The phosphoinositide-binding protein p40phox activates the NADPH oxidase during FcgammaIIA receptor-induced phagocytosis

Abstract

Superoxide produced by the phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is essential for host defense. Enzyme activation requires translocation of p67(phox), p47(phox), and Rac-GTP to flavocytochrome b558 in phagocyte membranes. To examine the regulation of phagocytosis-induced superoxide production, flavocytochrome b558, p47(phox), p67(phox), and the FcgammaIIA receptor were expressed from stable transgenes in COS7 cells. The resulting COS(phox)FcgammaR cells produce high levels of superoxide when stimulated with phorbol ester and efficiently ingest immunoglobulin (Ig)G-coated erythrocytes, but phagocytosis did not activate the NADPH oxidase. COS7 cells lack p40(phox), whose role in the NADPH oxidase is poorly understood. p40(phox) contains SH3 and phagocyte oxidase and Bem1p (PB1) domains that can mediate binding to p47(phox) and p67(phox), respectively, along with a PX domain that binds to phosphatidylinositol-3-phosphate (PI(3)P), which is generated in phagosomal membranes. Expression of p40(phox) was sufficient to activate superoxide production in COS(phox)FcgammaR phagosomes. FcgammaIIA-stimulated NADPH oxidase activity was abrogated by point mutations in p40(phox) that disrupt PI(3)P binding, or by simultaneous mutations in the SH3 and PB1 domains. Consistent with an essential role for PI(3)P in regulating the oxidase complex, phagosome NADPH oxidase activation in primary macrophages ingesting IgG-coated beads was inhibited by phosphatidylinositol 3 kinase inhibitors to a much greater extent than phagocytosis itself. Hence, this study identifies a role for p40(phox) and PI(3)P in coupling FcgammaR-mediated phagocytosis to activation of the NADPH oxidase.

Figure 1.

Interactions between p47^phox, p67^phox, and p40^phox subunits of the phagocyte NADPH oxidase. Structural motifs and identified interactions between p47^phox, p67^phox, and p40^phox are shown schematically. The p47^phox subunit contains a PX domain, two SH3 domains, and a C-terminal PRR. A domain containing four tetratricopeptide repeat (TPR) motifs comprises the N terminus of p67^phox, followed by an SH3, PB1, and second SH3 domain. The p67^phox subunit also contains a PRR adjacent to the N-terminal SH3 domain. p40^phox also contains a PX and PB1 domain, along with an intervening SH3 domain. In the p47^phox–p67^phox–p40^phox complex, p47^phox associates with p67^phox via a high-affinity tail-to-tail interaction involving the C-terminal PRR and SH3 domains in p47^phox and p67^phox, respectively, whereas p40^phox is tethered to p67^phox via a back-to-front interaction between their PB1 domains.

Figure 2.

Expression of FcγIIA receptor, phagocytosis, and NADPH oxidase activity. (A) Analysis of FcγIIA expression by flow cytometry using an FITC-conjugated antibody against CD32 is shown for COS7 and COS^phoxFcγR cells (left) and human peripheral blood monocytes (right) as indicated. The gray histogram indicates staining of either COS^phoxFcγR or monocytes with an FITC-conjugated isotype control mAb. (B and C) Phagocytosis of IgG–sheep RBCs in media containing NBT. (B) COS^phoxFcγR cells incubated with IgG-RBCs for 30 min at 37°C. Many of the ingested IgG-RBCs, which appear tan, are indicated by arrows. (C) Murine bone marrow–derived macrophages incubated with IgG-RBCs for 10 min at 37°C. Formazan-stained phagosomes, indicative of intraphagosomal superoxide production, are indicated by arrows. (D) COS^phoxFcγR cells incubated with 100 ng/ml phorbol myristate acetate for 30 min, showing diffuse formazan deposits. Bar, 30 μm.

Figure 3.

