Quantitative measurement of mast cell degranulation using a novel flow cytometric annexin-V binding assay

Background: Mast cells are primary mediators of allergic inflammation. Anti gen-mediated crosslinking of their cell surface immunoglobulin E (IgE) rece ptors 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 cros slinking or ionophore treatment. Exogenously added annexin-V was used to st ain exocytosing granules, and the extent of binding was measured now cytome trically. 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-T i binding assay. Results: Annexin-V specifically bound to mast cell granules exposed after s timulation in proportion to the extent of degranulation. Annexin-V binding was calcium dependent and was blocked by phosphatidylserine containing lipo somes, consistent with specific binding to this membrane Lipid. Visualizati on of annexin-V staining showed granular cell surface patches that colocali zed 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 mutan t protein. Conclusions: The annexin-V binding assay represents a powerful new flow cyt ometric method to monitor mast cell degranulation for functional analysis. (C) 1999 Wiley-Liss, Inc.