Cytosolic free calcium elevation mediates the phagosome-lysosome fusion during phagocytosis in human neutrophils

Abstract

Cytosolic free calcium ([Ca2+](i)) and fusion of secondary granules with the phagosomal membrane (phagosome-lysosome fusion, P-L fusion) were assessed in single adherent human neutrophils during phagocytosis of C3bi-opsonized yeast particles. Neutrophils were loaded with the fluorescent dye fura2/AM and [Ca2+](i) was assessed by dual excitation microfluorimetry. Discharge of lactoferrin, a secondary granule marker into the phagosome was verified by immunostaining using standard epifluorescence, confocal laser scanning and electron microscopy. In Ca2+-containing medium, upon contact with a yeast particle, a rapid rise in [Ca2+](i) was observed, followed by one or more Ca2+ peaks (maximal value 1,586 nM and median duration 145 s): P-L fusion was detected in 80% of the cells after 5-10 min. In Ca2+-free medium the amplitude, frequency and duration of the [Ca2+](i) transients were decreased (maximal value 368 nM, mostly one single Ca2+ peak and median duration 75 s): P-L fusion was decreased to 52%. Increasing the cytosolic Ca2+ buffering capacity by loading the cells with MAPT/AM led to a dose-dependent inhibition both of [Ca2+](i) elevations and P-L fusion. Under conditions where basal [Ca2+](i) was reduced to <20 nM and intracellular Ca2+ stores were depleted, P-L fusion was drastically inhibited while the cells ingested yeast particles normally. P-L fusion could be restored in Ca2+-buffered cells containing ingested particles by elevating [Ca2+](i) with the Ca2+-ionophore ionomycin. The present findings directly indicate that although the ingestion step of phagocytosis is a Ca2+-independent event, [Ca2+](i) transients triggered upon contact with opsonized particles are necessary to control the subsequent fusion or secondary granules with the phagosomal membrane.