Neutrophils are capable of producing significant amounts of reactive oxygen species (ROS) by the phagocyte NADPH oxidase, which consists of membrane-bound and cytoplasmic subunits that assemble during activation. Neutrophils harbor two distinct pools of the membrane-localized oxidase components, one expressed in the plasma membrane and one in the membranes of intracellular granules. Assembly of active oxidase at either type of membrane leads to release of extracellular ROS or to the production of ROS inside intracellular compartments, respectively. The cytoplasmic NADPH oxidase subunit p40phox seems selectively critical for the ability to generate intracellular ROS, and the recent characterization of patients with p40phox deficiency implies that selective loss of intracellular neutrophil ROS leads to disease with pronounced hyperinflammatory features, suggesting that these ROS are critical for regulation of inflammation. This study aimed at characterizing two pharmacological NADPH oxidase inhibitors, the newly described GSK2795039 and the widely used diphenyleneiodonium (DPI), focusing on their abilities to inhibit human neutrophil ROS production extra- and intracellularly. Whereas GSK2795039 blocked extra- and intracellular NADPH oxidase activity equally, DPI was found to selectively interfere with intracellular ROS production. Selectivity for the intracellular NADPH oxidase was evident as a lower IC50 value, faster onset, and irreversibility of inhibition. We found no evidence of direct interactions between DPI and p40phox, but the selectivity of DPI confirms that regulation of NADPH oxidase activity in neutrophils differs depending on the subcellular localization of the enzyme. This information may be used to pharmacologically mimic p40phox deficiency and to further our understanding of how intracellular ROS contribute to health and disease.

Neutrophils are phagocytic leukocytes capable of generating large amounts of reactive oxygen species (ROS) by the activation of a specialized electron transporting enzyme system, the phagocyte NADPH oxidase. The ROS formed by this NADPH oxidase are known to participate in microbial killing but are also increasingly recognized as regulators of inflammatory signaling and adaptive immune processes (13). The phagocyte NADPH oxidase is a multicomponent enzyme consisting of membrane-bound as well as cytosolic subunits. The membrane-localized part of the enzyme, referred to as cytochrome b558, is a heterodimer of the subunits p22phox (phox is an acronym for phagocyte oxidase) and gp91phox (also known as NOX2). The cytoplasmic subunits are p40phox, p47phox, and p67phox. In a resting cell, the membrane-bound cytochrome b558 and the cytosolic components are separated, but during activation, the cytoplasmic subunits translocate to the membrane and assemble a functional electron transport machinery (4). When active, electrons from cytoplasmic NADPH get transported across the membrane to molecular oxygen that is subsequently reduced to superoxide.

Neutrophil granulocytes are filled with different intracellular storage organelles (granules/vesicles), of which at least four distinct types can be distinguished by their content of marker molecules: myeloperoxidase (MPO) containing azurophil (primary) granules, lactoferrin containing specific (secondary) granules, and gelatinase containing gelatinase (tertiary) granules along with secretory vesicles. The cytochrome b558 is present in the plasma membrane of neutrophils, but the major part is, in fact, expressed in membranes of intracellular granules, most notably the specific granules (5). Depending on which pool of the cytochrome b558 is engaged in the assembly of a functional NADPH oxidase, the superoxide produced will be released extracellularly or retained intracellularly (6, 7). For efficient microbial killing, the obvious intracellular site in which intracellular ROS are produced is the membrane-enclosed phagosome formed following ingestion of particles (typically a microbe), to which specific and azurophil granules fuse when this compartment matures to a phagolysosome (8). However, it is becoming increasingly clear that neutrophils are capable of generating intracellular ROS even in the complete absence of phagocytosis (6). The exact identity of the ROS-producing intracellular nonphagosomal site/organelle is not known, but it is likely a result of granule–granule fusion events (9).

