NADPH oxidase controls phagosomal pH and antigen cross-presentation in human dendritic cells - PubMed (original) (raw)

NADPH oxidase controls phagosomal pH and antigen cross-presentation in human dendritic cells

Adriana R Mantegazza et al. Blood. 2008.

Abstract

The phagocyte NADPH oxidase (NOX2) is critical for the bactericidal activity of phagocytic cells and plays a major role in innate immunity. We showed recently that NOX2 activity in mouse dendritic cells (DCs) prevents acidification of phagosomes, promoting antigen cross-presentation. In order to investigate the role of NOX2 in the regulation of the phagosomal pH in human DCs, we analyzed the production of reactive oxygen species (ROS) and the phagosomal/endosomal pH in monocyte-derived DCs and macrophages (M(diameter)s) from healthy donors or patients with chronic granulomatous disease (CGD). As expected, we found that human M(diameter)s acidify their phagosomes more efficiently than human DCs. Accordingly, the expression of the vacuolar proton ATPase (V-H(+)-ATPase) was higher in M(diameter)s than in DCs. Phagosomal ROS production, however, was also higher in M(diameter)s than in DCs, due to higher levels of gp91phox expression and recruitment to phagosomes. In contrast, in the absence of active NOX2, the phagosomal and endosomal pH decreased. Both in the presence of a NOX2 inhibitor and in DCs derived from patients with CGD, the cross-presentation of 2 model tumor antigens was impaired. We conclude that NOX2 activity participates in the regulation of the phagosomal and endosomal pH in human DCs, and is required for efficient antigen cross-presentation.

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Figures

Figure 1

Figure 1

Phagosomal pH increases in human DCs, in contrast to human MØs, after latex bead phagocytosis. (A) DC phagosomal pH kinetics after a 15-minute phagocytosis pulse and the indicated time points of chase. (B) MØ phagosomal pH kinetics after a 15-minute phagocytosis pulse and the indicated time points of chase. Each dot represents a different blood donor. Slashes show average pH. Statistics were performed using the nonparametric Wilcoxon test for paired data. *P < .05; **P < .01. (C) Phagosomal membrane expression of gp91_phox_ and V-H+-ATPase assessed by Western blot in human DCs and MØs, after different time points of chase following magnetic bead phagocytosis and phagosome isolation. Purity of the fractions was assessed by evaluating the expression of gp96, Erk-2, and NF-κB.

Figure 2

Figure 2

ROS production in human DCs and MØs. ROS production was measured over time using luminol-dependent chemiluminiscence, after stimulation with PMA and/or phagocytosis of luminol-coated beads in DCs (left panels) and MØs (right panels) generated in parallel from a series of healthy volunteers. Thick lines show the average of all experiments. (A) Total ROS production after cell stimulation with 0.5 μg/mL PMA in DCs or MØs from the same donors. (B) DC and MØ phagosomal ROS production after a 15-minute pulse with luminol-coated beads. (C) DC and MØ phagosomal ROS production after a 15-minute pulse with luminol-coated beads followed by the addition of 0.5 μg/mL PMA.

Figure 3

Figure 3

Reduced alkalinization of the phagosomal pH in the absence of NOX2 activity. (A) DC (left) and MØ (right) phagosomal pH kinetics in the presence or absence of 10 μM DPI. The average and standard deviation (SD) of 3 independent experiments (3 different donors) are shown. (B) Control DC (left) and CGD-DC (right) phagosomal pH kinetics after a 15-minute phagocytosis pulse and the indicated time points of chase. Dots represent different blood donors. Slashes show average pH. Statistics were performed using the nonparametric Wilcoxon test for paired data. *P < .05; **P < .01

Figure 4

Figure 4

Efficient cross-presentation of melanoma particulate antigens requires NOX2 activity. (A) Control HLA-A2–positive DCs (gray) and DPI-treated DCs (white) were pulsed with either MelanA/MART-1 long peptides coated to latex beads (top left panel) or MelanA/MART-1 short peptides (bottom panel). DCs were pretreated with 10 μM DPI 30 minutes before the pulse and washed before the coculture with the CTL clone. The percentage of phagocytosis of FITC/FluoProbes 647 latex beads by control and DPI-treated DCs was assessed by FACS (top right panel). Cross-presentation was evaluated by measuring the production of IFN-γ by the HLA-A2–restricted CTL clone LT12 after DC-CTL coculture. (B) HLA-A2–negative Mel888 melanoma cells were treated overnight with oxaliplatin to induce apoptosis. Apoptosis and necrosis were quantified using annexin V–FITC and propidum iodide, respectively, by FACS. (C) Expression of MelanA/MART-1 in whole-cell lysates from apoptotic and nontreated Mel888 cells, assessed by Western blot. (D) Control HLA-A2–positive DCs (black) and DPI-treated DCs (white) were pulsed with either MelanA/MART-1 short peptides (left panel) or different numbers of apoptotic Mel888 cells, as described in “Methods.” Cross-presentation was evaluated as indicated.

Figure 5

Figure 5

Endosomal pH decreases in normal and CGD-DCs after dextran endocytosis. (A) Colocalization of FITC-dextran (green) and p47 (red) on endosomes of DCs, after a 10-minute endocytic pulse, as detected using confocal microscopy. (B) Endosomal ROS production measured by FACS. Control and DPI-treated DCs were pulsed with Oxyburst Green H2DCF coupled to BSA. Oxidation of the conjugate by endosomal ROS was detected by FACS. (C) DC endosomal pH kinetics after a 10-minute endocytosis pulse and the indicated time points of chase in the presence or absence of 10 μM DPI. The average and SD of 3 independent experiments are shown. Statistics were performed using the nonparametric Wilcoxon test for impaired data. **P < .01. (D) Control DC (blue) and CGD-DC (red) endosomal pH kinetics after a 10-minute endocytosis pulse and the indicated time points of chase. Statistics were performed using the nonparametric Wilcoxon test for paired data. *P < .05. (E) Endosomal membrane expression of Rab 5, lamp-1, gp91_phox_, and V-H+-ATPase after different time points of chase at 37°C following a 30-minute endocytic pulse at 4°C, as described in “Methods.” Purity of the fractions was assessed by evaluating the expression of gp96, Erk-2, and NF-κB.

Figure 6

Figure 6

Antigen cross-presentation of melanoma soluble antigens is dependent on NOX2 activity. (A) Control HLA-A2–positive DCs (black) and DPI-treated DCs (white) were pulsed with the indicated amounts of short or long MelanA/MART1 peptides. Cross-presentation was evaluated by measuring the production of IFN-γ by the HLA-A2–restricted CTL clone LT12 after DC-CTL coculture. The average and SD of 4 independent experiments are shown. (B) Control HLA-A2–positive DCs (black) and DPI-treated DCs (white) were pulsed with the indicated amounts of short or long gp100 peptides, as described in “Methods.” Cross-presentation was evaluated by measuring the production of IFN-γ by the HLA-A2–restricted CTL clone LT47 after DC-CTL coculture. The average and SD of 4 independent experiments are shown. (C) Control HLA-A2–positive DCs (black) and CGD-DCs (white) were pulsed with the indicated amounts of short or long MelanA/MART1 peptides, as described in “Methods.” Cross-presentation was evaluated by measuring the production of IFN-γ by the HLA-A2–restricted CTL clone LT12 after DC-CTL coculture. The average and SD of 4 independent experiments are shown for control and CGD-DCs. Statistics were performed using the nonparametric Wilcoxon test for nonpaired data. *P < .05; **P < .01.

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