INTRAPHAGOSOMAL CHLORINATION DYNAMICS AND YIELDS DETERMINED USING UNIQUE FLUORESCENT BACTERIAL MIMICS

Fluorescein was covalently attached through a cyst;amine linker group to carboxy-derivatized polyacrylamide microspheres to generate phagocy tosable particles containing fluorescent reporter groups. A unique fea ture of these heads is that the dye was recoverable in near quantitati ve yield from intracellular environments by thiol reduction of the cys tamine disulfide bond. Fluorescence microscopy indicated that individu al neutrophils could bind as many as similar to 20 serum-opsonized bea ds, although no appreciable cellular association was observed for unop sonized beads. By using methyl viologen to quench external fluorescenc e, it was demonstrated that 70-90% of the neutrophil-associated fluore scein on opsonized beads was inaccessible to the medium. The particle- bound fluorescein underwent near-stoichiometric conversion to chlorina ted derivatives when reacted with HOCl or the cell-free myeloperoxidas e (MPO)-H2O2-Cl- system; products were identified by KPLC separation a nd electrospray ionization mass spectrometry of the recovered dye. Flu orescence changes accompanying phagocytosis were consistent vpi th chl orination of the dye; fluorescence spectrometric and chemical trapping measurements indicated that intraphagosomal chlorination was far more extensive than extracellular chlorination. Yields of recovered chloro fluoresceins determined by HPLC indicated that sufficient HOCl had bee n produced intracellularly to kill entrapped bacteria. Fluorescein chl orination coincided approximately with phagocytosis and stimulated upt ake of O-2 by the cells, Demonstration that HOCl is produced within ph agosomes in sufficient concentrations to kill bacteria on a time scale associated with death constitutes strong evidence in support of a pri mary role for HOCl in the microbicidal action of neutrophils.