The importance of phagocyte ROS for human health, as well as the basic structure of the NADPH oxidase, has been clarified in primarily through studies of patients suffering from rare genetic defects leading to chronic granulomatous disease (CGD), which is clinically characterized by increased susceptibility to bacterial and fungal infections. CGD phagocytes are unable to generate ROS because of missing or nonfunctional oxidase components. In addition to repeated severe infections, CGD patients suffer also from a wide variety of inflammatory disorders (10), suggesting that phagocyte ROS are vital mediators of signals that limit or terminate inflammatory reactions. Neutrophils from patients with the classical forms of CGD lack the ability to form extra- as well as intracellular ROS, but experimental data interestingly demonstrate that one of the cytoplasmic NADPH oxidase subunits, p40phox, seems specifically important for oxidase activation at intracellular membranes (1113). In line with this, a recent clinical and experimental characterization of a novel nonclassical subgroup of CGD, deficient for p40phox, was recently described with a selective deficiency in the generation of intracellular ROS (7, 14, 15).

Because neutrophils are terminally differentiated, nondividing cells, in vitro genetic manipulations are not possible to use to establish a CGD cell line of functionally mature neutrophils. To mimic CGD cells, researchers are instead using a variety of pharmacological inhibitors of the NADPH oxidase. One of the most widely used inhibitors, diphenyleneiodonium (DPI), is a general blocker of flavoproteins, and as such, it is not specific for the phagocyte NADPH oxidase; DPI reportedly interferes also with other oxidases as well as with xanthine oxidase and proteins of the mitochondrial electron transport chain (16, 17). Similar drawbacks are common for many inhibitors, but recently, a small molecule named GSK2795039 [GSK; N-(1-isopropyl-3-(1-methylindolin-6-yl)-1H-pyrrolo[2,3-b]pyridin-4-yl)-1-methyl-1H-pyrazole-3-sulfonamide] was shown to be a quite specific inhibitor of the cytochrome b558–containing phagocyte oxidase (16).

We set out to characterize and compare DPI and GSK regarding their ability to interfere with NADPH oxidase activity at different sites in primary human neutrophils. Both inhibitors were effective blockers of neutrophil ROS production in general, but DPI surprisingly displayed a selective action against NADPH oxidase activation at intracellular sites. Not only were significantly lower DPI doses needed to suppress intracellular, as opposed to extracellular, ROS production, but the time required for inhibition was also shorter. In addition, whereas the inhibitory effect on the plasma membrane–localized NADPH oxidase was reversible and could be easily washed off, the inhibition of the NADPH oxidase in internal membranes was manifest even after extensive washing of the cells. Using a basic cell-free system, we did not find any evidence of direct interactions between DPI and p40phox that could explain DPI’s selective action. All in all, these data suggest that the molecular details of the NADPH oxidase complex (and its activation) are not identical for the two pools of cytochrome b558 in human neutrophils. They also imply that the mode of action for DPI is different for NADPH oxidase activation at the cell surface versus at intracellular sites. Our data may be of use for researchers wanting a pharmacological tool for selective suppression of intracellular neutrophil ROS.

Human neutrophils were isolated from buffy coats from healthy donors, or from peripheral blood, essentially as described by Boyum (18). Cells were washed in Krebs–Ringer phosphate buffer (KRG) containing glucose (10 mM) and Mg2+ (1.5 mM), resuspended in KRG supplemented with Ca2+ (1 mM), and stored on melting ice until use. Buffy coats and blood samples were obtained from the hospital blood bank after deidentification, and according to the Swedish legislation section code 4§ 3p SFS 2003:460, no informed consent is needed.

In 180 μl of KRG with 1 mM Ca2+ per well, 106 neutrophils were incubated with 1.5 mg of cytochrome c (Sigma) and indicated doses of the respective inhibitor (DPI from Sigma and GSK from MedChem Express). Samples were equilibrated for 5 min at 37°C before 20 μl of PMA (50 nM final concentration) was added, and absorbance was monitored at 550 nm for 30 min in a plate reader (CLARIOstar; BMG Labtech). The assay was performed in triplicates in a 96-well plate. Cytochrome c is cell-impermeable and exclusively reacts with extracellular superoxide (19). PMA-stimulated samples containing superoxide dismutase (SOD; 50 U/ml; Worthington) were included as negative controls. Absorbance values from these controls were subtracted from all samples when constructing the dose–response graph.

