scopus (1270) Google Scholar), NE disappeared from the cytosol 120 min after activation, as it translocated to the nucleus. In contrast, in ΔMPO neutrophils, NE was not detected in the cytosol. NE proteolytic activity in the cytosol from naive and activated neutrophils was also detected by adding purified recombinant histone H4 to these fractions. H4 is the relevant NE substrate during NETosis. Background partial H4 cleavage was detected in cytosol from naive neutrophils, which may be due to cytosolic proteases. Notably, H4 was completely degraded when incubated with cytosol from control neutrophils stimulated with PMA for 30 min (Figure 1C). This is consistent with the presence of active NE in the cytosol, since we previously showed that this protease degrades soluble H4 processively (Papayannopoulos et al., 2010Papayannopoulos V. Metzler K.D. Hakkim A. Zychlinsky A. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps.J. Cell Biol. 2010; 191: 677-691Crossref PubMed Scopus (1270) Google Scholar). The peak of processive H4 degradation coincided with the highest cytosolic NE concentration detected by ELISA (Figure 1C). Recombinant H4 degradation was blocked by the small-molecule, cell-permeable NE inhibitor (NEi) GW311616A (Macdonald et al., 2001Macdonald S.J.F. Dowle M.D. Harrison L.A. Shah P. Johnson M.R. Inglis G.G.A. Clarke G.D.E. Smith R.A. Humphreys D. Molloy C.R. et al.The discovery of a potent, intracellular, orally bioavailable, long duration inhibitor of human neutrophil elastase—GW311616A a development candidate.Bioorg. Med. Chem. Lett. 2001; 11: 895-898Crossref PubMed Scopus (52) Google Scholar), but not by an inhibitor of the related azurophilic granule protease, cathepsin G (CGi; Figure S1E), indicating that H4 was degraded by active NE in the cytosol. In contrast, only background protease activity was detected in the cytosol of ΔMPO neutrophils, suggesting that MPO is required for the release of proteolytically active NE from the granules into the cytosol during NET formation. Since the final destination of NE during NETosis is the nucleus, where it targets core histones, we examined the degradation of endogenous neutrophil nuclear H4 in activated control and ΔMPO neutrophils. Histone H4 was not cleaved in ΔMPO neutrophils stimulated with C. albicans (Figure 1D) or ΔMPO and CGD neutrophils stimulated with PMA (Figures S1C and S1D). Thus, ROS and MPO are required for the release of NE to the cytosol and its subsequent translocation to the nucleus during NETosis. Importantly, the route of NE translocation via the cytosol hinted that the translocation was driven by a novel MPO-dependent mechanism that does not involve membrane fusion. We also examined whether this mechanism is implicated in the delivery of NE to the phagosome, which involves membrane fusion. Notably, NE cleaves bacterial virulence factors such as the Shigella flexneri IpaB protein inside the phagosome, preventing microbial escape from the phagosome (Weinrauch et al., 2002Weinrauch Y. Drujan D. Shapiro S.D. Weiss J. Zychlinsky A. Neutrophil elastase targets virulence factors of enterobacteria.Nature. 2002; 417: 91-94Crossref PubMed Scopus (251) Google Scholar). Therefore, we tested whether MPO is required for NE function in the phagosome by incubating neutrophils with S. flexneri and examining the cleavage of the phagocytosed IpaB. IpaB was cleaved equally well by control neutrophils in the absence and presence of a pharmacological MPO inhibitor (ABAH; Figure S1F). Furthermore, neutrophils derived from a ΔMPO donor degraded IpaB with comparable efficiency. As expected, IpaB degradation was prevented in control and ΔMPO neutrophils when NE activity was inhibited pharmacologically by NEi (Macdonald et al., 2001Macdonald S.J.F. Dowle M.D. Harrison L.A. Shah P. Johnson M.R. Inglis G.G.A. Clarke G.D.E. Smith R.A. Humphreys D. Molloy C.R. et al.The discovery of a potent, intracellular, orally bioavailable, long duration inhibitor of human neutrophil elastase—GW311616A a development candidate.Bioorg. Med. Chem. Lett. 2001; 11: 895-898Crossref PubMed Scopus (52) Google Scholar). Thus, this mechanism for NE release appears to be specific to NET formation and not phagocytosis. Next, we investigated whether the factors that mediate NE release are contained in azurophilic granules. We isolated intact azurophilic granules by nitrogen cavitation and discontinuous Percoll density gradient centrifugation, which separates this granule subtype from other neutrophil granules and cytosol (Kjeldsen et al., 1994Kjeldsen L. Sengeløv H. Lollike K. Nielsen M.H. Borregaard N. Isolation and characterization of gelatinase granules from human neutrophils.Blood. 