STIMULATED RELEASE OF FLUORESCENTLY LABELED IGE FRAGMENTS THAT EFFICIENTLY ACCUMULATE IN SECRETORY GRANULES AFTER ENDOCYTOSIS IN RBL-2H3 MAST-CELLS

Sensitization of RBL-2H3 mast cells with monomeric fluorescein-5-isoth iocyanate (FITC)-labeled immunoglobulin E (IgE) results in slow but hi ghly efficient accumulation of labeled IgE fragments in a pool of acid ic peripheral vesicles that are visible by fluorescence microscopy aft er raising endosomal pH with ammonium chloride. Stimulation of cells c ontaining these FITC-IgE fragments by aggregation of high affinity rec eptors for IgE (Fc epsilon RI) or by Ca2+ ionophore and phorbol 12-myr istate 13-acetate results in release of FITC fluorescence from the cel ls, which can be monitored continuously with a spectrofluorometer. The fluorescence release process corresponds to cellular degranulation: i t is prevented under conditions that prevent stimulated beta-hexosamin idase release, and these two processes exhibit the same antigen dose-d ependence and kinetics. Pulse-chase labeling reveals that aggregation of FITC-IgE bound to Fc epsilon RI at the cell surface causes internal ization and delivery to the regulated secretory vesicles with a high e fficiency similar to monomeric IgE-Fc epsilon RI, but more rapidly. Bi nding of Cy3-modified IgE to Fc epsilon RI results in labeling of the same secretory vesicles as in FITC-IgE-sensitized cells, and these Cy3 -labeled vesicles can be observed by fluorescence microscopy without n eutralization of intracellular compartments. Simultaneous three-photon microscopy of serotonin fluorescence and two-photon microscopy of Cy3 fluorescence reveals that these Cy3-labeled vesicles coincide with se rotonin-labeled secretory granules. After stimulation of the cells via aggregation of IgE-Fc epsilon RI or addition of Ca2+ ionophore and ph orbol 12-myristate 13-acetate, depletion of the Cy3 label from the int racellular vesicles is observed with confocal microscopy, These result s provide strong evidence for the lysosomal nature of secretory granul es in these cells. In addition, they provide the basis for a direct, r eal-time method for monitoring single cell degranulation.