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Eosinophil Granule Proteins: Form and Function

  • K. Ravi Acharya
    Correspondence
    To whom correspondence may be addressed. Tel.: 44-1225-386238
    Affiliations
    Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, United Kingdom
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  • Steven J. Ackerman
    Correspondence
    Recipient of a David Parkin Visiting Professorship from the University of Bath. To whom correspondence may be addressed: Dept. of Biochemistry and Molecular Genetics, the University of Illinois at Chicago, College of Medicine, 900 S. Ashland Ave., Chicago, IL 60607. Tel.: 312-996-6149
    Affiliations
    the Department of Biochemistry and Molecular Genetics, College of Medicine, The University of Illinois, Chicago, Illinois 60607
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Open AccessPublished:May 06, 2014DOI:https://doi.org/10.1074/jbc.R113.546218
      Experimental and clinical data strongly support a role for the eosinophil in the pathogenesis of asthma, allergic and parasitic diseases, and hypereosinophilic syndromes, in addition to more recently identified immunomodulatory roles in shaping innate host defense, adaptive immunity, tissue repair/remodeling, and maintenance of normal tissue homeostasis. A seminal finding was the dependence of allergic airway inflammation on eosinophil-induced recruitment of Th2-polarized effector T-cells to the lung, providing a missing link between these innate immune effectors (eosinophils) and adaptive T-cell responses. Eosinophils come equipped with preformed enzymatic and nonenzymatic cationic proteins, stored in and selectively secreted from their large secondary (specific) granules. These proteins contribute to the functions of the eosinophil in airway inflammation, tissue damage, and remodeling in the asthmatic diathesis. Studies using eosinophil-deficient mouse models, including eosinophil-derived granule protein double knock-out mice (major basic protein-1/eosinophil peroxidase dual gene deletion) show that eosinophils are required for all major hallmarks of asthma pathophysiology: airway epithelial damage and hyperreactivity, and airway remodeling including smooth muscle hyperplasia and subepithelial fibrosis. Here we review key molecular aspects of these eosinophil-derived granule proteins in terms of structure-function relationships to advance understanding of their roles in eosinophil cell biology, molecular biology, and immunobiology in health and disease.

      Introduction

      Over the past three decades, the role of the eosinophil in human health and disease has received considerable attention (
      • Rosenberg H.F.
      • Dyer K.D.
      • Foster P.S.
      Eosinophils: changing perspectives in health and disease.
      ,
      • Hogan S.P.
      • Rosenberg H.F.
      • Moqbel R.
      • Phipps S.
      • Foster P.S.
      • Lacy P.
      • Kay A.B.
      • Rothenberg M.E.
      Eosinophils: biological properties and role in health and disease.
      ). The eosinophil has a vital role in allergic inflammatory processes that include asthma (
      • Lee J.J.
      • Dimina D.
      • Macias M.P.
      • Ochkur S.I.
      • McGarry M.P.
      • O'Neill K.R.
      • Protheroe C.
      • Pero R.
      • Nguyen T.
      • Cormier S.A.
      • Lenkiewicz E.
      • Colbert D.
      • Rinaldi L.
      • Ackerman S.J.
      • Irvin C.G.
      • Lee N.A.
      Defining a link with asthma in mice congenitally deficient in eosinophils.
      ,
      • Humbles A.A.
      • Lloyd C.M.
      • McMillan S.J.
      • Friend D.S.
      • Xanthou G.
      • McKenna E.E.
      • Ghiran S.
      • Gerard N.P.
      • Yu C.
      • Orkin S.H.
      • Gerard C.
      A critical role for eosinophils in allergic airways remodeling.
      ). Evidence implicates the eosinophil and its granule proteins in host resistance to parasites, particularly helminths, but also antimicrobial activities toward bacterial, viral, and protozoan pathogens, and as mediators of hypersensitivity diseases. This evidence consists of associations between elevated levels of eosinophils in blood and the occurrence of disease; correlations between disease severity and degree of eosinophilia; findings that the cationic eosinophil-derived granule proteins (EDGPs)
      The abbreviations used are:
      EDGP
      eosinophil-derived granule protein
      MBP
      major basic protein
      EPX
      eosinophil peroxidase
      ECP
      eosinophil cationic protein
      EDN
      eosinophil-derived neurotoxin
      CLC/Gal-10
      Charcot-Leyden crystal protein/Galectin-10
      Treg
      regulatory T cell
      EETs
      eosinophil extracellular DNA traps
      EAR
      eosinophil-associated ribonuclease
      NMS
      N-methyl scopolamine
      DC
      dendritic cell
      LPLase
      lysophospholipase.
      are toxic to cells and human tissues, producing changes mimicking those associated with disease (e.g. in bronchial asthma); deposition of the toxic EDGPs in diseased tissue; and observations that glucocorticoids suppress eosinophilia as part of their therapeutic effect (
      ).
      The eosinophil is rich in cationic granule proteins, shows a striking respiratory burst with the production of toxic oxygen radicals, brominates tissue and proteins, presents antigen to T-cells, and expresses both hematopoietic and inflammatory cytokines (
      • Hogan S.P.
      • Rosenberg H.F.
      • Moqbel R.
      • Phipps S.
      • Foster P.S.
      • Lacy P.
      • Kay A.B.
      • Rothenberg M.E.
      Eosinophils: biological properties and role in health and disease.
      ). These findings support the eosinophil as a key player in allergic inflammation and tissue homeostasis in hypersensitivity diseases (
      • Rosenberg H.F.
      • Dyer K.D.
      • Foster P.S.
      Eosinophils: changing perspectives in health and disease.
      ,
      • Hogan S.P.
      • Rosenberg H.F.
      • Moqbel R.
      • Phipps S.
      • Foster P.S.
      • Lacy P.
      • Kay A.B.
      • Rothenberg M.E.
      Eosinophils: biological properties and role in health and disease.
      ). Eosinophil-deficient mouse models (
      • Lee J.J.
      • Dimina D.
      • Macias M.P.
      • Ochkur S.I.
      • McGarry M.P.
      • O'Neill K.R.
      • Protheroe C.
      • Pero R.
      • Nguyen T.
      • Cormier S.A.
      • Lenkiewicz E.
      • Colbert D.
      • Rinaldi L.
      • Ackerman S.J.
      • Irvin C.G.
      • Lee N.A.
      Defining a link with asthma in mice congenitally deficient in eosinophils.
      ,
      • Humbles A.A.
      • Lloyd C.M.
      • McMillan S.J.
      • Friend D.S.
      • Xanthou G.
      • McKenna E.E.
      • Ghiran S.
      • Gerard N.P.
      • Yu C.
      • Orkin S.H.
      • Gerard C.
      A critical role for eosinophils in allergic airways remodeling.
      ,
      • Jacobsen E.A.
      • Lesuer W.E.
      • Willetts L.
      • Zellner K.R.
      • Mazzolini K.
      • Antonios N.
      • Beck B.
      • Protheroe C.
      • Ochkur S.I.
      • Colbert D.
      • Lacy P.
      • Moqbel R.
      • Appleton J.
      • Lee N.A.
      • Lee J.J.
      Eosinophil activities modulate the immune/inflammatory character of allergic respiratory responses in mice.
      ), including EDGP gene-deleted mice (
      • Denzler K.L.
      • Farmer S.C.
      • Crosby J.R.
      • Borchers M.
      • Cieslewicz G.
      • Larson K.A.
      • Cormier-Regard S.
      • Lee N.A.
      • Lee J.J.
      Eosinophil major basic protein-1 does not contribute to allergen-induced airway pathologies in mouse models of asthma.
      ,
      • Specht S.
      • Saeftel M.
      • Arndt M.
      • Endl E.
      • Dubben B.
      • Lee N.A.
      • Lee J.J.
      • Hoerauf A.
      Lack of eosinophil peroxidase or major basic protein impairs defense against murine filarial infection.
      ,
      • Doyle A.D.
      • Jacobsen E.A.
      • Ochkur S.I.
      • McGarry M.P.
      • Shim K.G.
      • Nguyen D.T.
      • Protheroe C.
      • Colbert D.
      • Kloeber J.
      • Neely J.
      • Shim K.P.
      • Dyer K.D.
      • Rosenberg H.F.
      • Lee J.J.
      • Lee N.A.
      Expression of the secondary granule proteins major basic protein 1 (MBP-1) and eosinophil peroxidase (EPX) is required for eosinophilopoiesis in mice.
      ), provide unique insights into the role of the eosinophil, in both tissue damage/repair/remodeling and novel immunomodulatory roles that bridge host innate and adaptive immune responses in allergic and parasitic diseases.
      Initial views of the eosinophil as providing direct antipathogen (helminth) activity are being supplanted by more nuanced views of the immunomodulatory functions of the eosinophil, including roles in supporting parasitic nematode survival (
      • Fabre V.
      • Beiting D.P.
      • Bliss S.K.
      • Gebreselassie N.G.
      • Gagliardo L.F.
      • Lee N.A.
      • Lee J.J.
      • Appleton J.A.
      Eosinophil deficiency compromises parasite survival in chronic nematode infection.
      ,
      • Gebreselassie N.G.
      • Moorhead A.R.
      • Fabre V.
      • Gagliardo L.F.
      • Lee N.A.
      • Lee J.J.
      • Appleton J.A.
      Eosinophils preserve parasitic nematode larvae by regulating local immunity.
      ); Appleton and colleagues (
      • Fabre V.
      • Beiting D.P.
      • Bliss S.K.
      • Gebreselassie N.G.
      • Gagliardo L.F.
      • Lee N.A.
      • Lee J.J.
      • Appleton J.A.
      Eosinophil deficiency compromises parasite survival in chronic nematode infection.
      ,
      • Gebreselassie N.G.
      • Moorhead A.R.
      • Fabre V.
      • Gagliardo L.F.
      • Lee N.A.
      • Lee J.J.
      • Appleton J.A.
      Eosinophils preserve parasitic nematode larvae by regulating local immunity.
      ) show that growth and survival of the muscle stage larvae of Trichinella spiralis require eosinophils, which promote accumulation of Th2 cells and inhibit the induction of inducible NOS by macrophages and neutrophils. Studies using a conditional eosinophil-deficient mouse strain (iPHIL) show that eosinophils modulate the immune and inflammatory character of inducible allergic responses in the lung (
      • Jacobsen E.A.
      • Lesuer W.E.
      • Willetts L.
      • Zellner K.R.
      • Mazzolini K.
      • Antonios N.
      • Beck B.
      • Protheroe C.
      • Ochkur S.I.
      • Colbert D.
      • Lacy P.
      • Moqbel R.
      • Appleton J.
      • Lee N.A.
      • Lee J.J.
      Eosinophil activities modulate the immune/inflammatory character of allergic respiratory responses in mice.
      ). Clinical trials using anti-interleukin-5 (IL-5) antibodies to ablate eosinophils in the bone marrow and blood, as well as reduce tissue eosinophils in patients with the eosinophilic but not neutrophilic phenotype of asthma, show efficacy in reversing eosinophil-mediated tissue damage, remodeling, fibrosis, and airway dysfunction (
      • Nair P.
      • Pizzichini M.M.
      • Kjarsgaard M.
      • Inman M.D.
      • Efthimiadis A.
      • Pizzichini E.
      • Hargreave F.E.
      • O'Byrne P.M.
      Mepolizumab for prednisone-dependent asthma with sputum eosinophilia.
      ), as well as end-organ damage in the hypereosinophilic syndrome (
      • Rothenberg M.E.
      • Klion A.D.
      • Roufosse F.E.
      • Kahn J.E.
      • Weller P.F.
      • Simon H.U.
      • Schwartz L.B.
      • Rosenwasser L.J.
      • Ring J.
      • Griffin E.F.
      • Haig A.E.
      • Frewer P.I.
      • Parkin J.M.
      • Gleich G.J.
      Treatment of patients with the hypereosinophilic syndrome with mepolizumab.
      ), highlighting the complex proinflammatory and immunomodulatory activities of the eosinophil in shaping the pathogenesis of these diseases.

