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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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Determinants of phagosomal pH in M1 and M2 macrophages. (A) Macrophages were challenged with FITC-SOZ in the presence of 10 μM DPI, and phagosomal pH was determined every 30 s for the first 30 min of maturation, as described in Materials and Methods. The pH traces show the mean ± SEM of 4–16 independent determinations. (B) M1 and M2 macrophages were challenged with FITC-SOZ. Between 15 and 20 min after phagosome sealing, macrophages were treated with DPI and CcA, and the pH was monitored until a stable baseline was achieved. Then cells were pulsed with NH4+, and the change in pH was determined, followed by an in situ calibration as detailed in Materials and Methods. The graph shown is representative of multiple buffering capacity determinations in M1 and M2 macrophages. (C) Collated phagosomal buffering capacity (βC) determinations obtained as in B. Data are means ± SEM from 11–15 independent determinations for each type. (D) Macrophages were labeled with FM4-64 before (left, top) or after (left, bottom) treatment with PLY. To determine whether PLY lysed internal membranes, cells were challenged with FITC-SOZ in the presence of fluid-phase dextran or pulsed for 1 h with dextran, which was then chased to lysosomes for 4 h, followed by treatment with PLY. Images were obtained by confocal microscopy. Scale bars, 10 μm. (E, G) M1 and M2 cells were challenged with FITC-SOZ, and 15–20 min after phagosome sealing, determination of H+ leak flux was performed in two steps. In step 1, cells were treated with 2 μM CcA and permeabilized with PLY at pH 6.8, and the phagosome was allowed to equilibrate to pH 6.8. After acquisition of baseline readings at pH 6.8, the cells were transferred to buffer pH 7.2 with PLY (step2). The change in pH over time was recorded and used to determine the H+ leak flux. The graph in F is representative of multiple similar experiments. (G) Collated determinations of H+ leak flux. Data are means ± SEM of ≥11 independent determinations in M1 and M2 macrophages. (H) Phagosomal proton-pumping activity was determined by challenging macrophages with FITC-SOZ and allowing acidification to proceed in the presence of 10 μM DPI. Between 10 and 15 min after phagosome sealing, 1 μM CCCP was added. After the phagosomal pH equilibrated near neutral pH, CCCP was washed out with 2% fat-free BSA, and the ensuing pH change was recorded to determine pump activity. Where indicated, 2 μM CcA was added after BSA to assess the contribution of the V-ATPase. (H) A representative trace. (I) Means ± SEM from eight or nine independent determinations for each cell type. *p < 0.05, ***p ≤ 0.001.
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Figure 5: Determinants of phagosomal pH in M1 and M2 macrophages. (A) Macrophages were challenged with FITC-SOZ in the presence of 10 μM DPI, and phagosomal pH was determined every 30 s for the first 30 min of maturation, as described in Materials and Methods. The pH traces show the mean ± SEM of 4–16 independent determinations. (B) M1 and M2 macrophages were challenged with FITC-SOZ. Between 15 and 20 min after phagosome sealing, macrophages were treated with DPI and CcA, and the pH was monitored until a stable baseline was achieved. Then cells were pulsed with NH4+, and the change in pH was determined, followed by an in situ calibration as detailed in Materials and Methods. The graph shown is representative of multiple buffering capacity determinations in M1 and M2 macrophages. (C) Collated phagosomal buffering capacity (βC) determinations obtained as in B. Data are means ± SEM from 11–15 independent determinations for each type. (D) Macrophages were labeled with FM4-64 before (left, top) or after (left, bottom) treatment with PLY. To determine whether PLY lysed internal membranes, cells were challenged with FITC-SOZ in the presence of fluid-phase dextran or pulsed for 1 h with dextran, which was then chased to lysosomes for 4 h, followed by treatment with PLY. Images were obtained by confocal microscopy. Scale bars, 10 μm. (E, G) M1 and M2 cells were challenged with FITC-SOZ, and 15–20 min after phagosome sealing, determination of H+ leak flux was performed in two steps. In step 1, cells were treated with 2 μM CcA and permeabilized with PLY at pH 6.8, and the phagosome was allowed to equilibrate to pH 6.8. After acquisition of baseline readings at pH 6.8, the cells were transferred to buffer pH 7.2 with PLY (step2). The change in pH over time was recorded and used to determine the H+ leak flux. The graph in F is representative of multiple similar experiments. (G) Collated determinations of H+ leak flux. Data are means ± SEM of ≥11 independent determinations in M1 and M2 macrophages. (H) Phagosomal proton-pumping activity was determined by challenging macrophages with FITC-SOZ and allowing acidification to proceed in the presence of 10 μM DPI. Between 10 and 15 min after phagosome sealing, 1 μM CCCP was added. After the phagosomal pH equilibrated near neutral pH, CCCP was washed out with 2% fat-free BSA, and the ensuing pH change was recorded to determine pump activity. Where indicated, 2 μM CcA was added after BSA to assess the contribution of the V-ATPase. (H) A representative trace. (I) Means ± SEM from eight or nine independent determinations for each cell type. *p < 0.05, ***p ≤ 0.001.

Mentions: We next explored whether the increased NADPH oxidase activity of M1 macrophages was responsible for the maintenance of near-neutral pH in their phagosomes (Figure 1A). Treatment with diphenyleneiodonium (DPI), a potent oxidase inhibitor, unmasked an acidification of phagosomes (Figure 5A). However, despite the absence of oxidase activity, the phagosomes in M1 macrophages acidified more slowly than those in M2 cells (Figure 5A). Thus factors other than the rate of proton consumption by dismutation of superoxide must contribute to the differential behavior of M1 and M2 phagosomes.