Superoxide production at extra- or intracellular sites was detected with isoluminol- or luminol-amplified chemiluminescence (CL), respectively (as described in detail in Refs. 6 and 19). For extracellular superoxide production, the cell-impermeable isoluminol (56 μM; Sigma) was used in combination with HRP (4 U/ml; Roche). For intracellular measurements, the cell-permeable luminol (56 μM; Sigma) was used while scavenging all extracellular ROS with SOD (50 U/ml; Worthington) and catalase (2000 U/ml; Worthington), enzymes that cannot penetrate the cell membrane. The CL activity was recorded in a plate reader (CLARIOstar; BMG Labtech) with 5 × 105 neutrophils per well. Neither DPI nor GSK had any effect on the enzymatic activity of HRP (data not shown).

If not stated differently, cells were incubated in the presence of the respective inhibitor for 5 min at 37°C before the stimuli, PMA (50 nM), fMLF (Sigma; 10 nM), or serum-opsonized Staphylococcus aureus (106 CFU), were added.

To examine whether it was possible to reverse inhibition by washing cells in inhibitor-free buffer, 500 μl of cells (5 × 106 cells/ml) were incubated together with GSK or DPI (20 μM) for 5 min at 37°C. Afterward, the cells were pelleted, washed three times, resuspended in KRG supplemented with 1 mM Ca2+, stimulated with 50 nM PMA, and assayed for ROS production.

Neutrophil lysates (107 cells/ml treated with 0.1% Triton X-100) were used as a source of MPO and diluted in the presence or absence of DPI and incubated with the peroxidase substrate 1,2-phenylenediamine (0.4 mg/ml; DAKO) and H2O2 (0.015%) for 30 min in darkness. After addition of H2SO4 (0.5 mM), absorbance was measured at 492 nm.

Cytosolic Ca2+ levels were monitored essentially as described (20). In short, neutrophils (6 × 106) were first labeled with Fluo-3 and FuraRed (both from Invitrogen) in 1 ml of KRG with Ca2+ and 1% FCS for 30 min at 37°C. After two washes, cells were resuspended in 1 ml of KRG with Ca2+ and 1% FCS and stored protected from light on ice until analyzed. Samples were equilibrated at 37°C for 5 min in the presence or absence of GSK (40 μM) or DPI (40 μM) before analyses started. Cells were collected by flow cytometry (Accuri C6; BD) during a total of 3 min. After 20 s, 30 μl of the chemotactic formylpeptide fMLF (final concentration of 10 nM) was added as a stimulus using gel-loading tips. After gating on the granulocyte population by forward and side scatter, fluorescent intensities of Fluo-3 and FuraRed were measured in FL-1 and FL-3, respectively; intracellular Ca2+ fluctuations were plotted as FL-1/FL-3 ratios over time using FlowJo software (version 10; Tree Star, Ashland, OR) and GraphPad Prism (version 8.2.0; GraphPad Software, La Jolla, CA).

Five micrograms of recombinant p40phox protein (with His and T7 tags; LifeSpan Bioscience, Seattle, WA) was incubated with 25 μM DPI in the presence and absence of 10 mM DTT for 15 min at 37°C. Control (only p40phox) or p40phox plus DPI with and without DTT samples were digested with trypsin using the filter-aided sample preparation method (21). The trypsin-digested peptides were purified, dried on SpeedVac, and reconstituted in 3% acetonitrile with 0.2% formic acid for analysis. Each fraction was analyzed on an Orbitrap Fusion Tribrid mass spectrometer interfaced with the Easy-nLC 1200 nanoflow liquid chromatography (LC) system (Thermo Fisher Scientific).

The database search was mostly performed using the Proteome Discoverer version 2.2 (Thermo Fisher Scientific) against the p40phox sequence from UniProt/SwissProt database (accession number Q15080; www.uniprot.org/uniprot/Q15080), with or without the 73 common proteomic contaminant database; Mascot 2.5.1 (Matrix Science) was used as a search engine with precursor mass tolerance of 10 ppm and fragment mass tolerance of 0.02 Da; trypsin or semitrypsin was set as the digestion rule with two missed cleavages allowed, mono-oxidation on methionine and pyroglutamate formation on the N-terminal glutamine was set as a variable modification for all searches; and separate searches were performed with the additional deamidation on N/Q or with the hypothetical DPI modification [C(12)H(7)I, 277.9592 Da] on any amino acid residue. The Fixed Value PSM Validator in Proteome Discoverer 2.2 was used to validate the search results. Precursor ion abundances were calculated in each search using the Minora feature detection node in Proteome Discoverer 2.2.