      Eosinophil-derived Granule Proteins

      With expectations that understanding the properties, activities, and secretion of eosinophil proteins in disease states would provide insights into cellular function, the cationic components of the large crystalloid-containing specific (secondary) granule of the eosinophil (Fig. 1) were extensively studied. These proteins include major basic protein-1 and -2 (MBP-1, MBP-2), eosinophil peroxidase (EPX), eosinophil cationic protein (ECP), and eosinophil-derived neurotoxin (EDN) (Fig. 1). The Charcot-Leyden crystal protein/Galectin-10 (CLC/Gal-10), although not cationic, is a hydrophobic autocrystallizing protein comprising ∼7–10% of total eosinophil protein (Fig. 1). EPX and MBP-2 are the only EDGPs uniquely expressed by the eosinophil and not other cells; the other EDGPs are variably expressed in ∼10–100-fold lesser amounts by other blood leukocytes, tissues, and cells including basophils (MBP-1 (
      • Leiferman K.M.
      • Gleich G.J.
      • Kephart G.M.
      • Haugen H.S.
      • Hisamatsu K.
      • Proud D.
      • Lichtenstein L.M.
      • Ackerman S.J.
      Differences between basophils and mast cells: failure to detect Charcot-Leyden crystal protein (lysophospholipase) and eosinophil granule major basic protein in human mast cells.
      ,
      • Ackerman S.J.
      • Kephart G.M.
      • Habermann T.M.
      • Greipp P.R.
      • Gleich G.J.
      Localization of eosinophil granule major basic protein in human basophils.
      ), EDN (
      • Abu-Ghazaleh R.I.
      • Dunnette S.L.
      • Loegering D.A.
      • Checkel J.L.
      • Kita H.
      • Thomas L.L.
      • Gleich G.J.
      Eosinophil granule proteins in peripheral blood granulocytes.
      ), CLC/Gal-10 (
      • Golightly L.M.
      • Thomas L.L.
      • Dvorak A.M.
      • Ackerman S.J.
      Charcot-Leyden crystal protein in the degranulation and recovery of activated basophils.
      ,
      • Ackerman S.J.
      • Weil G.J.
      • Gleich G.J.
      Formation of Charcot-Leyden crystals by human basophils.
      )), neutrophils (EDN, ECP) (
      • Rosenberg H.F.
      • Tenen D.G.
      • Ackerman S.J.
      Molecular cloning of the human eosinophil-derived neurotoxin: a member of the ribonuclease gene family.
      ,
      • Rosenberg H.F.
      • Ackerman S.J.
      • Tenen D.G.
      Human eosinophil cationic protein: molecular cloning of a cytotoxin and helminthotoxin with ribonuclease activity.
      ,
      • Sur S.
      • Glitz D.G.
      • Kita H.
      • Kujawa S.M.
      • Peterson E.A.
      • Weiler D.A.
      • Kephart G.M.
      • Wagner J.M.
      • George T.J.
      • Gleich G.J.
      • Leiferman K.M.
      Localization of eosinophil-derived neurotoxin and eosinophil cationic protein in neutrophilic leukocytes.
      ), liver (EDN) (
      • Sorrentino S.
      • Glitz D.G.
      • Hamann K.J.
      • Loegering D.A.
      • Checkel J.L.
      • Gleich G.J.
      Eosinophil-derived neurotoxin and human liver ribonuclease: identity of structure and linkage of neurotoxicity to nuclease activity.
      ), and regulatory T cells (Tregs) (
      • Kubach J.
      • Lutter P.
      • Bopp T.
      • Stoll S.
      • Becker C.
      • Huter E.
      • Richter C.
      • Weingarten P.
      • Warger T.
      • Knop J.
      • Müllner S.
      • Wijdenes J.
      • Schild H.
      • Schmitt E.
      • Jonuleit H.
      Human CD4+CD25+ regulatory T cells: proteome analysis identifies galectin-10 as a novel marker essential for their anergy and suppressive function.
      ,
      • Schmetterer K.G.
      • Neunkirchner A.
      • Pickl W.F.
      Naturally occurring regulatory T cells: markers, mechanisms, and manipulation.
      ) and TH2 central memory T cells (CLC/Gal-10) (
      • Wang Y.H.
      • Ito T.
      • Wang Y.H.
      • Homey B.
      • Watanabe N.
      • Martin R.
      • Barnes C.J.
      • McIntyre B.W.
      • Gilliet M.
      • Kumar R.
      • Yao Z.
      • Liu Y.J.
      Maintenance and polarization of human TH2 central memory T cells by thymic stromal lymphopoietin-activated dendritic cells.
      ). Although these EDGPs are expressed and may be secreted by these other cell types at sites of host innate and adaptive immune responses and inflammation, aside from expression of CLC/Gal-10 by regulatory T cells (
      • Kubach J.
      • Lutter P.
      • Bopp T.
      • Stoll S.
      • Becker C.
      • Huter E.
      • Richter C.
      • Weingarten P.
      • Warger T.
      • Knop J.
      • Müllner S.
      • Wijdenes J.
      • Schild H.
      • Schmitt E.
      • Jonuleit H.
      Human CD4+CD25+ regulatory T cells: proteome analysis identifies galectin-10 as a novel marker essential for their anergy and suppressive function.
      ), the roles and functions of these proteins beyond those of eosinophils have not been investigated, other than their potential use as biomarkers.
      Figure thumbnail gr1
      FIGURE 1Structural representations of the human eosinophil granule proteins showing their location and known functions. The cationic granule proteins shown are: MBP-1 (Protein Data Bank (PDB) code 1H8U); EDN (RNase-2) (PDB code 1HI2); ECP (RNase-3) (PDB code 1QMT); and EPX/EPO) (based on molecular modeling using a myeloperoxidase structure, PDB code 1D2V). Also shown is CLC/Gal-10 (PDB code 1LCL), which is mainly cytosolic but also present in a small residual population of primary large core-less granules in the mature eosinophil. CLC/Gal-10 forms the hexagonal bipyramidal crystals (arrow) considered a hallmark of eosinophilic inflammation in tissues and body fluids in eosinophil-associated diseases.
      Eosinophils form extracellular DNA traps (EETs), a component of innate antibacterial immune responses in a number of eosinophil-associated infectious, allergic, and autoimmune diseases (
      • Yousefi S.
      • Simon D.
      • Simon H.U.
      Eosinophil extracellular DNA traps: molecular mechanisms and potential roles in disease.
      ,
      • Yousefi S.
      • Gold J.A.
      • Andina N.
      • Lee J.J.
      • Kelly A.M.
      • Kozlowski E.
      • Schmid I.
      • Straumann A.
      • Reichenbach J.
      • Gleich G.J.
      • Simon H.U.
      Catapult-like release of mitochondrial DNA by eosinophils contributes to antibacterial defense.
      ). EETs consist of a meshwork of DNA fibers (formed from mitochondrial rather than nuclear DNA), and a number of EDGPs, including MBP-1 and ECP, co-localize in the EETs, suggesting that they participate in trapping and killing of bacteria by this mechanism (
      • Simon D.
      • Simon H.U.
      • Yousefi S.
      Extracellular DNA traps in allergic, infectious, and autoimmune diseases.
      ). EETs were identified in the lung in allergic asthma (
      • Dworski R.
      • Simon H.U.
      • Hoskins A.
      • Yousefi S.
      Eosinophil and neutrophil extracellular DNA traps in human allergic asthmatic airways.
      ) and in a number of allergic/reactive skin diseases including allergic contact dermatitis, ectoparasitoses, and larva migrans (
      • Simon D.
      • Hoesli S.
      • Roth N.
      • Staedler S.
      • Yousefi S.
      • Simon H.U.
      Eosinophil extracellular DNA traps in skin diseases.
      ). Multiple mechanisms of eosinophil priming and activation that include signaling through toll-like, cytokine, chemokine, and adhesion receptors initiate the formation of EETs containing the EDGPs (
      • Yousefi S.
      • Simon D.
      • Simon H.U.
      Eosinophil extracellular DNA traps: molecular mechanisms and potential roles in disease.
      ,
      • Morshed M.
      • Yousefi S.
      • Stöckle C.
      • Simon H.U.
      • Simon D.
      Thymic stromal lymphopoietin stimulates the formation of eosinophil extracellular traps.
      ) in a process requiring activation of NADPH oxidase. Although the specific role of the EDGPs in EETs has not been determined, their formation may provide a mechanism for bringing together these cationic toxins with their pathogen targets, or alternatively limit collateral tissue damage by the EDGPs (
      • Yousefi S.
      • Simon D.
      • Simon H.U.
      Eosinophil extracellular DNA traps: molecular mechanisms and potential roles in disease.
      ).

