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First published online May 4, 2004
doi: 10.1242/10.1242/jcs.01085

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Cryptic O₂^–-generating NADPH oxidase in dendritic cells

Sylvie Elsen^1,*, Jacques Doussière^1,*, Christian L. Villiers^2,*, Mathias Faure², Rolande Berthier², Anne Papaioannou², Nathalie Grandvaux^1,, Patrice N. Marche^2, and Pierre V. Vignais^1,

¹ Laboratoire de Biochimie et Biophysique des Systèmes Intégrés (UMR 5092 CNRS-CEA-UJF)
² Laboratoire d'Immunochimie (U548 INSERM-CEA-UJF), Département Réponse et Dynamique Cellulaires, CEA-Grenoble, 38054 Grenoble CEDEX 9, France

View larger version (104K): [in a new window]   Fig. 1. Effect of preincubation with LPS on the reduction of NBT by DCs challenged with PMA. General views of DCs (A-D) or sDCs (E). Left, phase contrast images; right, NBT staining. (A,E) Preincubation with LPS, followed by incubation with PMA. (B) No LPS in the preincubation medium, incubation with PMA. (C) Preincubation with LPS, no PMA in the incubation medium. (D) Control cells, no LPS in the preincubation medium, no PMA in the incubation medium. Micrographs F, G and H correspond to high magnification images of selected cells from plates A, B and E, respectively. (I) Time course of NBT reduction within DCs. Preincubation with LPS, followed by incubation with PMA (). Other conditions: no preincubation with LPS, PMA in the incubation medium (); Preincubation with LPS, no PMA in the incubation medium (); Control, no preincubation with LPS, no PMA in the incubation medium (). The results are representative of five separate experiments.

View larger version (8K): [in a new window]   Fig. 2. Effect of an overnight contact of DCs with different agonists (LPS, PSB, anti-CD40, poly I:C) on the increase in the PMA-dependent NBT reduction. The absorbance values are those obtained at the plateau. Three to five separate experiments were carried out to check the effect of the different stimuli. Data are means±s.d.

View larger version (26K): [in a new window]   Fig. 3. Immunodetection of the protein components of the O2–-generating NADPH oxidase in membrane and cytosolic fractions from murine DCs and bovine neutrophils. 5x106 cell equivalents of DC and neutrophil membrane and cytosol fractions were subjected to SDS-PAGE and then to western blot, using antibodies directed to the two subunits, Nox2 and p22phox, of the membrane-bound flavocytochrome b558 and to the cytosolic factors of oxidase activation, p67phox, p47phox and p40phox. (A) Immunoblots of murine DC membranes, sDC membranes and bovine neutrophil (N) membranes revealed with anti-Nox2 and anti-p22phox antibodies. In this experiment, sDC was obtained from spleens of mice pretreated with Flt-3L. Molecular mass markers (kDa) are indicated. (B) Immunoblots of murine DC cytosol revealed with anti-p67phox, anti-p47phox and anti-p40phox antibodies. (C) Immunoblots of DC membranes showing that p67phox, p47phox and p40phox are detected in crude DC membranes, but not in DC membranes purified on sucrose gradient.

View larger version (50K): [in a new window]   Fig. 4. Subcellular localization of the O2–-generating NADPH oxidase in DCs examined by confocal immunofluorescence microscopy. Cells were immunostained for different components of the NADPH oxidase using specific antibodies, followed by Cy3 donkey anti-rabbit antibodies (see Materials and Methods). Controls without specific antibodies were performed and found to be negative (not shown).