The open-modification (“Wildcard”) search was performed using the Byonic search engine (Protein Metrics, Cupertino, CA) against the p40phox sequence (Supplemental Table I). Precursor mass tolerance of 10 ppm and fragment mass tolerance of 20 ppm were used, trypsin with up to two missed cleavages was set as enzyme, and the Wildcard modification in the range from 50 to 800 Da was set as the sole modification. No false discovery ratefiltering was performed, and the spectra with the identified Wildcard modifications and the Byonic Score > 400 were manually compared between the DPI-treated samples and the untreated control sample.

Data analyses were performed using Excel or GraphPad Prism version 8.2.0 (GraphPad Software, San Diego, CA). Statistics were calculated by nonparametric two-way ANOVA followed by Sidak multiple comparisons test (Figs. 1B, 3A, 3B, 4A, 4B) if not stated otherwise. Statistically significant differences are indicated by NS, *p < 0.05, **p < 0.01, and ***p < 0.001.

Initially, we tested the effect of the two NADPH oxidase inhibitors on the generation of superoxide anions (O2) by primary human neutrophils using the gold standard cytochrome c reduction assay. This assay measures extracellular superoxide exclusively, and both inhibitors were able to completely suppress superoxide formation induced by PMA when present at >10 μM doses (Fig. 1A). Comparing dose–response curves for the two different inhibitors, it was clear that DPI was the more potent inhibitor (Fig. 1B). Importantly, high doses (up to 40 μM) of inhibitors were not cytotoxic, nor did they have general adverse effects on cell function, as inhibitor-treated neutrophils were fully able to mobilize intracellular Ca2+ in response to formyl peptide (fMLF) stimulation (Fig. 1C).

FIGURE 1.

Both DPI and GSK inhibit superoxide release from human neutrophils as measured by cytochrome c reduction.

Shown is a representative experiment (A) with cells preincubated for 5 min with buffer (KRG) or 27 μM indicated inhibitor before stimulation with PMA (50 nM; arrow), and a dose–response graph (B) displaying PMA-triggered superoxide release (as compared with samples in the absence of inhibitor) in the presence of indicated concentrations of DPI (triangles) or GSK (circles) as mean ± SD of three independent experiments. Negative control values obtained from PMA-stimulated samples in the presence of SOD were subtracted, and the 100% response value (no inhibitor) was set to x values of 10−9 M to be able to present the data on a logarithmic axis. Statistical differences between GSK and DPI are indicated. (C) Neither inhibitor caused general cell damage, as cells treated with high concentrations of inhibitors (40 μM) were perfectly capable of intracellular calcium signaling in response to chemoattractant stimulation. Representative calcium transients (out of at least three independent experiments) are shown, and arrows indicate the time points for addition of fMLF (10 nM). ***p < 0.001.

FIGURE 1.

Both DPI and GSK inhibit superoxide release from human neutrophils as measured by cytochrome c reduction.

Shown is a representative experiment (A) with cells preincubated for 5 min with buffer (KRG) or 27 μM indicated inhibitor before stimulation with PMA (50 nM; arrow), and a dose–response graph (B) displaying PMA-triggered superoxide release (as compared with samples in the absence of inhibitor) in the presence of indicated concentrations of DPI (triangles) or GSK (circles) as mean ± SD of three independent experiments. Negative control values obtained from PMA-stimulated samples in the presence of SOD were subtracted, and the 100% response value (no inhibitor) was set to x values of 10−9 M to be able to present the data on a logarithmic axis. Statistical differences between GSK and DPI are indicated. (C) Neither inhibitor caused general cell damage, as cells treated with high concentrations of inhibitors (40 μM) were perfectly capable of intracellular calcium signaling in response to chemoattractant stimulation. Representative calcium transients (out of at least three independent experiments) are shown, and arrows indicate the time points for addition of fMLF (10 nM). ***p < 0.001.

