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. 2006 Aug 7;203(8):1927-37.
doi: 10.1084/jem.20052069. Epub 2006 Jul 31.

Neutrophils from p40phox-/- mice exhibit severe defects in NADPH oxidase regulation and oxidant-dependent bacterial killing

Chris D Ellson  1 , Keith Davidson, G John Ferguson, Rod O'Connor, Len R Stephens, Phillip T Hawkins
Affiliations

Neutrophils from p40phox-/- mice exhibit severe defects in NADPH oxidase regulation and oxidant-dependent bacterial killing

Chris D Ellson et al. J Exp Med. .

Abstract

The generation of reactive oxygen species (ROS) by the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex plays a critical role in the antimicrobial functions of the phagocytic cells of the immune system. The catalytic core of this oxidase consists of a complex between gp91(phox), p22(phox), p47(phox), p67(phox), p40(phox), and rac-2. Mutations in each of the phox components, except p40(phox), have been described in cases of chronic granulomatous disease (CGD), defining their essential role in oxidase function. We sought to establish the role of p40(phox) by investigating the NADPH oxidase responses of neutrophils isolated from p40(phox-/-) mice. In the absence of p40(phox), the expression of p67(phox) is reduced by approximately 55% and oxidase responses to tumor necrosis factor alpha/fibrinogen, immunoglobulin G latex beads, Staphylococcus aureus, formyl-methionyl-leucyl-phenylalanine, and zymosan were reduced by approximately 97, 85, 84, 75, and 30%, respectively. The defect in ROS production by p40(phox-/-) neutrophils in response to S. aureus translated into a severe, CGD-like defect in the killing of this organism both in vitro and in vivo, defining p40(phox) as an essential component in bacterial killing.

