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Contrasting phagosome pH regulation and maturation in human M1 and M2 macrophages.

Canton J, Khezri R, Glogauer M, Grinstein S - Mol. Biol. Cell (2014)

Bottom Line: The paucity of V-ATPases in M1 phagosomes was associated with, and likely caused by, delayed fusion with late endosomes and lysosomes.The delayed kinetics of maturation was, in turn, promoted by the failure of M1 phagosomes to acidify.By contrast, M2 phagosomes proceed to acidify immediately in order to clear apoptotic bodies rapidly and effectively.

View Article: PubMed Central - PubMed

Affiliation: Program in Cell Biology, Hospital for Sick Children, Toronto, ON M5G 0A4, Canada.

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The NADPH oxidase is retained on M1 phagosomes but rapidly lost from M2 phagosomes. (A–C) Macrophages were challenged with SOZ for 1 min in the presence of NBT at 1 or 10 μg/ml. Excess SOZ was washed off with PBS, followed by incubation at 37°C in HEPES-buffered RPMI with 1 or 10 μg/ml NBT, acquiring pictures every 30 s for 30 min. (A) Images of individual phagosomes acquired at the indicated times during maturation for M1 (top) and M2 (bottom) macrophages. Scale bar, 5 μm. (B, C) Deposition of formazan was quantified using ImageJ and is depicted relative to the maximum value for each phagosome. Measurements for M1 macrophages were performed using 1 μg/ml NBT, as 10 μg/ml resulted in early signal saturation; for M2 macrophages, measurements were performed using 10 μg/ml NBT, as no signal could be detected at 1 μg/ml due to the low level of ROS production in these cells. Data represent deposition of formazan in a single phagosome, representative of three independent experiments. (D–H) Macrophages were challenged with biotinylated, FITC-labeled SOZ sedimented onto the cells by centrifugation for 1 min. Cells were incubated for 4 min at 37°C, then immediately placed on ice-cold PBS with streptavidin 647 to label incompletely internalized particles. The cells were then washed with ice-cold PBS, and the 5-min time-point coverslips were fixed with ice-cold methanol. The 20- and 90-min time-point coverslips were incubated for an additional 15 and 85 min, respectively, at 37°C and then fixed with ice-cold methanol. All coverslips were immunostained with a p22 monoclonal antibody and imaged by confocal microscopy. (D, E) Representative confocal images. (F) The fluorescence signal derived from the p22 monoclonal antibody at the phagosome membrane was quantified in images like those in D and E. Data in F represent the normalized mean fluorescence ± SEM from 30 phagosomes from three experiments. (G, H) Percentage of phagosomes with score 2, 1, or 0 (see Supplemental Figure S3 for scoring criteria) for p22 at the indicated time points in M1 (G) or M2 (H) macrophages. Data are means ± SEM from three independent experiments using cells from different donors.
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Figure 3: The NADPH oxidase is retained on M1 phagosomes but rapidly lost from M2 phagosomes. (A–C) Macrophages were challenged with SOZ for 1 min in the presence of NBT at 1 or 10 μg/ml. Excess SOZ was washed off with PBS, followed by incubation at 37°C in HEPES-buffered RPMI with 1 or 10 μg/ml NBT, acquiring pictures every 30 s for 30 min. (A) Images of individual phagosomes acquired at the indicated times during maturation for M1 (top) and M2 (bottom) macrophages. Scale bar, 5 μm. (B, C) Deposition of formazan was quantified using ImageJ and is depicted relative to the maximum value for each phagosome. Measurements for M1 macrophages were performed using 1 μg/ml NBT, as 10 μg/ml resulted in early signal saturation; for M2 macrophages, measurements were performed using 10 μg/ml NBT, as no signal could be detected at 1 μg/ml due to the low level of ROS production in these cells. Data represent deposition of formazan in a single phagosome, representative of three independent experiments. (D–H) Macrophages were challenged with biotinylated, FITC-labeled SOZ sedimented onto the cells by centrifugation for 1 min. Cells were incubated for 4 min at 37°C, then immediately placed on ice-cold PBS with streptavidin 647 to label incompletely internalized particles. The cells were then washed with ice-cold PBS, and the 5-min time-point coverslips were fixed with ice-cold methanol. The 20- and 90-min time-point coverslips were incubated for an additional 15 and 85 min, respectively, at 37°C and then fixed with ice-cold methanol. All coverslips were immunostained with a p22 monoclonal antibody and imaged by confocal microscopy. (D, E) Representative confocal images. (F) The fluorescence signal derived from the p22 monoclonal antibody at the phagosome membrane was quantified in images like those in D and E. Data in F represent the normalized mean fluorescence ± SEM from 30 phagosomes from three experiments. (G, H) Percentage of phagosomes with score 2, 1, or 0 (see Supplemental Figure S3 for scoring criteria) for p22 at the indicated time points in M1 (G) or M2 (H) macrophages. Data are means ± SEM from three independent experiments using cells from different donors.

Mentions: The net generation of ROS is much greater in M1 than in M2 macrophages not only because of their higher NOX2 content, but also because the oxidase remains active longer. This became apparent when monitoring the rate of superoxide generation over time in single phagosomes, incubating the cells in the presence of subsaturating concentrations of NBT (1 μg/ml for M1 and 10 μg/ml for M2 macrophages). Using bright-field microscopy, we could estimate the rate of formazan deposition by acquiring images at regular intervals and quantifying the progressive decrease in pixel intensity. As illustrated in Figure 3A and summarized in Figure 3, B and C, whereas M1 phagosomes generate ROS continuously for at least 30 min, the oxidase activity of M2 phagosomes becomes undetectable after 5–10 min.