Close modal

Human neutrophils are capable of ROS production also at intracellular sites where they would not be detected by the cytochrome c assay (19). Intracellular ROS production, of course, takes place in the phagosome after uptake of particulate prey but may, in fact, also be produced in the complete absence of phagosome formation (e.g., after PMA stimulation) (6). To distinguish extracellular ROS release from intracellular ROS production, we used two complementary CL systems (19). Extracellular ROS was measured with the membrane-impermeable probe isoluminol, whereas intracellular ROS was measured with the membrane-permeable probe luminol in the presence of extracellular scavengers (SOD and catalase) to neutralize extracellular ROS. The CL reactions detect peroxidase-processed ROS exclusively and HRP was added for extracellular measurements, whereas the intracellular system is dependent on endogenous MPO (22).

Stimulation of neutrophils with PMA resulted in ROS production both extracellularly and intracellularly, and the kinetics of responses were slightly different: the extracellular response had a faster onset but also declined more rapidly than the intracellular response (Fig. 2A).

FIGURE 2.

ROS production at distinct sites.

PMA stimulation of human neutrophils results in ROS production at two distinct sites, extracellular and intracellular, that can be measured independently by isoluminol- and luminol-enhanced CL, respectively (A). High doses (10 μM) of either GSK or DPI blocked EC as well as IC ROS production (B). Representative (out of at least 10 independent experiments) curves are shown (left), and bar diagrams (right) compare peak values (mean + SD; n = 3) after PMA stimulation in the presence or absence of inhibitors (10 μM), with statistical significance (using paired Student t tests) for each inhibitor as compared with controls with KRG buffer instead of inhibitor. Cells were incubated with inhibitors for 5 min at 37°C before stimulation with PMA. EC, extracellular; IC, intracellular. ***p < 0.001.

FIGURE 2.

ROS production at distinct sites.

PMA stimulation of human neutrophils results in ROS production at two distinct sites, extracellular and intracellular, that can be measured independently by isoluminol- and luminol-enhanced CL, respectively (A). High doses (10 μM) of either GSK or DPI blocked EC as well as IC ROS production (B). Representative (out of at least 10 independent experiments) curves are shown (left), and bar diagrams (right) compare peak values (mean + SD; n = 3) after PMA stimulation in the presence or absence of inhibitors (10 μM), with statistical significance (using paired Student t tests) for each inhibitor as compared with controls with KRG buffer instead of inhibitor. Cells were incubated with inhibitors for 5 min at 37°C before stimulation with PMA. EC, extracellular; IC, intracellular. ***p < 0.001.

Close modal

When the two NADPH oxidase inhibitors were tested at high doses in the CL systems, both compounds blocked extra- as well as intracellular ROS formation completely (Fig. 2B).

We next performed dose-titration experiments with the inhibitors using the two CL systems in parallel and found that GSK blocked extra- and intracellular ROS production with identical potency (Fig. 3A). In surprising contrast, DPI was considerably more potent regarding inhibition of intracellular compared to extracellular NADPH oxidase activity (Fig. 3B), with IC50 values differing around 30-fold (∼0.237 μM for extracellular and 0.007 μM for intracellular ROS production, respectively). Given this striking difference in potency, it was possible to use doses of DPI that more or less completely abolished intracellular ROS production, while the extracellular ROS release was largely unaffected. These data imply that DPI selectively inhibits intracellular NADPH oxidase activity.

FIGURE 3.

Inhibition of extracellular and intracellular ROS production induced by PMA.

Dose–response curves for inhibition of PMA-stimulated extracellular versus intracellular ROS production by GSK (A) or DPI (B). The 100% response value (no inhibitor) was set to x values of 10−10 M to be able to present the data on a logarithmic axis. Cells were incubated with inhibitors for 5 min at 37°C before stimulation with PMA. Shown are mean ROS peak values as percentages of control ± SD of three independent experiments performed in triplicate. Statistically significant differences between the EC and IC responses are indicated for each inhibitor dose. EC, extracellular; IC, intracellular. **p < 0.01, ***p < 0.001.

FIGURE 3.

Inhibition of extracellular and intracellular ROS production induced by PMA.

