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  • Title: Fusion protein vesicle-associated membrane protein 2 is implicated in IFN-gamma-induced piecemeal degranulation in human eosinophils from atopic individuals.
    Author: Lacy P, Logan MR, Bablitz B, Moqbel R.
    Journal: J Allergy Clin Immunol; 2001 Apr; 107(4):671-8. PubMed ID: 11295657.
    Abstract:
    BACKGROUND: Exocytosis is an integral event during IFN-gamma-induced piecemeal degranulation in eosinophils. In many tissues soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), including vesicle-associated membrane protein (VAMP), act as specific intracellular receptors to allow granule fusion with the membrane during degranulation. However, the mechanisms underlying eosinophil piecemeal degranulation induced by IFN-gamma are not well understood. OBJECTIVE: We sought to assess whether eosinophils express the vesicular SNARE protein VAMP-2 and to determine the involvement of VAMP-2 in IFN-gamma-induced piecemeal degranulation. METHODS: Human peripheral blood eosinophils (> or =97%) from atopic subjects were subjected to RT-PCR and sequence analysis with specific primers for VAMP-2 mRNA. Western blotting and flow cytometric analysis were carried out to confirm the identity of VAMP-2 and its susceptibility to cleavage by tetanus toxin. Confocal laser scanning microscopy imaging was conducted on double-labeled cytospin preparations of eosinophils at 0, 5, 10, 30, and 60 minutes and 16 hours of IFN-gamma (500 U/mL) stimulation. RESULTS: Eosinophils expressed VAMP-2 mRNA (n = 4 donors), which exhibited 100% homology with human VAMP-2 cDNA on sequencing. Eosinophils were also found to express tetanus toxin-sensitive VAMP-2 protein. RANTES and VAMP-2 immunofluorescence were observed to colocalize to similar intracellular structures by means of confocal imaging. IFN-gamma induced a rapid translocation of VAMP-2(+) organelles toward the cell membrane in correlation with RANTES. CONCLUSIONS: These findings suggest that exocytosis in human eosinophils is regulated by SNAREs, with a specific role indicated for VAMP-2 in piecemeal degranulation.
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