1994; 83: 1640-1649Crossref PubMed Google Scholar). To detect release of NE, we incubated granules with exogenous β-galactosidase and monitored its degradation by loss of β-galactosidase activity. To avoid variations in protease content between different granule preparations, we normalized the concentration of granules based on the content of NE and CG as measured by ELISA and immunoblotting. First, we tested whether H2O2 was sufficient to trigger NE release in this system. Several lines of evidence suggest that H2O2, the substrate of MPO, is a key ROS intermediate in NET formation, since it is sufficient to stimulate NET formation in neutrophils (Fuchs et al., 2007Fuchs T.A. Abed U. Goosmann C. Hurwitz R. Schulze I. Wahn V. Weinrauch Y. Brinkmann V. Zychlinsky A. Novel cell death program leads to neutrophil extracellular traps.J. Cell Biol. 2007; 176: 231-241Crossref PubMed Scopus (2237) Google Scholar) and in a cell-free assay where neutrophil nuclei are incubated with cytoplasmic extracts containing azurophilic granules in vitro (Figure S2A; Papayannopoulos et al., 2010Papayannopoulos V. Metzler K.D. Hakkim A. Zychlinsky A. Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps.J. Cell Biol. 2010; 191: 677-691Crossref PubMed Scopus (1270) Google Scholar). Moreover, catalase, which consumes H2O2, blocks NET formation (Fuchs et al., 2007Fuchs T.A. Abed U. Goosmann C. Hurwitz R. Schulze I. Wahn V. Weinrauch Y. Brinkmann V. Zychlinsky A. Novel cell death program leads to neutrophil extracellular traps.J. Cell Biol. 2007; 176: 231-241Crossref PubMed Scopus (2237) Google Scholar, Parker et al., 2012Parker H. Dragunow M. Hampton M.B. Kettle A.J. Winterbourn C.C. Requirements for NADPH oxidase and myeloperoxidase in neutrophil extracellular trap formation differ depending on the stimulus.J. Leukoc. Biol. 2012; 92: 841-849Crossref PubMed Scopus (295) Google Scholar). Azurophilic granules from control donors degraded β-galactosidase upon treatment with H2O2, indicating that active proteases were released and gained access to the substrate in the absence of detergent (Figures 2A and S2B). NEi and CGi together, but not individually, decreased β-galactosidase degradation, indicating the release of multiple active proteases (Figure S2C). Surprisingly, H2O2 did not disrupt the overall integrity of the granules, as reflected by the conservation of the granule signature in a CASY impedance counter, which measures membrane integrity by the exclusion of electrical current (Figure 2B). This observation suggested that the mechanism of NE release does not involve the dissolution of granule membranes, but rather a novel means of release from intact granules. Moreover, H2O2 failed to induce β-galactosidase degradation in ΔMPO granules, suggesting that MPO is required for NE release (Figures 2A and S2B). To examine whether H2O2 plays a role in activating proteases in this assay independently of their release, we tested β-galactosidase degradation after dissolving granule membranes with detergent to expose the substrate to the proteases. In control granules, addition of detergent did not induce β-galactosidase degradation, but proteolytic activity required H2O2 even in the absence of membranes (Figures 2A and S2B). In contrast, H2O2 treatment failed to activate proteases in ΔMPO granules treated with detergent. Therefore, the factors that drive protease activation and release in response to H2O2 are localized in azurophilic granules, and MPO is critical for both processes. To identify the factors that mediate NE release and activation, we probed for NE-binding partners in azurophilic granules by immunoprecipitation. We isolated azurophilic granules from control neutrophils, solubilized