Cargando…
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 a...
Formato: | Texto |
---|---|
Lenguaje: | English |
Publicado: |
The Rockefeller University Press
1990
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2200167/ https://www.ncbi.nlm.nih.gov/pubmed/2110568 |
_version_ | 1782148278171729920 |
---|---|
collection | PubMed |
description | 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. |
format | Text |
id | pubmed-2200167 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 1990 |
publisher | The Rockefeller University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-22001672008-05-01 Cytosolic free calcium elevation mediates the phagosome-lysosome fusion during phagocytosis in human neutrophils J Cell Biol Articles 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. The Rockefeller University Press 1990-05-01 /pmc/articles/PMC2200167/ /pubmed/2110568 Text en This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/4.0/). |
spellingShingle | Articles Cytosolic free calcium elevation mediates the phagosome-lysosome fusion during phagocytosis in human neutrophils |
title | Cytosolic free calcium elevation mediates the phagosome-lysosome fusion during phagocytosis in human neutrophils |
title_full | Cytosolic free calcium elevation mediates the phagosome-lysosome fusion during phagocytosis in human neutrophils |
title_fullStr | Cytosolic free calcium elevation mediates the phagosome-lysosome fusion during phagocytosis in human neutrophils |
title_full_unstemmed | Cytosolic free calcium elevation mediates the phagosome-lysosome fusion during phagocytosis in human neutrophils |
title_short | Cytosolic free calcium elevation mediates the phagosome-lysosome fusion during phagocytosis in human neutrophils |
title_sort | cytosolic free calcium elevation mediates the phagosome-lysosome fusion during phagocytosis in human neutrophils |
topic | Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2200167/ https://www.ncbi.nlm.nih.gov/pubmed/2110568 |