Fusion protein vesicle-associated membrane protein 2 is implicated in IFN-gamma-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 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.
Lacy
P
P
Pulmonary Research Group, Department of Medicine, University of Alberta, Edmonton, Alberta, Canada T6G 2S2.
Logan
M R
MR
Bablitz
B
B
Moqbel
R
R
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D015550:Cell Degranulation; D018946:Chemokine CCL5 / Q000032:analysis; D004804:Eosinophils / Q000502:physiology*; D006801:Humans; D006967:Hypersensitivity / Q000097:blood*; D007371:Interferon-gamma / Q000494:pharmacology*; D008565:Membrane Proteins / Q000032:analysis / Q000235:genetics / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D010980:Platelet Membrane Glycoproteins / Q000032:analysis; D050683:R-SNARE Proteins; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D050600:SNARE Proteins; D060149:Tetraspanin 30; D033921:Vesicular Transport Proteins
D015703:Antigens, CD; C554586:CD63 protein, human; C554587:Cd63 protein, mouse; D018946:Chemokine CCL5; D008565:Membrane Proteins; D010980:Platelet Membrane Glycoproteins; D050683:R-SNARE Proteins; D012333:RNA, Messenger; D050600:SNARE Proteins; D060149:Tetraspanin 30; D033921:Vesicular Transport Proteins; D007371:Interferon-gamma