      Eosinophil Major Basic Protein-1

      MBP-1 (13.8 kDa, extremely basic, pI = 11.4) is an abundant granule protein localized to the electron-dense crystalloid core of the secondary granule. The protein is initially expressed as a 25.2-kDa polypeptide (pre-pro form), comprising a highly acidic “pro-domain” and highly basic MBP-1 protein. The pro-domain is thought to neutralize the basic nature of MBP-1 during its synthesis, transport, and packaging in the eosinophil granule, but structural or functional studies are lacking. MBP-1 is highly toxic to mammalian cells in vitro and can damage helminths, bacteria, and mammalian cells (
      • Gleich G.J.
      • Adolphson C.R.
      • Leiferman K.M.
      The biology of the eosinophilic leukocyte.
      ). MBP-1 damages cells by disrupting the lipid bilayer membrane (
      • Gleich G.J.
      • Adolphson C.R.
      • Leiferman K.M.
      The biology of the eosinophilic leukocyte.
      ) or altering the activity of enzymes within tissues. MBP-1 stimulates mediator (histamine) release from basophils and mast cells, activates neutrophils and platelets, and augments superoxide generation by alveolar macrophages. MBP-1 levels are elevated in biological fluids (e.g. sputa and bronchoalveolar lavage fluids) from patients with asthma and other eosinophil-associated diseases. MBP-1 induces bronchoconstriction when administered into monkey lung and induces transient airway hyperreactivity when administered into rat, rabbit, or monkey trachea (
      • Gundel R.H.
      • Letts L.G.
      • Gleich G.J.
      Human eosinophil major basic protein induces airway constriction and airway hyperresponsiveness in primates.
      ). These effects could be neutralized by polyanions such as heparin or polyglutamic acids. MBP-1 has been implicated in tissue damage associated with eosinophil infiltration, notably to the respiratory epithelium in asthma. MBP-1 binds lactoferrin (
      • Thomas L.L.
      • Xu W.
      • Ardon T.T.
      Immobilized lactoferrin is a stimulus for eosinophil activation,.
      ) and is a potent inhibitor of recombinant and eosinophil heparanase (
      • Temkin V.
      • Aingorn H.
      • Puxeddu I.
      • Goldshmidt O.
      • Zcharia E.
      • Gleich G.J.
      • Vlodavsky I.
      • Levi-Schaffer F.
      Eosinophil major basic protein: first identified natural heparanase-inhibiting protein.
      ). Direct instillation of MBP-1 into the mouse airway induces airway remodeling, including induction of epithelial TGF-β and MMP1 expression and subepithelial fibrosis (
      • Pégorier S.
      • Wagner L.A.
      • Gleich G.J.
      • Pretolani M.
      Eosinophil-derived cationic proteins activate the synthesis of remodeling factors by airway epithelial cells.
      ). In a model of Duchenne muscular dystrophy and the mdx mouse model of Duchenne muscular dystrophy, eosinophils were shown to lyse muscle cells in vitro through release of MBP-1, and MBP-1 promoted fibrosis of dystrophin-deficient muscle and attenuated the cellular immune response to these cells (
      • Wehling-Henricks M.
      • Sokolow S.
      • Lee J.J.
      • Myung K.H.
      • Villalta S.A.
      • Tidball J.G.
      Major basic protein-1 promotes fibrosis of dystrophic muscle and attenuates the cellular immune response in muscular dystrophy.
      ).
      The role of MBP-1 in the development of allergen-induced pulmonary pathologies in asthma has been studied in mouse asthma models in which the MBP-1 gene was knocked out (MBP-1−/−) (
      • Denzler K.L.
      • Borchers M.T.
      • Crosby J.R.
      • Cieslewicz G.
      • Hines E.M.
      • Justice J.P.
      • Cormier S.A.
      • Lindenberger K.A.
      • Song W.
      • Wu W.
      • Hazen S.L.
      • Gleich G.J.
      • Lee J.J.
      • Lee N.A.
      Extensive eosinophil degranulation and peroxidase-mediated oxidation of airway proteins do not occur in a mouse ovalbumin-challenge model of pulmonary inflammation.
      ); MBP-1 deficiency had no effect on the development of allergen-induced airway histopathologies or inflammatory cell recruitment, nor any effects on airway hyperresponsiveness, which still developed in the absence of mouse MBP-1. Knock-out of the EPX gene (EPX−/−) and resulting EPX deficiency had no impact on asthma-associated pulmonary pathologies induced by allergen sensitization and provocation (
      • Ackerman S.J.
      To be, or not to be, an eosinophil: that is the ???.
      ), suggesting that the contributions of MBP-1 and EPX to disease pathology in allergic diseases likely occurred via their combined activities. Crossing MBP-1−/− and EPX−/− knock-out mice to address this issue in double knockouts unexpectedly generated a novel strain of mice with a highly specific deficiency in eosinophilopoiesis, and therefore eosinophils (
      • Doyle A.D.
      • Jacobsen E.A.
      • Ochkur S.I.
      • McGarry M.P.
      • Shim K.G.
      • Nguyen D.T.
      • Protheroe C.
      • Colbert D.
      • Kloeber J.
      • Neely J.
      • Shim K.P.
      • Dyer K.D.
      • Rosenberg H.F.
      • Lee J.J.
      • Lee N.A.
      Expression of the secondary granule proteins major basic protein 1 (MBP-1) and eosinophil peroxidase (EPX) is required for eosinophilopoiesis in mice.
      ). Although the mechanism for the eosinophil deficiency in these double knock-out mice remains to be determined, results suggest that: (i) granule protein gene expression and/or defective granulogenesis may be a checkpoint for survival of eosinophil progenitors, (ii) loss of concurrent expression of MBP-1 and EPX disrupts lineage-instructive gene regulatory mechanisms required for self-renewal or eosinophil progenitor survival, or (iii) most likely, dysfunctional granulogenesis leads to aberrant intracellular release of a toxicant such as mouse eosinophil-associated ribonucleases (EARs) capable of degrading intracellular RNAs, leading to the observed cell-autonomous defect (
      • Ackerman S.J.
      To be, or not to be, an eosinophil: that is the ???.
      ).