them with detergent, and immunoprecipitated proteins with an antibody against NE or a control mock antibody against matrix metalloproteinase 9 (MMP9), a protein that is stored in gelatinase granules. Anti-NE, but not the control antibody, selectively coimmunoprecipitated a granule protein complex containing MPO, azurocidin (AZU), CG, eosinophil cationic protein (ECP), defensin-1 (HD1), lysozyme (LYZ), and lactoferrin (LTF) (Figure 2C). LTF is primarily a specific granule protein, but it has also been found in azurophilic granules (Lominadze et al., 2005Lominadze G. Powell D.W. Luerman G.C. Link A.J. Ward R.A. McLeish K.R. Proteomic analysis of human neutrophil granules.Mol. Cell. Proteomics. 2005; 4: 1503-1521Crossref PubMed Scopus (261) Google Scholar). Western blot analysis confirmed the specific immunoprecipitation of several of these proteins (see Figures 4B–4D). To further confirm the specificity of the immunoprecipitation, we immunoblotted against an azurophilic granule protein that was not immunoprecipitated. The bactericidal/permeability-increasing protein (BPI) was detected only upon immunoprecipitation with an antibody against BPI, and not with an anti-NE antibody (Figure 2D). Interestingly, treatment of intact granules with H2O2 prior to solubilization and immunoprecipitation led to the dissociation of this complex, as significantly less protein was coimmunoprecipitated (Figure 2C). In order to investigate the effects of oxidation on the complex, we isolated azurophilic granules from peripheral blood neutrophils of healthy human donors and purified the complex by size-exclusion chromatography, probing the fractions for NE and MPO (Figures S3A and S3B). The complex eluted at a higher molecular weight than purified MPO (Figure S3B) and contained the same proteins that coprecipitated with NE as detected by mass spectrometry (Figure 2E). We also identified proteinase 3 (PR3), a related azurophilic granule protease with high homology to NE (Korkmaz et al., 2008Korkmaz B. Moreau T. Gauthier F. Neutrophil elastase, proteinase 3 and cathepsin G: physicochemical properties, activity and physiopathological functions.Biochimie. 2008; 90: 227-242Crossref PubMed Scopus (338) Google Scholar), in the purified complex. NE and MPO are present in the complex at a ratio of 2:1. Similarly to the immunoprecipitated complex (Figure 2C), the purified complex dissociated when pretreated with H2O2, as we did not detect these proteins in the complex-containing fractions by mass spectrometry, ELISA, or enzymatic activity (Figures S3C–S3E). This observation hinted that H2O2 may regulate the function of this azurophilic granule complex by modulating the association of its components. To facilitate the nomenclature, we refer to this azurophilic granule complex as the “azurosome.” To investigate the localization of the complex in neutrophils, we labeled MPO and NE with immunogold and performed transmission electron microscopy. We found three subsets of granules in naive and activated neutrophils of control and MPO-deficient neutrophils. In one subset, NE and MPO localized to the granule membrane in a radial pattern (Figure 3A , arrows). In the second subset of granules, NE and MPO were predominantly in the lumen (Figure 3B). In a third subpopulation, MPO and NE localized to both the membrane and the lumen (mixed). Our observations are consistent with similar findings in promyelocytes (Egesten et al., 1994Egesten A. Breton-Gorius J. Guichard J. Gullberg U. Olsson I. The heterogeneity of azurophil granules in neutrophil promyelocytes: immunogold localization of myeloperoxidase, cathepsin G, elastase, proteinase 3, and bactericidal/permeability increasing protein.Blood. 1994; 83: 2985-2994Crossref PubMed Google Scholar) and confirm the heterogeneity of azurophilic granules (Borregaard, 2010Borregaard N. Neutrophils, from marrow to microbes.Immunity. 