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

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

Determinants of phagosomal pH in M1 and M2 macrophages. (A) Macrophages were challenged with FITC-SOZ in the presence of 10 μM DPI, and phagosomal pH was determined every 30 s for the first 30 min of maturation, as described in Materials and Methods. The pH traces show the mean ± SEM of 4–16 independent determinations. (B) M1 and M2 macrophages were challenged with FITC-SOZ. Between 15 and 20 min after phagosome sealing, macrophages were treated with DPI and CcA, and the pH was monitored until a stable baseline was achieved. Then cells were pulsed with NH4+, and the change in pH was determined, followed by an in situ calibration as detailed in Materials and Methods. The graph shown is representative of multiple buffering capacity determinations in M1 and M2 macrophages. (C) Collated phagosomal buffering capacity (βC) determinations obtained as in B. Data are means ± SEM from 11–15 independent determinations for each type. (D) Macrophages were labeled with FM4-64 before (left, top) or after (left, bottom) treatment with PLY. To determine whether PLY lysed internal membranes, cells were challenged with FITC-SOZ in the presence of fluid-phase dextran or pulsed for 1 h with dextran, which was then chased to lysosomes for 4 h, followed by treatment with PLY. Images were obtained by confocal microscopy. Scale bars, 10 μm. (E, G) M1 and M2 cells were challenged with FITC-SOZ, and 15–20 min after phagosome sealing, determination of H+ leak flux was performed in two steps. In step 1, cells were treated with 2 μM CcA and permeabilized with PLY at pH 6.8, and the phagosome was allowed to equilibrate to pH 6.8. After acquisition of baseline readings at pH 6.8, the cells were transferred to buffer pH 7.2 with PLY (step2). The change in pH over time was recorded and used to determine the H+ leak flux. The graph in F is representative of multiple similar experiments. (G) Collated determinations of H+ leak flux. Data are means ± SEM of ≥11 independent determinations in M1 and M2 macrophages. (H) Phagosomal proton-pumping activity was determined by challenging macrophages with FITC-SOZ and allowing acidification to proceed in the presence of 10 μM DPI. Between 10 and 15 min after phagosome sealing, 1 μM CCCP was added. After the phagosomal pH equilibrated near neutral pH, CCCP was washed out with 2% fat-free BSA, and the ensuing pH change was recorded to determine pump activity. Where indicated, 2 μM CcA was added after BSA to assess the contribution of the V-ATPase. (H) A representative trace. (I) Means ± SEM from eight or nine independent determinations for each cell type. *p < 0.05, ***p ≤ 0.001.
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Figure 5: Determinants of phagosomal pH in M1 and M2 macrophages. (A) Macrophages were challenged with FITC-SOZ in the presence of 10 μM DPI, and phagosomal pH was determined every 30 s for the first 30 min of maturation, as described in Materials and Methods. The pH traces show the mean ± SEM of 4–16 independent determinations. (B) M1 and M2 macrophages were challenged with FITC-SOZ. Between 15 and 20 min after phagosome sealing, macrophages were treated with DPI and CcA, and the pH was monitored until a stable baseline was achieved. Then cells were pulsed with NH4+, and the change in pH was determined, followed by an in situ calibration as detailed in Materials and Methods. The graph shown is representative of multiple buffering capacity determinations in M1 and M2 macrophages. (C) Collated phagosomal buffering capacity (βC) determinations obtained as in B. Data are means ± SEM from 11–15 independent determinations for each type. (D) Macrophages were labeled with FM4-64 before (left, top) or after (left, bottom) treatment with PLY. To determine whether PLY lysed internal membranes, cells were challenged with FITC-SOZ in the presence of fluid-phase dextran or pulsed for 1 h with dextran, which was then chased to lysosomes for 4 h, followed by treatment with PLY. Images were obtained by confocal microscopy. Scale bars, 10 μm. (E, G) M1 and M2 cells were challenged with FITC-SOZ, and 15–20 min after phagosome sealing, determination of H+ leak flux was performed in two steps. In step 1, cells were treated with 2 μM CcA and permeabilized with PLY at pH 6.8, and the phagosome was allowed to equilibrate to pH 6.8. After acquisition of baseline readings at pH 6.8, the cells were transferred to buffer pH 7.2 with PLY (step2). The change in pH over time was recorded and used to determine the H+ leak flux. The graph in F is representative of multiple similar experiments. (G) Collated determinations of H+ leak flux. Data are means ± SEM of ≥11 independent determinations in M1 and M2 macrophages. (H) Phagosomal proton-pumping activity was determined by challenging macrophages with FITC-SOZ and allowing acidification to proceed in the presence of 10 μM DPI. Between 10 and 15 min after phagosome sealing, 1 μM CCCP was added. After the phagosomal pH equilibrated near neutral pH, CCCP was washed out with 2% fat-free BSA, and the ensuing pH change was recorded to determine pump activity. Where indicated, 2 μM CcA was added after BSA to assess the contribution of the V-ATPase. (H) A representative trace. (I) Means ± SEM from eight or nine independent determinations for each cell type. *p < 0.05, ***p ≤ 0.001.
Mentions: We next explored whether the increased NADPH oxidase activity of M1 macrophages was responsible for the maintenance of near-neutral pH in their phagosomes (Figure 1A). Treatment with diphenyleneiodonium (DPI), a potent oxidase inhibitor, unmasked an acidification of phagosomes (Figure 5A). However, despite the absence of oxidase activity, the phagosomes in M1 macrophages acidified more slowly than those in M2 cells (Figure 5A). Thus factors other than the rate of proton consumption by dismutation of superoxide must contribute to the differential behavior of M1 and M2 phagosomes.

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