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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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M1 macrophages produce more intraphagosomal reactive oxygen species than M2 macrophages. (A) Macrophages were challenged with SOZ for 30 min in the presence of NBT at 1 or 10 μg/ml, as indicated. Cells were then washed three times with PBS, fixed with 2% paraformaldehyde, and imaged by differential interference contrast microscopy. Dark formazan deposits indicate local production of superoxide. White arrows indicate SOZ-containing phagosomes that are negative for formazan deposits. Scale bars, 10 μm. (B, C) Macrophages were challenged with SOZ in the presence of 8 U/ml HRP. After 1 min of phagocytosis, excess SOZ was washed off and the cells bathed in PBS containing 10 mM glucose, 8 U/ml HRP, and 50 μM luminol. Luminescence was measured for 10 min (no treatment [NT]), and then superoxide dismutase (SOD; 50 U/ml) and catalase (2000 U/ml) were added to eliminate extracellular ROS. Luminescence was measured for an additional 80 min. (C) Luminescence determinations 20, 60, and 90 min after the addition of SOD and catalase. Data are displayed as relative luminescence units (RLUs) over time and represent the mean ± SEM from three independent experiments using cells from two donors. (D) Differentiated M1 and M2 macrophages were lysed and separated by 12% SDS–PAGE. Images show immunoblots for p22, gp91, and actin. (E) Quantitation of multiple experiments like that in D. The p22/actin and gp91/actin ratios are depicted. Data represent the mean ± SEM from three independent experiments using cells from two donors. *p < 0.05, ***p ≤ 0.001.
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Figure 2: M1 macrophages produce more intraphagosomal reactive oxygen species than M2 macrophages. (A) Macrophages were challenged with SOZ for 30 min in the presence of NBT at 1 or 10 μg/ml, as indicated. Cells were then washed three times with PBS, fixed with 2% paraformaldehyde, and imaged by differential interference contrast microscopy. Dark formazan deposits indicate local production of superoxide. White arrows indicate SOZ-containing phagosomes that are negative for formazan deposits. Scale bars, 10 μm. (B, C) Macrophages were challenged with SOZ in the presence of 8 U/ml HRP. After 1 min of phagocytosis, excess SOZ was washed off and the cells bathed in PBS containing 10 mM glucose, 8 U/ml HRP, and 50 μM luminol. Luminescence was measured for 10 min (no treatment [NT]), and then superoxide dismutase (SOD; 50 U/ml) and catalase (2000 U/ml) were added to eliminate extracellular ROS. Luminescence was measured for an additional 80 min. (C) Luminescence determinations 20, 60, and 90 min after the addition of SOD and catalase. Data are displayed as relative luminescence units (RLUs) over time and represent the mean ± SEM from three independent experiments using cells from two donors. (D) Differentiated M1 and M2 macrophages were lysed and separated by 12% SDS–PAGE. Images show immunoblots for p22, gp91, and actin. (E) Quantitation of multiple experiments like that in D. The p22/actin and gp91/actin ratios are depicted. Data represent the mean ± SEM from three independent experiments using cells from two donors. *p < 0.05, ***p ≤ 0.001.

Mentions: The rate and extent of deposition of formazan were considerably greater in M1 than in M2 phagosomes (Figure 2A). In fact, while formazan deposits were readily observed in M1 phagosomes when using 1 μg/ml NBT, they were only visible in a fraction of M2 phagosomes even when using a 10-fold higher concentration (10 μg/ml) of NBT (Figure 2A). The large difference in reactive oxygen species (ROS) generation was verified using luminol, adding superoxide dismutase and catalase 10 min after phagocytosis was initiated to selectively analyze intracellular (largely intraphagosomal) ROS generation (Dahlgren et al., 2007). As illustrated in Figure 2, B and C, ROS production was much greater and more sustained in M1 than in M2 phagosomes, in good agreement with the formazan deposition determinations.