      A homologue of MBP-1, called MBP-2, was identified and shown expressed exclusively by eosinophils. MBP-2 has ∼66% amino acid sequence identity with MBP-1 but is significantly less basic (pI = 8.7) (
      • Plager D.A.
      • Loegering D.A.
      • Weiler D.A.
      • Checkel J.L.
      • Wagner J.M.
      • Clarke N.J.
      • Naylor S.
      • Page S.M.
      • Thomas L.L.
      • Akerblom I.
      • Cocks B.
      • Stuart S.
      • Gleich G.J.
      A novel and highly divergent homolog of human eosinophil granule major basic protein.
      ). In vitro activities of MBP-1 and MBP-2 appear similar, e.g. cell destruction, induction of superoxide anion, IL-8 release from neutrophils, and induction of histamine and leukotriene C4 release from basophils, but human MBP-1 is more potent than MBP-2 in these activities (
      • Fredens K.
      • Dahl R.
      • Venge P.
      The Gordon phenomenon induced by the eosinophil cationic protein and eosinophil protein X.
      ). MBP-2, present only in eosinophils, may be a useful biomarker for eosinophil-associated diseases, but its utility has not been evaluated.
      MBP-1 is a monomer under physiologic conditions, but readily polymerizes in solution, forming insoluble aggregates due to the presence of five reactive thiol groups (in addition to four cysteines involved in disulfide bond formation) (
      • Oxvig C.
      • Gleich G.J.
      • Sottrup-Jensen L.
      Localization of disulfide bridges and free sulfhydryl groups in human eosinophil granule major basic protein.
      ). MBP-1 is synthesized as a precursor that is proteolytically processed to the mature granule form during packaging into the crystalloid core of the granule. The pro-domain removed in this process is heavily glycosylated with N-glycans, O-glycans, and glycosaminoglycans, raising the molecular mass to ∼30–50 kDa. MBP-1 does not exhibit high sequence similarity to other proteins aside from weak similarity (23–28%) to C-type lectin domains and the low affinity IgE receptor FcϵRII (
      • Patthy L.
      Homology of cytotoxic protein of eosinophilic leukocytes with IgE receptor Fcϵ RII: implications for its structure and function.
      ).
      The three-dimensional structure of MBP-1 (Fig. 1) (
      • Swaminathan G.J.
      • Weaver A.J.
      • Loegering D.A.
      • Checkel J.L.
      • Leonidas D.D.
      • Gleich G.J.
      • Acharya K.R.
      Crystal structure of the eosinophil major basic protein at 1.8 Å: an atypical lectin with a paradigm shift in specificity.
      ) shows that its overall topology is similar to that of C-type lectin domains, in particular to lithostathine, a glycoprotein expressed by exocrine pancreas. We showed that none of the amino acid residues involved in calcium binding in classical C-type lectins is conserved in MBP-1. The region corresponding to the carbohydrate-binding site in MBP-1 is highly basic and thus differs in structure from that of the other C-type lectins. The crystal structure of MBP-1 in complex with heparin disaccharide (Fig. 2A) showed that heparan sulfate may be a ligand (in agreement with data showing MBP-1 binding to heparin) (
      • Swaminathan G.J.
      • Myszka D.G.
      • Katsamba P.S.
      • Ohnuki L.E.
      • Gleich G.J.
      • Acharya K.R.
      Eosinophil-granule major basic protein, a C-type lectin, binds heparin.
      ). It is likely that heparan sulfate is not the sole physiologic ligand for MBP-1, and it may have the capacity to recognize a wide variety of sulfated ligands.
      Figure thumbnail gr2
      FIGURE 2Molecular details of the functional sites identified thus far. A, MBP-1 carbohydrate-binding region with bound heparin disaccharide and sulfate ions (PDB code 2BRS). B, EDN ribonucleolytic active site with bound inhibitor bis(adenosine)-5′-pentaphosphate (PDB code 1HI5). C, ECP ribonucleolytic active site with bound adenosine-2′,5′-diphosphate (PDB code 1H1H). D, CLC/Gal-10 carbohydrate recognition domain with bound mannose (PDB code 1QKQ).
      MBP-1 has been shown to function in vitro and in vivo as an endogenous allosteric antagonist of the inhibitory muscarinic M2 receptor (
      • Jacoby D.B.
      • Gleich G.J.
      • Fryer A.D.
      Human eosinophil major basic protein is an endogenous allosteric antagonist at the inhibitory muscarinic M2 receptor.
      ). Fryer and colleagues (
      • Jacoby D.B.
      • Gleich G.J.
      • Fryer A.D.
      Human eosinophil major basic protein is an endogenous allosteric antagonist at the inhibitory muscarinic M2 receptor.
      ) showed that MBP-1 potently inhibits binding of N-methyl scopolamine (NMS) to guinea pig M2, but not M3, receptors. MBP-1 was found to inhibit atropine-induced dissociation of NMS-receptor complexes, showing that MBP-1 interaction with the M2 receptor is allosteric, suggesting that it may function as an endogenous allosteric inhibitor of agonist binding to this inhibitory receptor. Inhibition of NMS binding by MBP-1 was reversible by heparin, which binds and neutralizes MBP-1, consistent with structural findings. Because eosinophils secrete MBP-1 during allergen-induced airway inflammation, and treatment of allergen-challenged or ozone-challenged guinea pigs with heparin or a neutralizing antibody to MBP-1 restores M2 receptor function (
      • Evans C.M.
      • Fryer A.D.
      • Jacoby D.B.
      • Gleich G.J.
      • Costello R.W.
      Pretreatment with antibody to eosinophil major basic protein prevents hyperresponsiveness by protecting neuronal M2 muscarinic receptors in antigen-challenged guinea pigs.
      ,
      • Fryer A.D.
      • Jacoby D.B.
      Function of pulmonary M2 muscarinic receptors in antigen-challenged guinea pigs is restored by heparin and poly-l-glutamate.
      ,
      • Yost B.L.
      • Gleich G.J.
      • Fryer A.D.
      Ozone-induced hyperresponsiveness and blockade of M2 muscarinic receptors by eosinophil major basic protein.
      ), eotaxin/CCR3-mediated eosinophil recruitment to and release of MBP-1 on airway nerves may contribute to M2 receptor dysfunction and vagally mediated bronchoconstriction in the asthmatic diathesis.