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Figures

Figure 1.
Figure 1.
Blood cell counts and phox protein expression in p40phox−/− mice. (A) Peripheral blood from p40phox+/+ and p40phox−/− animals was analyzed in a Vetabc animal blood cell counter. WBC, white blood cells; RBC, red blood cells; Plt, platelets, Lymph, lymphocytes; Mono, monocytes; Gran, granulocytes. Data represented are mean percentage of wild-type counts ± SE (n = 12). (B) 30 μg of clarified, homogenized tissues from p40phox+/+ and p40phox−/− animals was subjected to SDS-PAGE and immunoblotted for p40phox. Arrowheads indicate position of p40phox. We routinely observed immunoreactive bands in the spleen and thymus from p40phox−/− animals. We do not know whether they are products of alternative splicing of p40phox gene transcripts. (C) BMNs from p40phox+/+, p40phox+/−, and p40phox−/− animals were sonicated into SDS sample buffer, subjected to SDS-PAGE, and immunoblotted for p40phox, p47phox, and p67phox. Graphs represent quantitation of p40phox (D) and p67phox (E) levels in BMNs as mean percentage of wild-type samples ± SE (n = 10–13) from 6–10 independent experiments using three to six mice per preparation.
Figure 2.
Figure 2.
p40phox−/− neutrophils exhibit normal S. aureus phagocytosis and FMLP-induced signal transduction. (A) Primed p40phox+/+ and p40phox−/− BMNs were adhered to glass and allowed to phagocytose serum-opsonized, FITC-labeled S. aureus for 40 min. Coverslips were washed and fixed, and the mean number of internalized bacteria per neutrophil was determined. 300 neutrophils were examined (n = 2). (B) p40phox+/+ and p40phox−/− BMNs were incubated for 1min in the presence (+) or absence (−) of 10 μM FMLP. Resultant lysates were split between two blots and probed with phospho- (P) and total (T) antibodies against PKB, p38 MAPK, and p42/44 Erk. Graphs represent quantification of phospho-protein levels ± the range from a single experiment representative of three experiments. Black bars, +/+; white bars, −/−. Units are arbitrary.
Figure 3.
Figure 3.
p40phox−/− neutrophils have severe defects in ROS production in response to soluble stimuli. BMNs from p40phox+/+, p40phox+/−, and p40phox−/− animals were analyzed for soluble stimulus-induced ROS production using HRP-dependent, luminol-dependent chemiluminescence. Duplicate wells of BMNs (3.75 × 105/well) were stimulated in a luminometer and ROS production (measured in relative light units per second, rlu/sec) was followed over time. 10 μM of unprimed FMLP (A), 10 μM mTNF-α–primed FMLP (B), 10 μM mGM-CSF–primed FMLP (C), and 300 nM PMA (D). Response kinetics (top line graph), total integrated responses as percentage of wild-type (bar graph), and agonist dose-response curves (bottom graph) are shown. Dose-response curves are standardized to 100% with 300 nM PMA and 30 μM FMLP, respectively. Black, +/+; gray, +/−; white, −/−. All data are means ± SE (n = 6) from three independent experiments using three to six mice per preparation.
Figure 4.
Figure 4.
p40phox−/− neutrophils have differing defects in ROS production in response to particulate stimuli. BMNs from p40phox+/+ and p40phox−/− animals were analyzed for particulate stimulus-induced ROS production using luminol-dependent chemiluminescence (filled symbols, +/+; open symbols, −/−). Triplicate wells of mock-primed or mTNF-α–, mGM-CSF–primed BMNs (3.75 × 105 BMN/well for S. aureus and zymosan stimulations, 1.25 × 106/well for latex beads stimulations) were stimulated in a luminometer and ROS production (measured in relative light units per second, rlu/sec) was followed over time (A). Each horizontal panel of graphs is shown on the same scale. Data are means ± SD from a single experiment representative of two to five independent experiments. Particles were either unopsonized or opsonized with IgG or mouse serum. Particle/BMN ratios are as follows: zymosan, 5:1; S. aureus, 20:1; IgG latex beads, 50:1. (B) Total integrated responses of primed BMNs to different stimuli over 2 h are shown as a percentage of wild-type. Black, +/+; white, −/−. Data are means ± SE (n = 6–10) from two to five independent experiments using three to six mice per preparation.
Figure 5.
Figure 5.
NADPH oxidase responses of p40phox−/− neutrophils assessed by measuring oxygen consumption and phagosomal NBT deposition. Oxygen consumption of BMNs from p40phox+/+ and p40phox−/− animals, in response to various stimuli, was measured in a Clark-type oxygen electrode. Primed BMNs (5 × 106/ml) were prewarmed in the oxygen electrode chamber, stimuli were added, and oxygen consumption was followed over time. Line graphs (A) are typical examples of each stimulus: 1 μM PMA, IgG-Zym (20 particles per neutrophil), and heat-killed S. aureus (20 bacteria per neutrophil). S. aureus was heat killed before addition, as oxygen consumption by live bacteria dominated the neutrophil respiratory burst (unpublished data). BMNs were primed before IgG-Zym and S. aureus stimulation. (B) Quantification of rates of oxygen consumption as a percentage of wild-type, after deduction of prestimulus rate of oxygen consumption. Data are means ± SE (n = 3–5) from three to five independent experiments using three to six mice per preparation. (C) Primed BMNs were adhered to glass in the presence of NBT, IgG-Zym was added, and superoxide production (evident by the deposition of dark purple formazan precipitate) was imaged microscopically. Arrowheads indicate examples of NBT+ phagosomes. Images show typical results ∼30 min after IgG-Zym addition (n = 5 experiments using three to six mice per preparation).
Figure 6.
Figure 6.
p40phox−/− neutrophils have severe deficiencies in adhesion-dependent ROS production. BMNs from p40phox+/+ and p40phox−/− animals were analyzed for adhesion-dependent ROS production using HRP-dependent, luminol-dependent chemiluminescence on fibrinogen-coated plates (filled symbols, +/+; open symbols, −/−). Duplicate wells of BMNs (2.5 × 105/well) were stimulated in a luminometer and ROS production (measured in relative light units per second, rlu/sec) was followed over time. 20 ng/ml mTNF-α (A) and 18 μg/ml anti-CD18 (anti-β2 integrin) (B). Dotted lines are wild-type controls stimulated with buffer alone in A and IgG2a isotype control in B. Data are means ± range from a single experiment representative of three independent experiments. (C) Total integrated responses over 1 h as a percentage of wild-type. Data are means ± SE (n = 6) from three independent experiments. (D) Spreading responses on fibrinogen surfaces of TNF-α–stimulated neutrophils. Phase dark cells are considered spread. (E) Cell surface expression of CD18 in TNF-α–stimulated neutrophils. Solid lines are anti-CD18, and dotted lines are isotype controls. Black, +/+; gray, −/−. Data are from a single experiment representative of two independent experiments.
Figure 7.
Figure 7.
p40phox−/− neutrophils are severely deficient in killing of S. aureus in vitro and in vivo. (A and B) Primed BMNs from p40phox+/+ and p40phox−/− animals were incubated for 15 min with serum-opsonized S. aureus (approximate ratio of 1 bacterium to 4 neutrophils) (A) or 5 min with serum-opsonized E. coli (approximate ratio of 1 bacterium to 1 neutrophil) (B) with rapid mixing. Samples were added to ice-cold LB-saponin and sonicated to liberate ingested bacteria. Surviving bacteria were enumerated by plating and subsequent counting of colonies. “No neutrophil” controls and controls with 3 μM DPI were also run. Data are means ± SD (n = 3) and representative of two to five experiments using three to six mice per preparation. (C) A dose curve of DPI (0.003–10 μM) was applied to wild-type mouse neutrophils in parallel luminometer-based ROS production assays in response to S. aureus and S. aureus killing assays. Resultant NADPH oxidase activity and S. aureus survival values were plotted against each other (filled symbols) and a trendline was fitted, representing the dependence of killing on NADPH oxidase activity in wild-type neutrophils. The data point for p40phox−/− neutrophils in response to S. aureus (bacterial killing vs. NADPH oxidase activity) was plotted on the same graph (open symbol). Data are means ± SE (n = 6) from two to three independent experiments using three to six mice per preparation. (D) Mice were injected intraperitoneally with 5 × 107 live S. aureus. After 4 or 24 h, mice were killed and peritoneums were flushed with ice-cold buffer. Aliquots were added to ice-cold LB-saponin, sonicated to liberate ingested bacteria, diluted, and plated, and surviving bacteria were enumerated. Total numbers of surviving bacteria per animal were calculated for each time point. Black, p40phox+/+; white, p40phox−/−. Data are means ± SE (n = 3 mice per time point) from a single experiment representative of three independent experiments.
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