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

Abstract

Abstract. Cytosolic free calcium ([Ca2+]0 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+]~ 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+]~ was observed, followed by one or more Ca 2÷ 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 Ca ~÷-free medium the amplitude, frequency and duration of the [Ca2+]~ transients were decreased (maximal value 368 nM, mostly one single Ca ~+ peak and median duration 75 s): P-L fusion was decreased to 52%.Increasing the cytosolic Ca 2+ buffering capacity by loading the cells with MAPT/AM led to a dosedependent inhibition both of [Ca:+]i elevations and P-L fusion. Under conditions where basal [Ca2+]~ was reduced to <20 nM and intracellular Ca 2+ 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+]~ with the Ca 2+-ionophore ionomycin.The present findings directly indicate that although the ingestion step of phagocytosis is a Ca 2+-independent event, [Ca~+]~ transients triggered upon contact with opsonized particles are necessary to control the subsequent fusion of secondary granules with the phagosomal membrane.

Intracellular Calcium Waves Accompany Neutrophil Polarization, Formylmethionylleucylphenylalanine Stimulation, and Phagocytosis: A High Speed Microscopy Study

“…This is consistent with the reported intracellular origin of Ca 2ϩ signals mediating phagolysosome formation. Because phagocytosis takes place at very low Ca 2ϩ levels (37), we suggest that these Ca 2ϩ waves promote the formation of phagolysosomes, which does require Ca 2ϩ signaling (38,39). Although these Ca 2ϩ waves resemble those associated with chemotactic stimulation, they differ in their location.…”

VSOP/Hv1 proton channels sustain calcium entry, neutrophil migration, and superoxide production by limiting cell depolarization and acidification

“…Accordingly, we observed a thicker ring of polymerized actin around zymosan particles ingested by VSOP/Hv1 / neutrophils. The thicker cortical actin cytoskeleton might prevent the docking of oxidase subunits to the phagosome and the fusion of lysosomes with phagosomes (Jaconi et al, 1990). Whether lack of VSOP/Hv1 proton channels impairs the fusion of granules to phagosomes remains to be established, but we document here that other Ca 2+ -dependent functions of neutrophils are altered.…”

“…This signaling defect can have important consequences for the maturation of the phagosome and for the ability of neutrophils to kill microbes. Global Ca 2+ elevations accelerate phagocytosis by activating calpain (Dewitt and Hallett, 2002), whereas local Ca 2+ elevations around the phagosome are required for the fusion of lysosomes with phagosomes (Jaconi et al, 1990). The periphagosomal increase in ionomycin (Fig.…”

Selective Inhibition of IgG-Mediated Phagocytosis in Gelsolin-Deficient Murine Neutrophils

“…Intracellular Ca 2ϩ stores and granules also translocate to the phagosome and phagolysosome fusion takes place (11). In neutrophils, this is a calcium-dependent process (12).…”