      Eosinophil-derived Neurotoxin

      EDN is a small, basic protein that belongs to the ribonuclease A (RNase A) superfamily (
      • Rosenberg H.F.
      • Tenen D.G.
      • Ackerman S.J.
      Molecular cloning of the human eosinophil-derived neurotoxin: a member of the ribonuclease gene family.
      ). It is localized to the matrix of the secondary granule of the eosinophil (
      • Ackerman S.J.
      • Loegering D.A.
      • Venge P.
      • Olsson I.
      • Harley J.B.
      • Fauci A.S.
      • Gleich G.J.
      Distinctive cationic proteins of the human eosinophil granule: major basic protein, eosinophil cationic protein, and eosinophil-derived neurotoxin.
      ) and is also known as RNase-2, nonsecretory RNase, and RNase-Us, the latter based on its specificity toward uridine-containing nucleotides. EDN is one of the most abundant RNases in humans and has been isolated from a wide variety of sources including eosinophil, placenta, liver, and urine. There is ∼3.3 μg of EDN/106 eosinophils (
      • Abu-Ghazaleh R.I.
      • Dunnette S.L.
      • Loegering D.A.
      • Checkel J.L.
      • Kita H.
      • Thomas L.L.
      • Gleich G.J.
      Eosinophil granule proteins in peripheral blood granulocytes.
      ). The initial identification of EDN was based on its induction of ataxia, incoordination, spasmodic paralysis, muscle stiffness, and killing of cerebellar Purkinje cells when injected intrathecally into rabbits, a paralytic syndrome termed the Gordon phenomenon (
      • Fredens K.
      • Dahl R.
      • Venge P.
      The Gordon phenomenon induced by the eosinophil cationic protein and eosinophil protein X.
      ,
      • Gleich G.J.
      • Loegering D.A.
      • Bell M.P.
      • Checkel J.L.
      • Ackerman S.J.
      • McKean D.J.
      Biochemical and functional similarities between human eosinophil-derived neurotoxin and eosinophil cationic protein: homology with ribonuclease.
      ).
      Although EDN shares 67% amino acid sequence identity with ECP, its sequence identity with RNase A is only 36%. The ribonucleolytic activity of EDN is ∼3–30-fold lower than that of RNase A, depending on the substrate (
      • Sorrentino S.
      • Glitz D.G.
      • Hamann K.J.
      • Loegering D.A.
      • Checkel J.L.
      • Gleich G.J.
      Eosinophil-derived neurotoxin and human liver ribonuclease: identity of structure and linkage of neurotoxicity to nuclease activity.
      ). This enzymatic activity is a prerequisite for its cytotoxic, neurotoxic, and antiviral activities (
      • Sorrentino S.
      • Glitz D.G.
      • Hamann K.J.
      • Loegering D.A.
      • Checkel J.L.
      • Gleich G.J.
      Eosinophil-derived neurotoxin and human liver ribonuclease: identity of structure and linkage of neurotoxicity to nuclease activity.
      ,
      • Domachowske J.B.
      • Dyer K.D.
      • Bonville C.A.
      • Rosenberg H.F.
      Recombinant human eosinophil-derived neurotoxin/RNase 2 functions as an effective antiviral agent against respiratory syncytial virus,.
      ,
      • Newton D.L.
      • Nicholls P.J.
      • Rybak S.M.
      • Youle R.J.
      Expression and characterization of recombinant human eosinophil-derived neurotoxin and eosinophil-derived neurotoxin-anti-transferrin receptor sFv.
      ). The primary sequence of EDN contains a Trp-X-X-Trp motif between residues 7 and 10, specifying an unusual C-mannosylation of Trp-7. This involves attachment of an R-mannosyl residue via a C–C link to the indole moiety of Trp-7, the first example of this post-translational modification (
      • Hofsteenge J.
      • Müller D.R.
      • de Beer T.
      • Löffler A.
      • Richter W.J.
      • Vliegenthart J.F.
      New type of linkage between a carbohydrate and a protein: C-glycosylation of a specific tryptophan residue in human RNase Us,.
      ). Unlike the other EDGPs, EDN is a poor cationic toxin with limited toxicity for helminth parasites and mammalian cells at high concentrations (
      • Hamann K.J.
      • Barker R.L.
      • Loegering D.A.
      • Gleich G.J.
      Comparative toxicity of purified human eosinophil granule proteins for newborn larvae of Trichinella spiralis.
      ,
      • Hamann K.J.
      • Gleich G.J.
      • Checkel J.L.
      • Loegering D.A.
      • McCall J.W.
      • Barker R.L.
      In vitro killing of microfilariae of Brugia pahangi and Brugia malayi by eosinophil granule proteins.
      ). However, as a ribonuclease, it is considerably more effective against single-stranded RNA viruses (
      • Domachowske J.B.
      • Dyer K.D.
      • Bonville C.A.
      • Rosenberg H.F.
      Recombinant human eosinophil-derived neurotoxin/RNase 2 functions as an effective antiviral agent against respiratory syncytial virus,.
      ).
      EDN activates human dendritic cells (DCs), leading to their expression of a variety of inflammatory chemokines, cytokines, growth factors, and soluble receptors (
      • Yang D.
      • Chen Q.
      • Rosenberg H.F.
      • Rybak S.M.
      • Newton D.L.
      • Wang Z.Y.
      • Fu Q.
      • Tchernev V.T.
      • Wang M.
      • Schweitzer B.
      • Kingsmore S.F.
      • Patel D.D.
      • Oppenheim J.J.
      • Howard O.M.
      Human ribonuclease A superfamily members, eosinophil-derived neurotoxin and pancreatic ribonuclease, induce dendritic cell maturation and activation,.
      ). EDN also induces both phenotypic and functional maturation of DCs, as well as acts as an alarmin that activates the TLR2-MyD88 signaling pathway in DCs, enhancing Th2 immune responses (
      • Yang D.
      • Chen Q.
      • Su S.B.
      • Zhang P.
      • Kurosaka K.
      • Caspi R.R.
      • Michalek S.M.
      • Rosenberg H.F.
      • Zhang N.
      • Oppenheim J.J.
      Eosinophil-derived neurotoxin acts as an alarmin to activate the TLR2-MyD88 signal pathway in dendritic cells and enhances Th2 immune responses.
      ).
      The crystal structure of recombinant EDN (
      • Leonidas D.D.
      • Boix E.
      • Prill R.
      • Suzuki M.
      • Turton R.
      • Minson K.
      • Swaminathan G.J.
      • Youle R.J.
      • Acharya K.R.
      Mapping the ribonucleolytic active site of eosinophil-derived neurotoxin (EDN): high resolution crystal structures of EDN complexes with adenylic nucleotide inhibitors.
      ) showed that the topology of the molecule includes the RNase A fold (Fig. 1) and that the core ribonucleolytic active site architecture (Fig. 2B) is conserved, although both ECP (
      • Boix E.
      • Leonidas D.D.
      • Nikolovski Z.
      • Nogués M.V.
      • Cuchillo C.M.
      • Acharya K.R.
      Crystal structure of eosinophil cation protein at 2.4 Å resolution.
      ) and EDN exhibit significant differences at the peripheral substrate-binding sites (
      • Boix E.
      • Nikolovski Z.
      • Moiseyev G.P.
      • Rosenberg H.F.
      • Cuchillo C.M.
      • Nogués M.V.
      Kinetic and product distribution analysis of human eosinophil cationic protein indicates a subsite arrangement that favors exonuclease-type activity.
      ). High-resolution crystal structures in complex with nucleotide inhibitors (
      • Leonidas D.D.
      • Boix E.
      • Prill R.
      • Suzuki M.
      • Turton R.
      • Minson K.
      • Swaminathan G.J.
      • Youle R.J.
      • Acharya K.R.
      Mapping the ribonucleolytic active site of eosinophil-derived neurotoxin (EDN): high resolution crystal structures of EDN complexes with adenylic nucleotide inhibitors.
      ) present a detailed picture of differences and flexibility between EDN and RNase A in substrate recognition (Fig. 2B).
      In the mouse, the related family of EARs, initially identified by Lee and colleagues (
      • Larson K.A.
      • Olson E.V.
      • Madden B.J.
      • Gleich G.J.
      • Lee N.A.
      • Lee J.J.
      Two highly homologous ribonuclease genes expressed in mouse eosinophils identify a larger subgroup of the mammalian ribonuclease superfamily.
      ), exhibits highly divergent properties both from one another and from human EDN and ECP. Zhang et al. (
      • Zhang J.
      • Dyer K.D.
      • Rosenberg H.F.
      Evolution of the rodent eosinophil-associated RNase gene family by rapid gene sorting and positive selection.
      ) showed that there is a striking similarity between the evolutionary patterns of the mouse EAR genes and those of the major histocompatibility complex, immunoglobulin, and T cell receptor genes, enabling them to hypothesize that host defense and generation of diversity are among the primary physiological functions of the murine EARs. The discovery of a large number of divergent EARs suggests the intriguing possibility that these proteins have been specifically tailored through evolution to fight against distinct mouse pathogens (
      • Zhang J.
      • Dyer K.D.
      • Rosenberg H.F.
      Evolution of the rodent eosinophil-associated RNase gene family by rapid gene sorting and positive selection.
      ).