Contrasting phagosome pH regulation and maturation in human M1 and M2 macrophages.

Canton J, Khezri R, Glogauer M, Grinstein S - Mol. Biol. Cell (2014)

The NADPH oxidase is retained on M1 phagosomes but rapidly lost from M2 phagosomes. (A–C) Macrophages were challenged with SOZ for 1 min in the presence of NBT at 1 or 10 μg/ml. Excess SOZ was washed off with PBS, followed by incubation at 37°C in HEPES-buffered RPMI with 1 or 10 μg/ml NBT, acquiring pictures every 30 s for 30 min. (A) Images of individual phagosomes acquired at the indicated times during maturation for M1 (top) and M2 (bottom) macrophages. Scale bar, 5 μm. (B, C) Deposition of formazan was quantified using ImageJ and is depicted relative to the maximum value for each phagosome. Measurements for M1 macrophages were performed using 1 μg/ml NBT, as 10 μg/ml resulted in early signal saturation; for M2 macrophages, measurements were performed using 10 μg/ml NBT, as no signal could be detected at 1 μg/ml due to the low level of ROS production in these cells. Data represent deposition of formazan in a single phagosome, representative of three independent experiments. (D–H) Macrophages were challenged with biotinylated, FITC-labeled SOZ sedimented onto the cells by centrifugation for 1 min. Cells were incubated for 4 min at 37°C, then immediately placed on ice-cold PBS with streptavidin 647 to label incompletely internalized particles. The cells were then washed with ice-cold PBS, and the 5-min time-point coverslips were fixed with ice-cold methanol. The 20- and 90-min time-point coverslips were incubated for an additional 15 and 85 min, respectively, at 37°C and then fixed with ice-cold methanol. All coverslips were immunostained with a p22 monoclonal antibody and imaged by confocal microscopy. (D, E) Representative confocal images. (F) The fluorescence signal derived from the p22 monoclonal antibody at the phagosome membrane was quantified in images like those in D and E. Data in F represent the normalized mean fluorescence ± SEM from 30 phagosomes from three experiments. (G, H) Percentage of phagosomes with score 2, 1, or 0 (see Supplemental Figure S3 for scoring criteria) for p22 at the indicated time points in M1 (G) or M2 (H) macrophages. Data are means ± SEM from three independent experiments using cells from different donors.
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Figure 3: The NADPH oxidase is retained on M1 phagosomes but rapidly lost from M2 phagosomes. (A–C) Macrophages were challenged with SOZ for 1 min in the presence of NBT at 1 or 10 μg/ml. Excess SOZ was washed off with PBS, followed by incubation at 37°C in HEPES-buffered RPMI with 1 or 10 μg/ml NBT, acquiring pictures every 30 s for 30 min. (A) Images of individual phagosomes acquired at the indicated times during maturation for M1 (top) and M2 (bottom) macrophages. Scale bar, 5 μm. (B, C) Deposition of formazan was quantified using ImageJ and is depicted relative to the maximum value for each phagosome. Measurements for M1 macrophages were performed using 1 μg/ml NBT, as 10 μg/ml resulted in early signal saturation; for M2 macrophages, measurements were performed using 10 μg/ml NBT, as no signal could be detected at 1 μg/ml due to the low level of ROS production in these cells. Data represent deposition of formazan in a single phagosome, representative of three independent experiments. (D–H) Macrophages were challenged with biotinylated, FITC-labeled SOZ sedimented onto the cells by centrifugation for 1 min. Cells were incubated for 4 min at 37°C, then immediately placed on ice-cold PBS with streptavidin 647 to label incompletely internalized particles. The cells were then washed with ice-cold PBS, and the 5-min time-point coverslips were fixed with ice-cold methanol. The 20- and 90-min time-point coverslips were incubated for an additional 15 and 85 min, respectively, at 37°C and then fixed with ice-cold methanol. All coverslips were immunostained with a p22 monoclonal antibody and imaged by confocal microscopy. (D, E) Representative confocal images. (F) The fluorescence signal derived from the p22 monoclonal antibody at the phagosome membrane was quantified in images like those in D and E. Data in F represent the normalized mean fluorescence ± SEM from 30 phagosomes from three experiments. (G, H) Percentage of phagosomes with score 2, 1, or 0 (see Supplemental Figure S3 for scoring criteria) for p22 at the indicated time points in M1 (G) or M2 (H) macrophages. Data are means ± SEM from three independent experiments using cells from different donors.
Mentions: The net generation of ROS is much greater in M1 than in M2 macrophages not only because of their higher NOX2 content, but also because the oxidase remains active longer. This became apparent when monitoring the rate of superoxide generation over time in single phagosomes, incubating the cells in the presence of subsaturating concentrations of NBT (1 μg/ml for M1 and 10 μg/ml for M2 macrophages). Using bright-field microscopy, we could estimate the rate of formazan deposition by acquiring images at regular intervals and quantifying the progressive decrease in pixel intensity. As illustrated in Figure 3A and summarized in Figure 3, B and C, whereas M1 phagosomes generate ROS continuously for at least 30 min, the oxidase activity of M2 phagosomes becomes undetectable after 5–10 min.

Bottom Line: The paucity of V-ATPases in M1 phagosomes was associated with, and likely caused by, delayed fusion with late endosomes and lysosomes.The delayed kinetics of maturation was, in turn, promoted by the failure of M1 phagosomes to acidify.By contrast, M2 phagosomes proceed to acidify immediately in order to clear apoptotic bodies rapidly and effectively.

View Article: PubMed Central - PubMed

Affiliation: Program in Cell Biology, Hospital for Sick Children, Toronto, ON M5G 0A4, Canada.

Show MeSH
Related in: MedlinePlus