2010; 33: 657-670Abstract Full Text Full Text PDF PubMed Scopus (972) Google Scholar). Quantitation of electron micrographs showed that MPO and NE were localized exclusively in the membrane in 50% of labeled granules and exclusively in the lumen in 25%. In the remaining 25%, the proteins were localized in both the membrane and the lumen (Figure 3C). These data are consistent with the idea that azurosome components localize in the membrane in a subset of azurophilic granules, but they do not constitute a quantitative assessment of protein association and abundance. To determine whether the azurosome is exposed on granule membranes, we incubated isolated native azurophilic granules with an antibody against MPO or a control antibody against BPI, a protein that is not found in the azurosome (Figure 2D) and is not expected to be on the membrane. We centrifuged the mixture of granules and antibodies over a discontinuous Percoll gradient and isolated the intact azurophilic granules from the appropriate gradient fraction. Only the antibody against MPO was detected in the azurophilic granule fraction, and the antibody against BPI did not cosediment, confirming that the granule membranes were intact and undamaged, shielding BPI from antibody recognition (Figure 3D). Neither of the two antibodies sedimented in the absence of granules. These results indicated that MPO is exposed on the surface of azurophilic granules and is accessible to antibodies added externally. Consistently, treatment of azurophilic granules from naive control neutrophils with proteinase K in the absence of detergent partially degraded MPO and AZU, indicating that in naive neutrophils, a fraction of MPO and AZU are exposed at the surface of granules (Figure 3E). AZU was better protected than MPO, suggesting that the former may be less exposed. BPI was completely protected from degradation, corroborating that the granules were intact and the exposure of MPO and AZU was not due to damaged granule membranes. This was further confirmed by impedance measurement of membrane integrity (not shown). Together, these data suggest that naive neutrophils contain a subset of azurophilic granules that harbor the azurosome on their membranes and are poised to release proteases upon oxidative stimulation. The association with membranes does not seem to require MPO, since NE localizes to granule membranes in both control and ΔMPO neutrophils (Figure 3A). Rather, MPO is required for the ability of the complex to release proteins across membranes. The results shown in Figure 2C suggested that oxidants may regulate NE release from the granule by modulating the newly identified membrane-associated complex. To address whether H2O2 is required for NE release during NETosis in neutrophils, we tested whether depleting intracellular H2O2 with PEG-catalase, which is taken up by the cells and consumes H2O2, would block NE release into the cytosol. PEG-catalase completely blocked NE release, indicating that H2O2 regulates this process and is required for NETosis (Figure 4A). Next, we investigated how H2O2 triggers NE release. We previously found that during NETosis, NE translocates to the nucleus while MPO remains in the granules (Papayannopoulos et al., 2010Papayannopoulos V. Metzler K.D. Hakkim A. Zychlinsky A. Neutrophil elastase and myeloperoxidase regulat" @default.
• W2011145349 created "2016-06-24" @default.
• W2011145349 creator A5023986734 @default.
• W2011145349 creator A5028505633 @default.
• W2011145349 creator A5042380681 @default.
• W2011145349 creator A5056416821 @default.
• W2011145349 creator A5088622582 @default.
• W2011145349 date "2014-08-01" @default.
• W2011145349 modified "2023-10-17" @default.
• W2011145349 title "A Myeloperoxidase-Containing Complex Regulates Neutrophil Elastase Release and Actin Dynamics during NETosis" @default.
• W2011145349 cites W1216511763 @default.
• W2011145349 cites W1578854153 @default.
• W2011145349 cites W1820769215 @default.
• W2011145349 cites W1964457547 @default.
• W2011145349 cites W1980734840 @default.
• W2011145349 cites W1991952977 @default.
• W2011145349 cites W2004121149 @default.
• W2011145349 cites W2004536682 @default.
• W2011145349 cites W2014678245 @default.
• W2011145349 cites W2019916852 @default.
• W2011145349 cites W2030193400 @default.
• W2011145349 cites W2031729422 @default.
• W2011145349 cites W2038859753 @default.
• W2011145349 cites W2041912799 @default.
• W2011145349 cites W2043756611 @default.
• W2011145349 cites W2067022007 @default.
