Direct measurement of free chloride concentrations in the phagolysosomes of human neutrophils

Human neutrophils synthesize hypochlorous acid, a non-radical oxidant, as one of the antimicrobial agents to kill phagocytosed pathogens within phagolysosomes. The production of HOCl is catalyzed by myeloperoxidase using chloride anions (Cl-). Even though various approaches have been documented to measure cytosolic Cl- concentrations, direct detection of phagolysosomal free Cl- in a real-time fashion is not available. Here we report the development of a new dual-labeled fluorescent probe by conjugation of the chloride-sensitive 6-methoxyquinoline and chloride-insensitive tetramethylrhodamine to the zymosan particles. On the surface of the zymosan particles, the chloride-sensitive fluorophore responded linearly to chloride concentrations when plotted according to the Stern-Volmer collisional quenching equation and the chloride-insensitive fluorophore served as an internal reference. After phagocytosis into neutrophils, the probe was able to monitor intraphagosomal chloride levels authentically. Human neutrophils in the medium containing 122 mM chloride had a phagolysosomal chloride level of 73.3 +/- 12.2 mM (N = 5). In contrast, the neutrophils in a chloride-free isoosmotic medium had a chloride level of 6.6 +/- 1.9 mM (N = 5) in the phagolysosomal compartment. These data clearly demonstrated that the extracellular chloride levels affect intraphagolysosomal chloride levels in human neutrophils. Moreover, when the extracellular chloride concentration was switched from high level (122 mM) to low level (0 mM) or vice versa, the probe demonstrated complete reversibility. The data indicate that the new zymosan-conjugated chloride probe described here can be used as an indicator for measurement of the free chloride level within the phagolysosomes of neutrophils and other phagocytic cell types.