Dose–response curves for inhibition of PMA-stimulated extracellular versus intracellular ROS production by GSK (A) or DPI (B). The 100% response value (no inhibitor) was set to x values of 10−10 M to be able to present the data on a logarithmic axis. Cells were incubated with inhibitors for 5 min at 37°C before stimulation with PMA. Shown are mean ROS peak values as percentages of control ± SD of three independent experiments performed in triplicate. Statistically significant differences between the EC and IC responses are indicated for each inhibitor dose. EC, extracellular; IC, intracellular. **p < 0.01, ***p < 0.001.

Close modal

PMA is a useful tool in that it induces extra- and intracellular ROS production simultaneously, and because it is a soluble stimulus, intracellular activity cannot be ascribed to phagosomal ROS generation. We next tested whether the selective inhibition of DPI was evident also for intracellular ROS production taking place in phagosomes. DPI inhibited intracellular ROS production in response to serum-opsonized S. aureus (Fig. 4A), with very similar IC50 values and dose–response profile to those obtained when intracellular activity was triggered by PMA (Fig. 3B). Phagocytosis of S. aureus only resulted in minute levels of extracellular ROS release (data not shown), so we instead triggered extracellular NADPH oxidase activation with the chemoattractant formyl peptide fMLF. The IC50 and dose–response profile for inhibition of fMLF-induced ROS release (Fig. 4A) were similar to those of PMA-induced extracellular activity (Fig. 3B) but distinct from intracellular ROS production triggered with phagocytosis (Fig. 4A) or PMA (Fig. 3B). Together, these data show that the selective action of DPI on intracellular NADPH oxidase activity is evident with different stimuli and is not dependent on PMA as the activating trigger.

FIGURE 4.

Inhibition of extracellular and intracellular ROS production induced by fMLF or phagocytosis.

Dose–response curves for inhibition of fMLF-stimulated extracellular versus S. aureus–triggered intracellular ROS production by DPI (A). Cells were incubated with inhibitor for 5 min at 37°C before stimulation. Shown are mean ROS peak values as percentages of control ± SEM of three independent experiments in triplicate. Statistically significant differences between the EC and IC responses are indicated for each inhibitor dose. The 100% response value (no inhibitor) was set to x values of 10−10 M to be able to present the data on a logarithmic axis. (B) DPI (10 μM) did not decrease MPO activity in neutrophil lysates. Shown are mean + SD of three independent experiments. EC, extracellular; IC, intracellular. ***p < 0.001.

FIGURE 4.

Inhibition of extracellular and intracellular ROS production induced by fMLF or phagocytosis.

Dose–response curves for inhibition of fMLF-stimulated extracellular versus S. aureus–triggered intracellular ROS production by DPI (A). Cells were incubated with inhibitor for 5 min at 37°C before stimulation. Shown are mean ROS peak values as percentages of control ± SEM of three independent experiments in triplicate. Statistically significant differences between the EC and IC responses are indicated for each inhibitor dose. The 100% response value (no inhibitor) was set to x values of 10−10 M to be able to present the data on a logarithmic axis. (B) DPI (10 μM) did not decrease MPO activity in neutrophil lysates. Shown are mean + SD of three independent experiments. EC, extracellular; IC, intracellular. ***p < 0.001.

Close modal

As mentioned, intracellular CL activity is completely dependent on endogenous MPO activity (22), and the selective effect of DPI on intracellular CL could, thus, potentially be due to inhibition of MPO activity rather than inhibition of ROS generation by the NADPH oxidase. However, the presence of DPI (10 μM) did not diminish MPO activity in neutrophil lysates (Fig. 4B).

To better understand the selective action of DPI, we next evaluated the kinetics of inhibition of the extra- and intracellular NADPH oxidase, respectively, by injecting DPI after stimulation with PMA (at the peak of responses, i.e., 8 min for extracellular and 22 min for intracellular CL) and monitoring the drop in CL signals. With DPI, the onset of inhibition of the intracellular CL signal was significantly faster (p < 0.05, comparing the time postinjection that the CL signal had dropped below 50% by an unpaired, nonparametric Mann–Whitney U test) than the onset of inhibition of extracellular ROS production (Fig. 5A); a 50% decrease in intracellular CL was achieved after 3.1 ± 1.1 s (mean ± SD; n = 4), whereas 50% reduction in extracellular CL was reached after 9.2 ± 2.7 s (mean ± SD; n = 4). In general, GSK was faster than DPI regarding onset of inhibition, but GSK was equally fast in inhibiting extra- and intracellular NADPH oxidase activity in this experimental setting (Fig. 5B).