Localization of EYFP-p40PX and full-length EYFP-p40^phox in PLB-985 granulocytes and COS^phoxFcγR cells during phagocytosis. (A, C, E, G, I, and K) EYFP fluorescence (green). (B, D, F, H, J, and L) EYFP and Alexa Fluor 555 (IgG-RBCs or IgG-beads, red). Regions in which red and green labels overlap appear orange or yellow. (A–D) PLB-985 granulocytes. (E–L) COS^phoxFcγR cells. (E and F) EYFP-p40PX, IgG-RBCs. (G and H) Full-length EYFP-p40^phox, IgG-RBCs. (A–D and I–L) Full-length EYFP-p40^phox IgG beads without (A, B, I, and J) and with (C, D, K, and L) wortmannin. Images are representative of two to three independent experiments. Arrowheads point to representative phagosomes. Bars: A, 5 μm; C and L, 20 μm. Insets are magnified twofold relative to the adjacent panels.

Figure 4.

Expression of p40^phox in COS^phoxFcγR cells and FcγR-elicited NADPH oxidase activity. Data shown is representative of at least three independent experiments. (A) Immunoblots of human neutrophil and COS7 cell lysates (10 μg protein per lane) probed with antibodies for p40^phox, p47^phox, and p67^phox. COS^phoxFcγR cells were transfected with either a stable p40^phox transgene or with varying amounts of p40pRK5 for transient expression as indicated. (B) IgG-RBC–elicited NADPH oxidase activity in COS^phoxFcγR cells expressing p40^phox. Multiple formazan-stained phagosomes (arrows) in COS^phoxFcγR transfected with 0.05 μg p40pRK5 (representative photomicrograph; bar, 30 μm). Similar numbers of formazan-stained phagosomes were present in COS^phoxFcγR cells transfected with larger amounts of plasmid or expressing p40^phox from a stable transgene. (C) Flow cytometry analysis of COSFcγR cell lines incubated with Fc OxyBURST Green–opsonized zymosan for 30 min. Fluorescence intensity is shown on the x axis.

Figure 5.

Expression of p40^phox mutants in COS^phoxFcγR cells and effect on IgG–sheep RBC–elicited NADPH oxidase activity. Data shown is representative of at least three independent experiments. (A) Immunoblot of cell lysates from COS^phoxFcγR cells transfected with 0.67 μg of either empty pRK5 or pRK5 containing cDNAs for either wild-type or mutant p40^phox. Blots were probed with antibodies for p40^phox, p47^phox, and p67^phox. (B) COS^phoxFcγR cells were transfected as in A and incubated with IgG-RBCs in the presence of NBT for 30 min at 37°C. The percentage of cells with NBT⁺ phagosomes is shown as the mean ± SD (n = 4 except for W207R/D289A, where n = 3). (C) COS^phoxFcγR cells were transfected as in A for expression of YFP-tagged wild-type or mutant derivatives of p40^phox as indicated or a YFP-tagged PX domain of p40^phox and incubated with IgG–sheep RBCs or with IgG latex beads (*) without or with 50 nM wortmannin, followed by confocal microscopy. Individual phagosomes were scored for either the presence (black bars) or absence of YFP-p40^phox or YFP-p40PX translocation. The number of phagosomes scored for each construct is also shown. Data was collected from two to four independent experiments.

Figure 6.

Effects of PI3 kinase inhibitor on macrophage phagocytosis and NADPH oxidase activity elicited by IgG-opsonized latex beads. Murine PEMs were incubated with varying concentrations of wortmannin or LY294002 for 30 min at 37°C or with DMSO vehicle (control) before adding IgG-opsonized latex beads (3.3 μm). Data is the mean ± SD (n ≥ 3 experiments). (A) The percentage of macrophages with internalized beads (black bars) and the phagocytic index (white bars; data normalized as the percentage of the phagocytic index for vehicle-treated control macrophages, which was ∼400–600) are shown. (B) NADPH oxidase activity during phagocytosis of IgG beads, as measured by lucigenin-dependent chemiluminescence integrated over 60 min. Data is the mean ± SD (n ≥ 3 experiments). The background signal from gp91^phox-null PEM samples run in parallel was 90.0 ± 27.8 and has been subtracted from the wild-type PEM signal.