      Eosinophil Cationic Protein

      ECP also belongs to the RNase A superfamily and is known as RNase-3 (
      • Rosenberg H.F.
      • Ackerman S.J.
      • Tenen D.G.
      Human eosinophil cationic protein: molecular cloning of a cytotoxin and helminthotoxin with ribonuclease activity.
      ,
      • Rosenberg H.F.
      • Domachowske J.B.
      Eosinophils, eosinophil ribonucleases, and their role in host defense against respiratory virus pathogens.
      ,
      • Boix E.
      • Carreras E.
      • Nikolovski Z.
      • Cuchillo C.M.
      • Nogués M.V.
      Identification and characterization of human eosinophil cationic protein by an epitope-specific antibody,.
      ). Mature ECP is a small cationic polypeptide of 133 residues. Similar to EDN, it is located in the matrix of the specific granule of the eosinophil, but as compared with EDN (pI = 8.9), ECP is considerably more cationic (pI = 10.8). There is ∼5.3 μg of ECP/106 eosinophils (
      • Abu-Ghazaleh R.I.
      • Dunnette S.L.
      • Loegering D.A.
      • Checkel J.L.
      • Kita H.
      • Thomas L.L.
      • Gleich G.J.
      Eosinophil granule proteins in peripheral blood granulocytes.
      ). Like EDN, ECP induces the neurotoxic Gordon phenomenon (
      • Fredens K.
      • Dahl R.
      • Venge P.
      The Gordon phenomenon induced by the eosinophil cationic protein and eosinophil protein X.
      ). ECP has marked toxicity for a variety of helminth parasites, hemoflagellates, bacteria, single-stranded RNA viruses, and host tissues (
      • Rosenberg H.F.
      Recombinant human eosinophil cationic protein: ribonuclease activity is not essential for cytotoxicity.
      ). Serum ECP levels can be used as a clinical tool for estimating eosinophil inflammatory activity in asthma and other allergic diseases, and levels are related to disease severity. The antibacterial activity and parasitic toxicity of ECP are greater than EDN (
      • Rosenberg H.F.
      Recombinant human eosinophil cationic protein: ribonuclease activity is not essential for cytotoxicity.
      ). In vitro, ECP can function as an antiviral agent and may participate in host defense against the single-stranded RNA respiratory syncytial virus (
      • Domachowske J.B.
      • Dyer K.D.
      • Adams A.G.
      • Leto T.L.
      • Rosenberg H.F.
      Eosinophil cationic protein/RNase 3 is another RNase A-family ribonuclease with direct antiviral activity.
      ). The toxicity of ECP for bacteria and helminths does not appear related to its RNase activity (
      • Rosenberg H.F.
      Recombinant human eosinophil cationic protein: ribonuclease activity is not essential for cytotoxicity.
      ), whereas RNase activity is required for its antiviral (
      • Domachowske J.B.
      • Dyer K.D.
      • Adams A.G.
      • Leto T.L.
      • Rosenberg H.F.
      Eosinophil cationic protein/RNase 3 is another RNase A-family ribonuclease with direct antiviral activity.
      ) and neurotoxic activities (
      • Fredens K.
      • Dahl R.
      • Venge P.
      The Gordon phenomenon induced by the eosinophil cationic protein and eosinophil protein X.
      ). The RNase activity of ECP is 100-fold lower than EDN for most RNA substrates, and their in vivo substrates have not been identified (
      • Rosenberg H.F.
      • Ackerman S.J.
      • Tenen D.G.
      Human eosinophil cationic protein: molecular cloning of a cytotoxin and helminthotoxin with ribonuclease activity.
      ,
      • Hamann K.J.
      • Ten R.M.
      • Loegering D.A.
      • Jenkins R.B.
      • Heise M.T.
      • Schad C.R.
      • Pease L.R.
      • Gleich G.J.
      • Barker R.L.
      Structure and chromosome localization of the human eosinophil-derived neurotoxin and eosinophil cationic protein genes: evidence for intronless coding sequences in the ribonuclease gene superfamily.
      ).
      The crystal structure of ECP (
      • Boix E.
      • Leonidas D.D.
      • Nikolovski Z.
      • Nogués M.V.
      • Cuchillo C.M.
      • Acharya K.R.
      Crystal structure of eosinophil cation protein at 2.4 Å resolution.
      ) shows the “RNase fold” (Fig. 1), but with significant divergence from RNase A and EDN. The structure also shows how the cationic residues are distributed on the ECP surface, an observation that may have implications for understanding the considerable cytotoxicity of this enzyme. The structure of ECP in complex with adenosine-2′,5′-diphosphate revealed details of the active site (Fig. 2C) and a structural explanation for the lower substrate affinity and catalytic efficiency of ECP (
      • Mohan C.G.
      • Boix E.
      • Evans H.R.
      • Nikolovski Z.
      • Nogués M.V.
      • Cuchillo C.M.
      • Acharya K.R.
      The crystal structure of eosinophil cationic protein in complex with 2′,5′-ADP at 2.0 Å resolution reveals the details of the ribonucleolytic active site.
      ).
      Although eosinophils and their EDGPs are associated with host defense responses against helminth parasites, a number of the EDGPs also possess antibacterial activity. ECP has antibacterial activities not shared by EDN (
      • Boix E.
      • Salazar V.A.
      • Torrent M.
      • Pulido D.
      • Nogués M.V.
      • Moussaoui M.
      Structural determinants of the eosinophil cationic protein antimicrobial activity.
      ,
      • Sánchez D.
      • Moussaoui M.
      • Carreras E.
      • Torrent M.
      • Nogués V.
      • Boix E.
      Mapping the eosinophil cationic protein antimicrobial activity by chemical and enzymatic cleavage.
      ). Evidence is accumulating that eosinophils and the EDGPs may participate in host responses to certain bacterial infections (
      • Svensson L.
      • Wennerås C.
      Human eosinophils selectively recognize and become activated by bacteria belonging to different taxonomic groups.
      ). ECP is active in vitro against both Gram-negative and Gram-positive strains of bacteria, its mechanism of toxicity involving both the bacterial cell wall and the cytoplasmic membrane. Torrent et al. (
      • Torrent M.
      • Pulido D.
      • Nogués M.V.
      • Boix E.
      Exploring new biological functions of amyloids: bacteria cell agglutination mediated by host protein aggregation.
      ) propose and provide evidence for a novel molecular mechanism to explain the bacterial agglutinating activity of ECP, showing in situ formation of fibrillar, amyloid-like aggregates at the bacterial cell surface that bind amyloid diagnostic dyes. The agglutinating activity of ECP appears driven by the amyloid-like aggregation of the protein at the bacteria cell surface; elimination of the amyloidogenic behavior by a single point mutation (I13A) abolished both its agglutinating and its antimicrobial activities, the mutant being defective in triggering leakage and lipid vesicle aggregation. These findings support the novel concept that the amyloidogenic behavior of ECP, and possibly other EDGPs, participates in antibacterial host responses to infection, and suggest that the biophysical properties of bactericidal N-terminal peptides of ECP (amino acids 1–45) (
      • Torrent M.
      • Pulido D.
      • de la Torre B.G.
      • García-Mayoral M.F.
      • Nogués M.V.
      • Bruix M.
      • Andreu D.
      • Boix E.
      Refining the eosinophil cationic protein antibacterial pharmacophore by rational structure minimization.
      ) or other EDGPs might guide development of novel antimicrobials (
      • Pulido D.
      • Torrent M.
      • Andreu D.
      • Nogués M.V.
      • Boix E.
      Two human host defense ribonucleases against mycobacteria, the eosinophil cationic protein (RNase 3) and RNase 7.
      ).
      Native ECP purified to homogeneity from blood leukocytes or purified eosinophils shows considerable molecular heterogeneity, from multiple glycosylated isoforms to the nonglycosylated native protein, as well as functional heterogeneity of these glycoforms relative to nonglycosylated ECP with respect to its cytotoxic activity for mammalian cells (
      • Trulson A.
      • Byström J.
      • Engström A.
      • Larsson R.
      • Venge P.
      The functional heterogeneity of eosinophil cationic protein is determined by a gene polymorphism and post-translational modifications.
      ). A gene polymorphism in the coding region, ECP 434(G>C), determines the cytotoxicity of ECP for mammalian cells but has minor effects on fibroblast-mediated gel contraction (measure of fibroblast activation) and no effect on ECP RNase activity (
      • Rubin J.
      • Zagai U.
      • Blom K.
      • Trulson A.
      • Engström A.
      • Venge P.
      The coding ECP 434(G>C) gene polymorphism determines the cytotoxicity of ECP but has minor effects on fibroblast-mediated gel contraction and no effect on RNase activity.
      ). This polymorphism changes an arginine (base at 434 is G) at position 97 to threonine (base at 434 is C). The ECP 434(G>C) polymorphism correlates with the natural course of Schistosoma mansoni infection (
      • Eriksson J.
      • Reimert C.M.
      • Kabatereine N.B.
      • Kazibwe F.
      • Ireri E.
      • Kadzo H.
      • Eltahir H.B.
      • Mohamed A.O.
      • Vennervald B.J.
      • Venge P.
      The 434(G>C) polymorphism within the coding sequence of eosinophil cationic protein (ECP) correlates with the natural course of Schistosoma mansoni infection.
      ) and with inflammatory bowel disease in an age- and gender-dependent manner (
      • Blom K.
      • Rubin J.
      • Halfvarson J.
      • Törkvist L.
      • Rönnblom A.
      • Sangfelt P.
      • Lördal M.
      • Jönsson U.B.
      • Sjöqvist U.
      • Håkansson L.D.
      • Venge P.
      • Carlson M.
      Eosinophil associated genes in the inflammatory bowel disease 4 region: correlation to inflammatory bowel disease revealed.
      ). These ECP genotypes also show associations with the symptoms of allergy and asthma (
      • Jönsson U.B.
      • Byström J.
      • Stålenheim G.
      • Venge P.
      Polymorphism of the eosinophil cationic protein-gene is related to the expression of allergic symptoms.
      ,
      • Jönsson U.B.
      • Håkansson L.D.
      • Jõgi R.
      • Janson C.
      • Venge P.
      Associations of ECP (eosinophil cationic protein)-gene polymorphisms to allergy, asthma, smoke habits and lung function in two Estonian and Swedish sub cohorts of the ECRHS II study.
      ). Venge and colleagues (
      • Woschnagg C.
      • Rubin J.
      • Venge P.
      Eosinophil cationic protein (ECP) is processed during secretion.
      ) reported that the various ECP glycoforms are processed during eosinophil secretion; the modifications to secreted ECP by activated eosinophils is explained in part by differences in their degree of N-linked glycosylation, such that secreted ECP acquires the masses of the more cytotoxic, less glycosylated, isoforms, including the nonglycosylated species (
      • Woschnagg C.
      • Rubin J.
      • Venge P.
      Eosinophil cationic protein (ECP) is processed during secretion.
      ), explaining in part the structural and functional heterogeneity of ECP as reported in the literature.

