title: Intragranular vesiculotubular compartments are involved in piecemeal degranulation by activated human eosinophils.
abstract: Eosinophils, leukocytes involved in allergic, inflammatory and immunoregulatory responses, have a distinct capacity to rapidly secrete preformed granule-stored proteins through piecemeal degranulation (PMD), a secretion process based on vesicular transport of proteins from within granules for extracellular release. Eosinophil-specific granules contain cytokines and cationic proteins, such as major basic protein (MBP). We evaluated structural mechanisms responsible for mobilizing proteins from within eosinophil granules. Human eosinophils stimulated for 30-60 min with eotaxin, regulated on activation, normal, T-cell expressed and secreted (RANTES) or platelet activating factor exhibited ultrastructural features of PMD (e.g. losses of granule contents) and extensive vesiculotubular networks within emptying granules. Brefeldin A inhibited granule emptying and collapsed intragranular vesiculotubular networks. By immunonanogold ultrastructural labelings, CD63, a tetraspanin membrane protein, was localized within granules and on vesicles outside of granules, and mobilization of MBP into vesicles within and extending from granules was demonstrated. Electron tomography with three dimension reconstructions revealed granule internal membranes to constitute an elaborate tubular network able to sequester and relocate granule products upon stimulation. We provide new insights into PMD and identify eosinophil specific granules as organelles whose internal tubulovesicular networks are important for the capacity of eosinophils to secrete, by vesicular transport, their content of preformed and granule-stored cytokines and cationic proteins.
authors.affiliation: Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, UFJF, Juiz de Fora, MG, Brazil.
authors.forename: Rossana C N
authors.initials: RC
authors.lastname: Melo
authors.forename: Sandra A C
authors.initials: SA
authors.lastname: Perez
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Spencer
authors.forename: Ann M
authors.initials: AM
authors.lastname: Dvorak
authors.forename: Peter F
authors.initials: PF
authors.lastname: Weller
mesh_terms: D015703:Antigens, CD / Q000378:metabolism; D020126:Brefeldin A / Q000378:metabolism; D015550:Cell Degranulation; D054413:Chemokine CCL11; D018946:Chemokine CCL5 / Q000378:metabolism; D019742:Chemokines, CC / Q000378:metabolism; D002631:Chemotactic Factors, Eosinophil / Q000378:metabolism; D047091:Eosinophil Granule Proteins / Q000378:metabolism*; D047093:Eosinophil Major Basic Protein / Q000378:metabolism; D004804:Eosinophils / Q000378:metabolism* / Q000648:ultrastructure*; D006801:Humans; D021621:Imaging, Three-Dimensional; D007425:Intracellular Membranes / Q000378:metabolism*; D010972:Platelet Activating Factor / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000378:metabolism; D060149:Tetraspanin 30
chemical_list: D015703:Antigens, CD; C515456:CCL11 protein, human; C554586:CD63 protein, human; D054413:Chemokine CCL11; D018946:Chemokine CCL5; D019742:Chemokines, CC; D002631:Chemotactic Factors, Eosinophil; D047091:Eosinophil Granule Proteins; D010972:Platelet Activating Factor; D010980:Platelet Membrane Glycoproteins; D011500:Protein Synthesis Inhibitors; D060149:Tetraspanin 30; D020126:Brefeldin A; D047093:Eosinophil Major Basic Protein