Cytosolic free calcium elevation mediates the phagosome-lysosome fusion during phagocytosis in human neutrophils.
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 less than 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 of secondary granules with the phagosomal membrane.
Jaconi
M E
ME
Division of Infectious Diseases, Geneva University Hospital, Switzerland.
Lew
D P
DP
Carpentier
J L
JL
Magnusson
K E
KE
Sjögren
M
M
Stendahl
O
O
D001572:Benzofurans; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D003600:Cytosol / Q000378:metabolism; D004533:Egtazic Acid / Q000031:analogs & derivatives; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives*; D006801:Humans; D007150:Immunohistochemistry; D007781:Lactoferrin / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism / Q000502:physiology*; D008561:Membrane Fusion / Q000502:physiology; D008854:Microscopy, Electron; D009504:Neutrophils / Q000378:metabolism*; D010587:Phagocytosis / Q000502:physiology*; D010588:Phagosomes / Q000378:metabolism / Q000502:physiology*
D001572:Benzofurans; D005456:Fluorescent Dyes; C049925:fura-2-am; D004533:Egtazic Acid; C058180:5,5'-dimethyl-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate; D007781:Lactoferrin; D002118:Calcium; D016257:Fura-2