View larger version (23K): [in a new window]   Fig. 5. Titration of a low potential heme b in DC membranes. (A) Effect of sodium dithionite on the optical spectrum of DC membranes in the presence of phenosafranin as redox indicator. Conditions of titration are described in Materials and Methods. Selected traces (1 to 8) illustrate the effect of the dithionite-dependent reduction on the increase of the peaks of heme b at 426 nm and heme a3 at 444 nm, and on the decrease of the peak of phenosafranin at 525 nm. (B) Relationship between the spectral modifications of phenosafranin (525 nm) , heme b (426 nm) and heme a3 (444 nm) upon reduction by sodium dithionite. (C) Nernst plot of the redox titration of the low potential heme b in DC membranes. The ox/red ratio of the low potential heme b was calculated from the second wave of absorbance increase (at high concentration of sodium dithionite) at 426 nm. Forward titration () and backward titration ().

View larger version (19K): [in a new window]   Fig. 6. Inhibitory effect of DC membranes on the elicited neutrophil NADPH oxidase activity in a cell-free system. NADPH oxidase activity (nmol O2– produced per minute in each well of the microtiter plate) was determined after preincubation for 10 minutes at room temperature of bovine neutrophil membranes (6 µg protein/assay) or murine DC membranes (10.5 µg protein/assay) or a mixture of neutrophil membranes (6 µg protein/assay) and DC membranes (10.5 µg protein/assay) with neutrophil cytosol (60 µg protein), ATP, GTPS, MgSO4, and increasing amounts of arachidonic acid (AA) (final volume 30 µl). The preincubation step was followed by addition of cytochrome c, KCN and NADPH in PBS (see Materials and Methods). The figure shows the rate of production of O2– per minute at different concentrations of arachidonic acid. (A) Neutrophil membranes (); DC membranes (); neutrophil membranes mixed with DC membranes () (the shift of the optimal concentration of arachidonic acid towards higher values when DC membranes and neutrophil membranes are added together in the medium is due to the higher levels of membrane lipids functioning as an unspecific trap of arachidonic acid). (B) Dose-response of the inhibitory effect of DC membranes on the neutrophil oxidase activity at the optimal concentration of arachidonic acid (); DC membranes heated for 10 minutes at 100°C and mixed with neutrophil membranes (); DC membranes treated for 1 hour at 20°C with proteinase K (proteinase K/membrane protein: 1/100) and mixed with neutrophil membranes ().

View larger version (17K): [in a new window]   Fig. 7. Effects of pretreatment of neutrophil membranes and DC membranes by ß-OG on the elicited NADPH oxidase activity in cell-free system. Murine DC membranes (21 µg protein) and bovine neutrophil membranes (6 µg protein) were pretreated by 1% ß-OG for 3 minutes at 0°C. This was followed by the addition of bovine neutrophil cytosol, GTPS, ATP, KCN, MgSO4 and increasing amounts of arachidonic acid (AA) to activate the NADPH oxidase. The elicited oxidase activity was determined by the production of O2– (see Materials and Methods) and expressed in terms of heme b turnover. (A) neutrophil membranes, control (), pretreated by ß-OG (). (B) DC membranes, control (), pretreated by ß-OG ().

View larger version (26K): [in a new window]   Fig. 8. Differential effects of a KCl extract and a ß-OG extract of DC membranes on the NADPH oxidase activity of neutrophil membranes elicited in a cell-free system. Bovine neutrophil membranes (6.2 µg protein) were mixed with various amounts of KCl or ß-OG extracts from DC membranes, and their NADPH oxidase was activated in the cell-free system consisting of bovine neutrophil cytosol, GTPS, ATP, MgSO4, KCN and increasing amounts of arachidonic acid (AA). The elicited oxidase activity was determined by the production of O2– (see Materials and Methods) and expressed in terms of heme b turnover. Curve a, control (absence of DC membrane extract). Curves b, c and d correspond to 1.15 µg, 2.3 µg and 4.6 µg protein in the ß-OG extract of DC membranes, respectively. (Inset) Dose-response of the elicited oxidase activity of neutrophil membranes treated with a ß-OG extract of DC membranes (), with a ß-OG extract of neutrophil membranes (), and with a KCl extract (0.5 M KCl) of DC membranes ().