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

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

M1 macrophages produce more intraphagosomal reactive oxygen species than M2 macrophages. (A) Macrophages were challenged with SOZ for 30 min in the presence of NBT at 1 or 10 μg/ml, as indicated. Cells were then washed three times with PBS, fixed with 2% paraformaldehyde, and imaged by differential interference contrast microscopy. Dark formazan deposits indicate local production of superoxide. White arrows indicate SOZ-containing phagosomes that are negative for formazan deposits. Scale bars, 10 μm. (B, C) Macrophages were challenged with SOZ in the presence of 8 U/ml HRP. After 1 min of phagocytosis, excess SOZ was washed off and the cells bathed in PBS containing 10 mM glucose, 8 U/ml HRP, and 50 μM luminol. Luminescence was measured for 10 min (no treatment [NT]), and then superoxide dismutase (SOD; 50 U/ml) and catalase (2000 U/ml) were added to eliminate extracellular ROS. Luminescence was measured for an additional 80 min. (C) Luminescence determinations 20, 60, and 90 min after the addition of SOD and catalase. Data are displayed as relative luminescence units (RLUs) over time and represent the mean ± SEM from three independent experiments using cells from two donors. (D) Differentiated M1 and M2 macrophages were lysed and separated by 12% SDS–PAGE. Images show immunoblots for p22, gp91, and actin. (E) Quantitation of multiple experiments like that in D. The p22/actin and gp91/actin ratios are depicted. Data represent the mean ± SEM from three independent experiments using cells from two donors. *p < 0.05, ***p ≤ 0.001.
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Figure 2: M1 macrophages produce more intraphagosomal reactive oxygen species than M2 macrophages. (A) Macrophages were challenged with SOZ for 30 min in the presence of NBT at 1 or 10 μg/ml, as indicated. Cells were then washed three times with PBS, fixed with 2% paraformaldehyde, and imaged by differential interference contrast microscopy. Dark formazan deposits indicate local production of superoxide. White arrows indicate SOZ-containing phagosomes that are negative for formazan deposits. Scale bars, 10 μm. (B, C) Macrophages were challenged with SOZ in the presence of 8 U/ml HRP. After 1 min of phagocytosis, excess SOZ was washed off and the cells bathed in PBS containing 10 mM glucose, 8 U/ml HRP, and 50 μM luminol. Luminescence was measured for 10 min (no treatment [NT]), and then superoxide dismutase (SOD; 50 U/ml) and catalase (2000 U/ml) were added to eliminate extracellular ROS. Luminescence was measured for an additional 80 min. (C) Luminescence determinations 20, 60, and 90 min after the addition of SOD and catalase. Data are displayed as relative luminescence units (RLUs) over time and represent the mean ± SEM from three independent experiments using cells from two donors. (D) Differentiated M1 and M2 macrophages were lysed and separated by 12% SDS–PAGE. Images show immunoblots for p22, gp91, and actin. (E) Quantitation of multiple experiments like that in D. The p22/actin and gp91/actin ratios are depicted. Data represent the mean ± SEM from three independent experiments using cells from two donors. *p < 0.05, ***p ≤ 0.001.
Mentions: The rate and extent of deposition of formazan were considerably greater in M1 than in M2 phagosomes (Figure 2A). In fact, while formazan deposits were readily observed in M1 phagosomes when using 1 μg/ml NBT, they were only visible in a fraction of M2 phagosomes even when using a 10-fold higher concentration (10 μg/ml) of NBT (Figure 2A). The large difference in reactive oxygen species (ROS) generation was verified using luminol, adding superoxide dismutase and catalase 10 min after phagocytosis was initiated to selectively analyze intracellular (largely intraphagosomal) ROS generation (Dahlgren et al., 2007). As illustrated in Figure 2, B and C, ROS production was much greater and more sustained in M1 than in M2 phagosomes, in good agreement with the formazan deposition determinations.

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