title: Vesicle-mediated secretion of human eosinophil granule-derived major basic protein.
abstract: Major basic protein (MBP), the predominant cationic protein of human eosinophil specific granules, is stored within crystalloid cores of these granules. Secretion of MBP contributes to the immunopathogenesis of varied diseases. Prior electron microscopy (EM) of eosinophils in sites of inflammation noted losses of granule cores in the absence of granule exocytosis and suggested that eosinophil granule proteins might be released through piecemeal degranulation (PMD), a secretory process mediated by transport vesicles. Because release of eosinophil granule-derived MBP through PMD has not been studied, we evaluated secretion of this cationic protein by human eosinophils. Intracellular localizations of MBP were studied within nonstimulated and eotaxin-stimulated human eosinophils by both immunofluorescence and a pre-embedding immunonanogold EM method that enables optimal epitope preservation and antigen access to membrane microdomains. In parallel, quantification of transport vesicles was assessed in eosinophils from a patient with hypereosinophilic syndrome (HES). Our data demonstrate vesicular trafficking of MBP within eotaxin-stimulated eosinophils. Vesicular compartments, previously implicated in transport from granules to the plasma membrane, including large vesiculotubular carriers termed eosinophil sombrero vesicles (EoSVs), were found to contain MBP. These secretory compartments were significantly increased in numbers within HES eosinophils. Moreover, in addition to granule-stored MBP, even unstimulated eosinophils contained appreciable amounts of MBP within secretory vesicles, as evidenced by immunonanogold EM and immunofluorescent colocalizations of MBP and CD63. These data suggest that eosinophil MBP, with its multiple extracellular activities, can be mobilized from granules by PMD into secretory vesicles and both granule- and secretory vesicle-stored pools of MBP are available for agonist-elicited secretion of MBP from human eosinophils. The recognition of PMD as a secretory process to release MBP is important to understand the pathological basis of allergic and other eosinophil-associated inflammatory diseases.
authors.affiliation: Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, Juiz de Fora, Minas Gerais, Brazil.
authors.forename: Rossana C N
authors.initials: RC
authors.lastname: Melo
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Spencer
authors.forename: Sandra A C
authors.initials: SA
authors.lastname: Perez
authors.forename: Josiane S
authors.initials: JS
authors.lastname: Neves
authors.forename: Staci P
authors.initials: SP
authors.lastname: Bafford
authors.forename: Ellen S
authors.initials: ES
authors.lastname: Morgan
authors.forename: Ann M
authors.initials: AM
authors.lastname: Dvorak
authors.forename: Peter F
authors.initials: PF
authors.lastname: Weller
mesh_terms: D001693:Biological Transport, Active; D015550:Cell Degranulation; D054413:Chemokine CCL11 / Q000494:pharmacology; D047093:Eosinophil Major Basic Protein / Q000378:metabolism*; D004804:Eosinophils / Q000187:drug effects / Q000378:metabolism* / Q000502:physiology / Q000648:ultrastructure; D006801:Humans; D017681:Hypereosinophilic Syndrome / Q000503:physiopathology; D066298:In Vitro Techniques; D046529:Microscopy, Electron, Transmission; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D011994:Recombinant Proteins / Q000494:pharmacology; D022142:Secretory Vesicles / Q000378:metabolism / Q000648:ultrastructure
chemical_list: C515456:CCL11 protein, human; D054413:Chemokine CCL11; D011994:Recombinant Proteins; D047093:Eosinophil Major Basic Protein