      Eosinophil Peroxidase (EPX/EPO)

      During activation, eosinophils can generate potentially toxic reactive oxygen species, which unlike the neutrophil, are mainly directed extracellularly (
      • Lacy P.
      • Abdel-Latif D.
      • Steward M.
      • Musat-Marcu S.
      • Man S.F.
      • Moqbel R.
      Divergence of mechanisms regulating respiratory burst in blood and sputum eosinophils and neutrophils from atopic subjects.
      ). Oxidant production begins with the generation of superoxide by the membrane bound NADPH oxidase of eosinophils, which dismutates into hydrogen peroxide (H2O2). EPX, the most abundant cationic protein of the matrix of the specific granule, uses this H2O2 as an oxidizing substrate to generate potent oxidizing species, including hypohalous acids. In addition to bromide and chloride, EPX preferentially uses thiocyanate (SCN) ions to generate HOSCN, shown by Wang et al. (
      • Wang J.G.
      • Mahmud S.A.
      • Thompson J.A.
      • Geng J.G.
      • Key N.S.
      • Slungaard A.
      The principal eosinophil peroxidase product, HOSCN, is a uniquely potent phagocyte oxidant inducer of endothelial cell tissue factor activity: a potential mechanism for thrombosis in eosinophilic inflammatory states.
      ,
      • Wang J.G.
      • Mahmud S.A.
      • Nguyen J.
      • Slungaard A.
      Thiocyanate-dependent induction of endothelial cell adhesion molecule expression by phagocyte peroxidases: a novel HOSCN-specific oxidant mechanism to amplify inflammation.
      ) to exert considerable biologic activity, e.g. as a potent oxidant inducer of tissue factor activity in endothelial cells; the HOSCN generated by EPX from activated tissue eosinophils may induce the prothrombotic and proinflammatory endothelial and endocardial phenotypes responsible for thrombotic complications seen in the hypereosinophilic syndrome. EPX is structurally distinguished from the other EDGPs, being a two-chain hemoprotein (68 kDa); it is initially synthesized as a single chain precursor that is proteolytically processed to a 55-kDa heavy chain and 12.5-kDa light chain. EPX is highly cationic and similar to ECP and MBP-1 in this regard. Biochemical evidence suggests that EPX is structurally related to myeloperoxidase (Fig. 1) present in neutrophil-specific granules. Patients with myeloperoxidase deficiency have normal levels of EPX, indicating independent expression mechanisms for these peroxidases. There is ∼12 μg of EPX/106 eosinophils (
      • Abu-Ghazaleh R.I.
      • Dunnette S.L.
      • Loegering D.A.
      • Checkel J.L.
      • Kita H.
      • Thomas L.L.
      • Gleich G.J.
      Eosinophil granule proteins in peripheral blood granulocytes.
      ). EPX exerts some cytotoxic effects as a cationic toxin, being able to kill parasites (
      • Auriault C.
      • Capron M.
      • Capron A.
      Activation of rat and human eosinophils by soluble factor(s) released by Schistosoma mansoni schistosomula.
      ,
      • Locksley R.M.
      • Wilson C.B.
      • Klebanoff S.J.
      Role for endogenous and acquired peroxidase in the toxoplasmacidal activity of murine and human mononuclear phagocytes.
      ) and mammalian cells in the absence of H2O2 and a halide co-factor. Furthermore, EPX exerts both anti-inflammatory (
      • Henderson W.R.
      • Jörg A.
      • Klebanoff S.J.
      Eosinophil peroxidase-mediated inactivation of leukotrienes B4, C4, and D4.
      ) and pro-inflammatory (
      • Henderson W.R.
      • Jong E.C.
      • Klebanoff S.J.
      Binding of eosinophil peroxidase to mast cell granules with retention of peroxidatic activity.
      ) activities.
      All of the human EDGPs including EPX itself, MBP-1, EDN, and ECP, are post-translationally modified by EPX via nitration at specific tyrosine residues during their synthesis and packaging in the developing eosinophil (
      • Ulrich M.
      • Petre A.
      • Youhnovski N.
      • Prömm F.
      • Schirle M.
      • Schumm M.
      • Pero R.S.
      • Doyle A.
      • Checkel J.
      • Kita H.
      • Thiyagarajan N.
      • Acharya K.R.
      • Schmid-Grendelmeier P.
      • Simon H.U.
      • Schwarz H.
      • Tsutsui M.
      • Shimokawa H.
      • Bellon G.
      • Lee J.J.
      • Przybylski M.
      • Döring G.
      Post-translational tyrosine nitration of eosinophil granule toxins mediated by eosinophil peroxidase.
      ). High-resolution affinity-mass spectrometry showed single specific nitration sites at Tyr-349 in EPX and Tyr-33 in both EDN and ECP, and crystal structures of EDN and ECP, as well as structural models of EPX, suggest that these nitrated tyrosine residues are surface-exposed. Studies in EPX−/−, gp91phox−/−, and NOS−/− knock-out mice showed that tyrosine nitration of these cellular toxins and ribonucleases is mediated by EPX itself in the presence of H2O2 and small amounts of nitrogen oxide. Thus, EPX appears to nitrate itself via an autocatalytic mechanism. Tyrosine nitration of the EDGPs was shown to occur during eosinophil differentiation and was independent of inflammation. The specific roles of EPX-mediated nitrosylation of the EDGPs in eosinophil-mediated innate host immune defense mechanisms characterized by their secretion during cell activation, e.g. against parasites or during tissue damage in parasitic infections or allergic responses, have not been determined.

