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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 exhibit delayed phagosome-lysosome fusion kinetics. Macrophages were challenged with either FITC-labeled SOZ or tetramethyl rhodamine–labeled SOZ and centrifuged for 1 min, and unbound particles were washed with PBS. Cells were incubated for 4 min at 37°C, then immediately placed on ice-cold PBS with anti-human IgG 647 (A–D) or anti-human IgG 488 (E, F) to label incompletely internalized zymosan particles. Cells were then incubated at 37°C in RPMI for the indicated times, fixed with either ice-cold methanol (A–D) or 2% paraformaldehyde (E, F), and immunostained for either TfR (A, B) or LAMP-1 (B, C). Lysosomes in the cells in E and F were preloaded with dextran 647 as outlined in Materials and Methods. Arrowheads indicate phagosomes positive for TfR (A), LAMP-1 (C), or dextran (E). Arrows indicate phagosomes negative for TfR (A), LAMP-1 (B), or dextran (E). Scale bars, 5 μm. (B, D, F) Percentage of phagosomes positive for TfR, LAMP-1, or dextran at the indicated times. Data are means ± SEM from three independent experiments using cells from different donors. *p < 0.05; ***p ≤ 0.001.
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Figure 4: M1 macrophages exhibit delayed phagosome-lysosome fusion kinetics. Macrophages were challenged with either FITC-labeled SOZ or tetramethyl rhodamine–labeled SOZ and centrifuged for 1 min, and unbound particles were washed with PBS. Cells were incubated for 4 min at 37°C, then immediately placed on ice-cold PBS with anti-human IgG 647 (A–D) or anti-human IgG 488 (E, F) to label incompletely internalized zymosan particles. Cells were then incubated at 37°C in RPMI for the indicated times, fixed with either ice-cold methanol (A–D) or 2% paraformaldehyde (E, F), and immunostained for either TfR (A, B) or LAMP-1 (B, C). Lysosomes in the cells in E and F were preloaded with dextran 647 as outlined in Materials and Methods. Arrowheads indicate phagosomes positive for TfR (A), LAMP-1 (C), or dextran (E). Arrows indicate phagosomes negative for TfR (A), LAMP-1 (B), or dextran (E). Scale bars, 5 μm. (B, D, F) Percentage of phagosomes positive for TfR, LAMP-1, or dextran at the indicated times. Data are means ± SEM from three independent experiments using cells from different donors. *p < 0.05; ***p ≤ 0.001.

Mentions: After sealing, the phagosomal membrane undergoes extensive remodeling, a result of multiple fusion and fission events with other endomembrane compartments. The differential rate of clearance of the NADPH oxidase from M1 and M2 phagosomes suggests that maturation proceeds at different rates and/or by different routes in the two types of macrophages. To determine whether this was indeed the case, we measured the course of acquisition and loss of several endomembrane markers from M1 or M2 phagosomes. Macrophages were challenged with labeled SOZ, and after 5 min of phagocytosis, external or incompletely internalized particles were identified by addition of fluorescently labeled antibodies. By subsequently omitting from consideration the antibody-tagged particles, we were able to more precisely demarcate the initiation of the maturation period in the untagged (fully internalized) SOZ. As illustrated in Figure 4, A and B, transferrin receptors (TfRs), which are indicative of early/recycling endosomes, were acquired very rapidly by M2 phagosomes and disappeared shortly thereafter, as lysosome-associated membrane protein 1 (LAMP-1) was recruited. The later stages of acquisition of LAMP-1, a late endosome-lysosome marker, corresponded to the time of acquisition of labeled dextran, which was chased after pulsing (see Materials and Methods) to serve as a lysosomal marker. This pattern of rapid maturation is somewhat faster than that described for unpolarized primary and immortalized macrophages (Tsang et al., 2000; Sokolovska et al., 2013) and is compatible with the rapid acquisition of V-ATPases deduced from pH measurements. In stark contrast, M1 phagosomes retained TfR longer and acquired LAMP-1 and dextran more slowly and less completely than their M2 counterparts. The delayed maturation of M1 phagosomes is consistent with the prolonged residence of gp91 and p22 on their membrane and the sustained phagosomal oxidase activity.


