title: CD63 is tightly associated with intracellular, secretory events chaperoning piecemeal degranulation and compound exocytosis in human eosinophils.
abstract: Eosinophil activation leads to secretion of presynthesized, granule-stored mediators that determine the course of allergic, inflammatory, and immunoregulatory responses. CD63, a member of the transmembrane-4 glycoprotein superfamily (tetraspanins) and present on the limiting membranes of eosinophil-specific (secretory) granules, is considered a potential surface marker for eosinophil degranulation. However, the intracellular secretory trafficking of CD63 in eosinophils and other leukocytes is not understood. Here, we provide a comprehensive investigation of CD63 trafficking at high resolution within human eosinophils stimulated with inflammatory stimuli, CCL11 and tumor necrosis factor α, which induce distinctly differing secretory processes in eosinophils: piecemeal degranulation and compound exocytosis, respectively. By using different transmission electron microscopy approaches, including an immunonanogold technique, for enhanced detection of CD63 at subcellular compartments, we identified a major intracellular pool of CD63 that is directly linked to eosinophil degranulation events. Transmission electron microscopy quantitative analyses demonstrated that, in response to stimulation, CD63 is concentrated within granules undergoing secretion by piecemeal degranulation or compound exocytosis and that CD63 tracks with the movements of vesicles and granules in the cytoplasm. Although CD63 was observed at the cell surface after stimulation, immunonanogold electron microscopy revealed that a strong CD63 pool remains in the cytoplasm. It is remarkable that CCL11 and tumor necrosis factor α triggered increased formation of CD63(+) large vesiculotubular carriers (eosinophil sombrero vesicles), which fused with granules in the process of secretion, likely acting in the intracellular translocation of CD63. Altogether, we identified active, intracellular CD63 trafficking connected to eosinophil granule-derived secretory pathways. This is important for understanding the complex secretory activities of eosinophils underlying immune responses.
authors.affiliation: Laboratory of Cellular Biology, Department of Biology, ICB, Federal University of Juiz de Fora, UFJF, Juiz de Fora, Brazil;
authors.forename: Lívia A S
authors.initials: LA
authors.lastname: Carmo
authors.affiliation: Laboratory of Cellular Biology, Department of Biology, ICB, Federal University of Juiz de Fora, UFJF, Juiz de Fora, Brazil;
authors.forename: Kennedy
authors.initials: K
authors.lastname: Bonjour
authors.affiliation: Department of General Internal Medicine and Clinical Laboratory Medicine, Akita University Graduate School of Medicine, Akita, Japan;
authors.forename: Shigeharu
authors.initials: S
authors.lastname: Ueki
authors.affiliation: Institute of Biomedical Sciences, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil;
authors.forename: Josiane S
authors.initials: JS
authors.lastname: Neves
authors.affiliation: Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA; and.
authors.forename: Linying
authors.initials: L
authors.lastname: Liu
authors.affiliation: Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA; and.
authors.forename: Lisa A
authors.initials: LA
authors.lastname: Spencer
authors.affiliation: Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: Ann M
authors.initials: AM
authors.lastname: Dvorak
authors.affiliation: Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA; and.
authors.forename: Peter F
authors.initials: PF
authors.lastname: Weller
authors.affiliation: Laboratory of Cellular Biology, Department of Biology, ICB, Federal University of Juiz de Fora, UFJF, Juiz de Fora, Brazil; Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA; and rossana.melo@ufjf.du.br.
authors.forename: Rossana C N
authors.initials: RC
authors.lastname: Melo
mesh_terms: D001692:Biological Transport; D015550:Cell Degranulation / Q000502:physiology*; D054413:Chemokine CCL11 / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004804:Eosinophils / Q000378:metabolism*; D005089:Exocytosis / Q000502:physiology*; D006801:Humans; D021381:Protein Transport; D022142:Secretory Vesicles / Q000378:metabolism*; D060149:Tetraspanin 30 / Q000378:metabolism*
chemical_list: C515456:CCL11 protein, human; C554586:CD63 protein, human; D054413:Chemokine CCL11; D060149:Tetraspanin 30
keywords: cell secretion; immune responses; inflammation; transmission electron microscopy; vesicular trafficking