• W2011145349 cites W2080878773 @default.
• W2011145349 cites W2082827619 @default.
• W2011145349 cites W2089782191 @default.
• W2011145349 cites W2097560724 @default.
• W2011145349 cites W2099356583 @default.
• W2011145349 cites W2104934425 @default.
• W2011145349 cites W2108585076 @default.
• W2011145349 cites W2112543741 @default.
• W2011145349 cites W2115959782 @default.
• W2011145349 cites W2118217258 @default.
• W2011145349 cites W2129703385 @default.
• W2011145349 cites W2129794631 @default.
• W2011145349 cites W2134595043 @default.
• W2011145349 cites W2135915191 @default.
• W2011145349 cites W2141522272 @default.
• W2011145349 cites W2145019007 @default.
• W2011145349 cites W2147154305 @default.
• W2011145349 cites W2155178750 @default.
• W2011145349 cites W2314530439 @default.
• W2011145349 cites W4242509007 @default.
• W2011145349 doi "https://doi.org/10.1016/j.celrep.2014.06.044" @default.
• W2011145349 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4471680" @default.
• W2011145349 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/25066128" @default.
• W2011145349 hasPublicationYear "2014" @default.
• W2011145349 type Work @default.
• W2011145349 sameAs 2011145349 @default.
• W2011145349 citedByCount "473" @default.
• W2011145349 countsByYear W20111453492014 @default.
• W2011145349 countsByYear W20111453492015 @default.
• W2011145349 countsByYear W20111453492016 @default.
• W2011145349 countsByYear W20111453492017 @default.
• W2011145349 countsByYear W20111453492018 @default.
• W2011145349 countsByYear W20111453492019 @default.
• W2011145349 countsByYear W20111453492020 @default.
• W2011145349 countsByYear W20111453492021 @default.
• W2011145349 countsByYear W20111453492022 @default.
• W2011145349 countsByYear W20111453492023 @default.
• W2011145349 crossrefType "journal-article" @default.
• W2011145349 hasAuthorship W2011145349A5023986734 @default.
• W2011145349 hasAuthorship W2011145349A5028505633 @default.
• W2011145349 hasAuthorship W2011145349A5042380681 @default.
• W2011145349 hasAuthorship W2011145349A5056416821 @default.
• W2011145349 hasAuthorship W2011145349A5088622582 @default.
• W2011145349 hasBestOaLocation W20111453491 @default.
• W2011145349 hasConcept C121332964 @default.
• W2011145349 hasConcept C125705527 @default.
• W2011145349 hasConcept C134897094 @default.
• W2011145349 hasConcept C145912823 @default.
• W2011145349 hasConcept C181199279 @default.
• W2011145349 hasConcept C185592680 @default.
• W2011145349 hasConcept C198677920 @default.
• W2011145349 hasConcept C202751555 @default.
• W2011145349 hasConcept C203014093 @default.
• W2011145349 hasConcept C203565725 @default.
• W2011145349 hasConcept C24890656 @default.
• W2011145349 hasConcept C2776317360 @default.
• W2011145349 hasConcept C2776326425 @default.
• W2011145349 hasConcept C2776914184 @default.
• W2011145349 hasConcept C2781044401 @default.
• W2011145349 hasConcept C55493867 @default.
• W2011145349 hasConcept C86803240 @default.
• W2011145349 hasConcept C95444343 @default.
• W2011145349 hasConceptScore W2011145349C121332964 @default.
• W2011145349 hasConceptScore W2011145349C125705527 @default.
• W2011145349 hasConceptScore W2011145349C134897094 @default.
• W2011145349 hasConceptScore W2011145349C145912823 @default.
• W2011145349 hasConceptScore W2011145349C181199279 @default.
• W2011145349 hasConceptScore W2011145349C185592680 @default.
• W2011145349 hasConceptScore W2011145349C198677920 @default.
• W2011145349 hasConceptScore W2011145349C202751555 @default.
• W2011145349 hasConceptScore W2011145349C203014093 @default.
• W2011145349 hasConceptScore W2011145349C203565725 @default.

• next