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 exhibit delayed phagosome-lysosome fusion kinetics. Macrophages were challenged with either FITC-labeled SOZ or tetramethyl rhodamine–labeled SOZ and centrifuged for 1 min, and unbound particles were washed with PBS. Cells were incubated for 4 min at 37°C, then immediately placed on ice-cold PBS with anti-human IgG 647 (A–D) or anti-human IgG 488 (E, F) to label incompletely internalized zymosan particles. Cells were then incubated at 37°C in RPMI for the indicated times, fixed with either ice-cold methanol (A–D) or 2% paraformaldehyde (E, F), and immunostained for either TfR (A, B) or LAMP-1 (B, C). Lysosomes in the cells in E and F were preloaded with dextran 647 as outlined in Materials and Methods. Arrowheads indicate phagosomes positive for TfR (A), LAMP-1 (C), or dextran (E). Arrows indicate phagosomes negative for TfR (A), LAMP-1 (B), or dextran (E). Scale bars, 5 μm. (B, D, F) Percentage of phagosomes positive for TfR, LAMP-1, or dextran at the indicated times. Data are means ± SEM from three independent experiments using cells from different donors. *p < 0.05; ***p ≤ 0.001.
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Related In: Results  -  Collection

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Figure 4: M1 macrophages exhibit delayed phagosome-lysosome fusion kinetics. Macrophages were challenged with either FITC-labeled SOZ or tetramethyl rhodamine–labeled SOZ and centrifuged for 1 min, and unbound particles were washed with PBS. Cells were incubated for 4 min at 37°C, then immediately placed on ice-cold PBS with anti-human IgG 647 (A–D) or anti-human IgG 488 (E, F) to label incompletely internalized zymosan particles. Cells were then incubated at 37°C in RPMI for the indicated times, fixed with either ice-cold methanol (A–D) or 2% paraformaldehyde (E, F), and immunostained for either TfR (A, B) or LAMP-1 (B, C). Lysosomes in the cells in E and F were preloaded with dextran 647 as outlined in Materials and Methods. Arrowheads indicate phagosomes positive for TfR (A), LAMP-1 (C), or dextran (E). Arrows indicate phagosomes negative for TfR (A), LAMP-1 (B), or dextran (E). Scale bars, 5 μm. (B, D, F) Percentage of phagosomes positive for TfR, LAMP-1, or dextran at the indicated times. Data are means ± SEM from three independent experiments using cells from different donors. *p < 0.05; ***p ≤ 0.001.
Mentions: After sealing, the phagosomal membrane undergoes extensive remodeling, a result of multiple fusion and fission events with other endomembrane compartments. The differential rate of clearance of the NADPH oxidase from M1 and M2 phagosomes suggests that maturation proceeds at different rates and/or by different routes in the two types of macrophages. To determine whether this was indeed the case, we measured the course of acquisition and loss of several endomembrane markers from M1 or M2 phagosomes. Macrophages were challenged with labeled SOZ, and after 5 min of phagocytosis, external or incompletely internalized particles were identified by addition of fluorescently labeled antibodies. By subsequently omitting from consideration the antibody-tagged particles, we were able to more precisely demarcate the initiation of the maturation period in the untagged (fully internalized) SOZ. As illustrated in Figure 4, A and B, transferrin receptors (TfRs), which are indicative of early/recycling endosomes, were acquired very rapidly by M2 phagosomes and disappeared shortly thereafter, as lysosome-associated membrane protein 1 (LAMP-1) was recruited. The later stages of acquisition of LAMP-1, a late endosome-lysosome marker, corresponded to the time of acquisition of labeled dextran, which was chased after pulsing (see Materials and Methods) to serve as a lysosomal marker. This pattern of rapid maturation is somewhat faster than that described for unpolarized primary and immortalized macrophages (Tsang et al., 2000; Sokolovska et al., 2013) and is compatible with the rapid acquisition of V-ATPases deduced from pH measurements. In stark contrast, M1 phagosomes retained TfR longer and acquired LAMP-1 and dextran more slowly and less completely than their M2 counterparts. The delayed maturation of M1 phagosomes is consistent with the prolonged residence of gp91 and p22 on their membrane and the sustained phagosomal oxidase activity.

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