Quantitative measurement of mast cell degranulation using a novel flow cytometric annexin-V binding assay.
BACKGROUND
Mast cells are primary mediators of allergic inflammation. Antigen-mediated crosslinking of their cell surface immunoglobulin E (IgE) receptors results in degranulation and the release of proinflammatory mediators including histamine, tumor necrosis factor-alpha, and leukotrienes.


METHODS
Mast cells were stimulated to degranulate by using either IgE crosslinking or ionophore treatment. Exogenously added annexin-V was used to stain exocytosing granules, and the extent of binding was measured flow cytometrically. Release of the enzyme beta-hexosaminidase was used for population-based measurements of degranulation. Two known inhibitors of degranulation, the phosphatidylinositol 3 kinase inhibitor wortmannin and overexpression of a mutant rab3d protein, were used as controls to validate the annexin-V binding assay.


RESULTS
Annexin-V specifically bound to mast cell granules exposed after stimulation in proportion to the extent of degranulation. Annexin-V binding was calcium dependent and was blocked by phosphatidylserine containing liposomes, consistent with specific binding to this membrane lipid. Visualization of annexin-V staining showed granular cell surface patches that colocalized with the exocytic granule marker VAMP-green fluorescent protein (GFP). Wortmannin inhibited both annexin-V binding and beta-hexosaminidase release in RBL-2H3 cells, as did the expression of a dominant negative rab3d mutant protein.


CONCLUSIONS
The annexin-V binding assay represents a powerful new flow cytometric method to monitor mast cell degranulation for functional analysis.
Demo
S D
SD
Rigel Inc., South San Francisco, California, USA.
Masuda
E
E
Rossi
A B
AB
Throndset
B T
BT
Gerard
A L
AL
Chan
E H
EH
Armstrong
R J
RJ
Fox
B P
BP
Lorens
J B
JB
Payan
D G
DG
Scheller
R H
RH
Fisher
J M
JM
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D017304:Annexin A5 / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology; D015550:Cell Degranulation / Q000187:drug effects; D003594:Cytoplasmic Granules / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins; D008081:Liposomes / Q000494:pharmacology; D008164:Luminescent Proteins; D008407:Mast Cells / Q000502:physiology*; D051379:Mice; D008856:Microscopy, Fluorescence; D010718:Phosphatidylserines / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D000077191:Wortmannin; D001619:beta-N-Acetylhexosaminidases / Q000032:analysis; D020692:rab3 GTP-Binding Proteins
D000730:Androstadienes; D017304:Annexin A5; D008081:Liposomes; D008164:Luminescent Proteins; D010718:Phosphatidylserines; D049452:Green Fluorescent Proteins; D001619:beta-N-Acetylhexosaminidases; D019204:GTP-Binding Proteins; D020692:rab3 GTP-Binding Proteins; D002118:Calcium; D000077191:Wortmannin