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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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Phagosomes acidify in M2 macrophages but maintain near-neutral pH in M1 macrophages. (A) Macrophages were challenged with serum-opsonized fluorescein isothiocyanate-conjugated zymosan (FITC-SOZ), and upon particle binding, pH measurements were made every 30 s for 30 min by ratiometric fluorescence imaging, as detailed in Materials and Methods. M1 macrophages are shown in blue and M2 macrophages in red. (B) M2 macrophages were treated with 2 μM concanamycin A (CcA, green) or left untreated (red, replicated from A), and phagosomal pH measurements were performed as in A. (C) The graph compares phagosomal pH values obtained as in A, 30 min after completion of phagocytosis. Data represent the means ± SEM of 6–13 independent experiments using cells from at least three separate donors. ***p ≤ 0.001.
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Figure 1: Phagosomes acidify in M2 macrophages but maintain near-neutral pH in M1 macrophages. (A) Macrophages were challenged with serum-opsonized fluorescein isothiocyanate-conjugated zymosan (FITC-SOZ), and upon particle binding, pH measurements were made every 30 s for 30 min by ratiometric fluorescence imaging, as detailed in Materials and Methods. M1 macrophages are shown in blue and M2 macrophages in red. (B) M2 macrophages were treated with 2 μM concanamycin A (CcA, green) or left untreated (red, replicated from A), and phagosomal pH measurements were performed as in A. (C) The graph compares phagosomal pH values obtained as in A, 30 min after completion of phagocytosis. Data represent the means ± SEM of 6–13 independent experiments using cells from at least three separate donors. ***p ≤ 0.001.

Mentions: M1 and M2 macrophages exhibit differential expression of phagocytic receptors (Mantovani et al., 2004). To analyze phagosome acidification in M1 and M2 macrophages under comparable conditions, we sought and identified a phagocytic target that was taken up efficiently by both phenotypes, namely serum-opsonized zymosan (SOZ). Before opsonization, zymosan particles were labeled with a pH-sensitive fluorophore of pKa ≈ 6.3, suitable for measurements of phagosomal pH (Steinberg and Grinstein, 2007). A high-resolution system that combines fluorescence ratio imaging with continuous bright-field microscopy was used to identify macrophages that bound labeled SOZ and to measure the pH of the resulting phagosomes as described in Materials and Methods. Using this method, we observed striking differences in the behavior of M1 and M2 cells. Phagosomes formed by M1 macrophages remained near neutrality for at least 30 min after formation, with an average pH of 7.55 ± 0.16. In contrast, M2 phagosomes acidified rapidly, reaching a steady-state pH of 4.99 ± 0.18 within 10 min (Figure 1, A and C). The acidification recorded in M2 phagosomes was prevented by concanamycin A (CcA), confirming the involvement of V-ATPases (Figure 1, B and C). We next explored the mechanisms underlying the differential regulation of pH in these macrophage phenotypes.


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

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

Phagosomes acidify in M2 macrophages but maintain near-neutral pH in M1 macrophages. (A) Macrophages were challenged with serum-opsonized fluorescein isothiocyanate-conjugated zymosan (FITC-SOZ), and upon particle binding, pH measurements were made every 30 s for 30 min by ratiometric fluorescence imaging, as detailed in Materials and Methods. M1 macrophages are shown in blue and M2 macrophages in red. (B) M2 macrophages were treated with 2 μM concanamycin A (CcA, green) or left untreated (red, replicated from A), and phagosomal pH measurements were performed as in A. (C) The graph compares phagosomal pH values obtained as in A, 30 min after completion of phagocytosis. Data represent the means ± SEM of 6–13 independent experiments using cells from at least three separate donors. ***p ≤ 0.001.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Figure 1: Phagosomes acidify in M2 macrophages but maintain near-neutral pH in M1 macrophages. (A) Macrophages were challenged with serum-opsonized fluorescein isothiocyanate-conjugated zymosan (FITC-SOZ), and upon particle binding, pH measurements were made every 30 s for 30 min by ratiometric fluorescence imaging, as detailed in Materials and Methods. M1 macrophages are shown in blue and M2 macrophages in red. (B) M2 macrophages were treated with 2 μM concanamycin A (CcA, green) or left untreated (red, replicated from A), and phagosomal pH measurements were performed as in A. (C) The graph compares phagosomal pH values obtained as in A, 30 min after completion of phagocytosis. Data represent the means ± SEM of 6–13 independent experiments using cells from at least three separate donors. ***p ≤ 0.001.
Mentions: M1 and M2 macrophages exhibit differential expression of phagocytic receptors (Mantovani et al., 2004). To analyze phagosome acidification in M1 and M2 macrophages under comparable conditions, we sought and identified a phagocytic target that was taken up efficiently by both phenotypes, namely serum-opsonized zymosan (SOZ). Before opsonization, zymosan particles were labeled with a pH-sensitive fluorophore of pKa ≈ 6.3, suitable for measurements of phagosomal pH (Steinberg and Grinstein, 2007). A high-resolution system that combines fluorescence ratio imaging with continuous bright-field microscopy was used to identify macrophages that bound labeled SOZ and to measure the pH of the resulting phagosomes as described in Materials and Methods. Using this method, we observed striking differences in the behavior of M1 and M2 cells. Phagosomes formed by M1 macrophages remained near neutrality for at least 30 min after formation, with an average pH of 7.55 ± 0.16. In contrast, M2 phagosomes acidified rapidly, reaching a steady-state pH of 4.99 ± 0.18 within 10 min (Figure 1, A and C). The acidification recorded in M2 phagosomes was prevented by concanamycin A (CcA), confirming the involvement of V-ATPases (Figure 1, B and C). We next explored the mechanisms underlying the differential regulation of pH in these macrophage phenotypes.

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