Fusion protein vesicle-associated membrane protein 2 is implicated in IFN-γ-induced piecemeal degranulation in human eosinophils from atopic individuals

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 eosinaphil 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 (greater than or equal to 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.