      Charcot-Leyden Crystal Protein

      CLC protein forms distinctive bipyramidal hexagonal crystals, hallmarks of eosinophil participation in allergic and related inflammatory reactions. These crystals, found at sites of eosinophil infiltration in tissues and in body fluids and secretions, were identified more than 150 years ago. CLC is a small, slightly acidic (pI ∼5.1–5.7) 142-amino acid protein of 16.5 kDa (
      • Ackerman S.J.
      • Corrette S.E.
      • Rosenberg H.F.
      • Bennett J.C.
      • Mastrianni D.M.
      • Nicholson-Weller A.
      • Weller P.F.
      • Chin D.T.
      • Tenen D.G.
      Molecular cloning and characterization of human eosinophil Charcot-Leyden crystal protein (lysophospholipase): similarities to IgE binding proteins and the S-type animal lectin superfamily.
      ). It was initially identified as an eosinophil lysophospholipase (LPLase) (
      • Weller P.F.
      • Bach D.S.
      • Austen K.F.
      Biochemical characterization of human eosinophil Charcot-Leyden crystal protein (lysophospholipase).
      ) but has since been assigned to the galectin superfamily of S-type animal lectins as the 10th member (Galectin-10) based on amino acid sequence (
      • Ackerman S.J.
      • Corrette S.E.
      • Rosenberg H.F.
      • Bennett J.C.
      • Mastrianni D.M.
      • Nicholson-Weller A.
      • Weller P.F.
      • Chin D.T.
      • Tenen D.G.
      Molecular cloning and characterization of human eosinophil Charcot-Leyden crystal protein (lysophospholipase): similarities to IgE binding proteins and the S-type animal lectin superfamily.
      ), three-dimensional structure (CLC/Gal-10, Fig. 1) (
      • Leonidas D.D.
      • Elbert B.L.
      • Zhou Z.
      • Leffler H.
      • Ackerman S.J.
      • Acharya K.R.
      Crystal structure of human Charcot-Leyden crystal protein, an eosinophil lysophospholipase, identifies it as a new member of the carbohydrate-binding family of galectins.
      ), and genomic organization (
      • Dyer K.D.
      • Handen J.S.
      • Rosenberg H.F.
      The genomic structure of the human Charcot-Leyden crystal protein gene is analogous to those of the galectin genes.
      ). Importantly, we showed that CLC/Gal-10 lacks any LPLase activity and that the weak enzymatic activity initially associated with the purified protein was due to contamination by a highly active 75-kDa pancreatic LPLase also expressed by eosinophils (
      • Ackerman S.J.
      • Liu L.
      • Kwatia M.A.
      • Savage M.P.
      • Leonidas D.D.
      • Swaminathan G.J.
      • Acharya K.R.
      Charcot-Leyden crystal protein (galectin-10) is not a dual function galectin with lysophospholipase activity but binds a lysophospholipase inhibitor in a novel structural fashion.
      ).
      The crystal structure of CLC/Gal-10 provides details of a carbohydrate recognition domain with both similarities to and important differences from other members of the galectin family (
      • Leonidas D.D.
      • Elbert B.L.
      • Zhou Z.
      • Leffler H.
      • Ackerman S.J.
      • Acharya K.R.
      Crystal structure of human Charcot-Leyden crystal protein, an eosinophil lysophospholipase, identifies it as a new member of the carbohydrate-binding family of galectins.
      ). Structural studies showed that CLC/Gal-10 does not bind β-galactosides, but can bind mannose in the crystal in a unique manner different from the binding of lactosamine carbohydrates by other related galectins (Fig. 2D) (
      • Swaminathan G.J.
      • Leonidas D.D.
      • Savage M.P.
      • Ackerman S.J.
      • Acharya K.R.
      Selective recognition of mannose by the human eosinophil Charcot-Leyden crystal protein (galectin-10): a crystallographic study at 1.8 Å resolution.
      ). The physiologic significance of this mannose binding remains equivocal. The partial conservation of residues involved in carbohydrate binding leads to significant changes in the topology and chemical nature of the carbohydrate recognition domain and has implications for glycan recognition by CLC/Gal-10 in vivo. These findings are beginning to provide clues toward identifying a physiologically relevant ligand for CLC/Gal-10 and understanding its potential intracellular and extracellular roles in eosinophil biology.
      Our recent experiments using Southwestern (ligand) blotting, co-purification, co-immunoprecipitation, and confocal microscopy show that CLC/Gal-10 interacts in vitro and intracellularly in activated eosinophils with the two glycosylated human eosinophil granule cationic ribonucleases, EDN and ECP.
      C. B. Doyle and S. J. Ackerman, unpublished results.
      Studies in human blood eosinophils using immunofluorescence confocal microscopy show that interferon-γ activation induces the rapid co-localization of CLC/Gal-10 with EDN and CD63.4 Because CLC/Gal-10 does not inhibit the ribonuclease activity of EDN, it may function instead as a carrier for the sequestration and vesicular transport of these ribonucleases, during granulogenesis in the differentiating eosinophil, and during piecemeal degranulation (Fig. 3) in the activated eosinophil, enabling their extracellular functions in host defense and allergic inflammation without intracellular damage to the eosinophil itself during their secretion.
      Figure thumbnail gr3
      FIGURE 3Mechanisms of eosinophil degranulation. Eosinophils release their secondary (specific) granule contents, including the granule cationic proteins and other immunomodulatory mediators, by four different mechanisms. In classical exocytosis, mainly seen at sites of bacterial infection, the contents of single granules are released by fusion of the granule membrane with the plasma membrane lipid bilayer. In compound exocytosis, mainly seen with eosinophil degranulation onto helminth parasites, a number of granules first coalesce and fuse, and the cationic protein contents are then released through a single fusion pore at the plasma membrane. In piecemeal degranulation, mainly seen in eosinophil inflammatory responses in human tissues, secretory vesicles bud from granules, capturing granule matrix and/or core contents, and are targeted to the plasma membrane in a fashion analogous to the release of neurotransmitters from neurons. Differential secretion of the cationic proteins from the matrix (EPX, EDN/RNase-2, ECP/RNase-3) or core (MBP-1, MBP-2) of the granule has been shown to occur, leaving coreless granules with intact matrix or intact cores with no matrix in the cell. In cytolysis, intact whole granules are deposited in tissues and body fluids after disruption of the plasma membrane due to eosinophil necrosis.
      In addition to its considerable expression at both mRNA and protein levels in eosinophils and basophils, CLC/Gal-10 was identified as a constituent of human regulatory T cells. A global proteomics analysis of highly purified human CD4+CD25+ Tregs identified CLC/Gal-10 as a novel biomarker, shown by siRNA knockdown to be essential for maintaining Treg anergy and suppressive functions on T cell activation (
      • Kubach J.
      • Lutter P.
      • Bopp T.
      • Stoll S.
      • Becker C.
      • Huter E.
      • Richter C.
      • Weingarten P.
      • Warger T.
      • Knop J.
      • Müllner S.
      • Wijdenes J.
      • Schild H.
      • Schmitt E.
      • Jonuleit H.
      Human CD4+CD25+ regulatory T cells: proteome analysis identifies galectin-10 as a novel marker essential for their anergy and suppressive function.
      ). The mechanism by which CLC/Gal-10 participates, through galectin-type interaction with a glycan ligand(s) or protein-protein interaction, in maintaining the CD4+CD25+ Treg phenotype has not been established.
      A number of studies identify CLC/Gal-10 as a potentially useful biomarker of eosinophil involvement in asthma, allergic rhinitis, and other eosinophil-associated diseases. Elevated levels of CLC/Gal-10 have been measured by one of us, by ELISA, as a biomarker of active eosinophilic inflammation that is highly correlated with the number of tissue (esophageal) eosinophils in eosinophilic esophagitis (
      • Furuta G.T.
      • Kagalwalla A.F.
      • Lee J.J.
      • Alumkal P.
      • Maybruck B.T.
      • Fillon S.
      • Masterson J.C.
      • Ochkur S.
      • Protheroe C.
      • Moore W.
      • Pan Z.
      • Amsden K.
      • Robinson Z.
      • Capocelli K.
      • Mukkada V.
      • Atkins D.
      • Fleischer D.
      • Hosford L.
      • Kwatia M.A.
      • Schroeder S.
      • Kelly C.
      • Lovell M.
      • Melin-Aldana H.
      • Ackerman S.J.
      The oesophageal string test: a novel, minimally invasive method measures mucosal inflammation in eosinophilic oesophagitis.
      ), a rare immune-mediated food allergic disease of increasing incidence. CLC/Gal-10 was also identified as a potentially useful biomarker of eosinophilic airway inflammation in induced sputum for identifying the eosinophilic phenotype of asthma to guide treatment considerations (
      • Chua J.C.
      • Douglass J.A.
      • Gillman A.
      • O'Hehir R.E.
      • Meeusen E.N.
      Galectin-10, a potential biomarker of eosinophilic airway inflammation.
      ). In celiac disease, CLC/Gal-10 expression was found related to both disease activity (histologic grade) and numbers of tissue eosinophils in intestinal lesions, suggesting it as a novel biomarker for evaluating tissue damage and eosinophil involvement in the pathogenesis of this gluten intolerance. Finally, genetic variations (SNPs) in the promoter region of the CLC gene were identified as potential susceptibility biomarkers for allergic rhinitis (
      • Bryborn M.
      • Halldén C.
      • Säll T.
      • Cardell L.O.
      CLC: a novel susceptibility gene for allergic rhinitis?.
      ), with the pattern of variation compatible with a recessive inheritance model and observed increased levels of CLC/Gal-10 protein in the nasal fluid of patients with allergic rhinitis during the allergy season (
      • Ghafouri B.
      • Irander K.
      • Lindbom J.
      • Tagesson C.
      • Lindahl M.
      Comparative proteomics of nasal fluid in seasonal allergic rhinitis.
      ).

      Perspectives

      With a goal toward understanding the roles and specific functions of the EDGPs in the normal and pathologic activities of the eosinophil, significant progress has been made by determining the three-dimensional structures of four of these mediators using x-ray crystallographic approaches. These studies provided novel insights and vital clues toward understanding the structural basis for their unique biologic and enzymatic activities at a molecular level, paving the way for future “form and function” analyses to better define their unique biochemical properties, biologic activities, and pathologic contributions to eosinophil-mediated inflammatory responses, tissue damage and repair, remodeling, and fibrosis, as well as innate and adaptive host immune responses to infectious agents including parasitic helminths, bacteria, and viruses.

      Acknowledgments

      We thank Drs. Nethaji Thiyagarajan and Geoffrey Masuyer for FIGURE 1, FIGURE 2.

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