FIGURE 5.

Kinetics of NADPH oxidase inhibition.

Kinetics of the inhibitory effects of DPI (A) and GSK (B) on the extracellular (solid lines) and intracellular (dotted lines) NADPH oxidase. Each inhibitor (20 μM) was injected (arrows) during PMA-triggered ROS production, and the CL responses were followed; note that x-axes display s. Shown are representative curves out of at least four independent experiments. EC, extracellular; IC, intracellular.

FIGURE 5.

Kinetics of NADPH oxidase inhibition.

Kinetics of the inhibitory effects of DPI (A) and GSK (B) on the extracellular (solid lines) and intracellular (dotted lines) NADPH oxidase. Each inhibitor (20 μM) was injected (arrows) during PMA-triggered ROS production, and the CL responses were followed; note that x-axes display s. Shown are representative curves out of at least four independent experiments. EC, extracellular; IC, intracellular.

Close modal

We next tested whether the effects of the NADPH oxidase inhibitors were reversible by measuring ROS production in inhibitor-treated cells that had been washed and resuspended in inhibitor-free buffer prior to stimulation. The effects of GSK could be fully reversed, and after washing, the cells responded to PMA stimulation with ROS production extra- as well as intracellularly (Fig. 6A). When DPI-treated cells were tested, the ability to produce extracellular ROS was fully regained by the washing procedure, similar to GSK-treated cells. In contrast to GSK, DPI-treated cells were completely unable to produce intracellular ROS in response to PMA even after extensive washing (Fig. 6A). The reversibility of DPI on extracellular ROS production was evident also using the cytochrome c reduction assay (Fig. 6B).

FIGURE 6.

Reversibility of ROS production.

Reversibility of ROS production after washing away GSK (black) or DPI (white). (A) Cells were incubated with each inhibitor (20 μM) for 5 min and then washed three times with inhibitor-free buffer before being assayed for extracellular (left) and intracellular (right) ROS production in response to PMA. Shown are mean + SD of ROS peak values obtained from four independent experiments. The response of control cells incubated with buffer (KRG) before being subjected to the same washing procedure and PMA stimulation is set to 100%. Statistical significance between GSK and DPI was calculated by paired Student t tests. (B) That the inhibitory effect of DPI on extracellular ROS production could be washed away was ascertained by cytochrome c reduction assay. Cells were incubated with DPI (20 μM) for 5 min and then washed and stimulated with PMA in the presence or absence of new DPI (20 μM). The graph displays mean Δ values (max OD − initial OD) + SD from two independent experiments performed in triplicate. Statistical significance was calculated by an unpaired t test. *p < 0.05, ***p < 0.001.

FIGURE 6.

Reversibility of ROS production.

Reversibility of ROS production after washing away GSK (black) or DPI (white). (A) Cells were incubated with each inhibitor (20 μM) for 5 min and then washed three times with inhibitor-free buffer before being assayed for extracellular (left) and intracellular (right) ROS production in response to PMA. Shown are mean + SD of ROS peak values obtained from four independent experiments. The response of control cells incubated with buffer (KRG) before being subjected to the same washing procedure and PMA stimulation is set to 100%. Statistical significance between GSK and DPI was calculated by paired Student t tests. (B) That the inhibitory effect of DPI on extracellular ROS production could be washed away was ascertained by cytochrome c reduction assay. Cells were incubated with DPI (20 μM) for 5 min and then washed and stimulated with PMA in the presence or absence of new DPI (20 μM). The graph displays mean Δ values (max OD − initial OD) + SD from two independent experiments performed in triplicate. Statistical significance was calculated by an unpaired t test. *p < 0.05, ***p < 0.001.

Close modal

Given the new information about the particular importance of p40phox for activation of the intracellular NADPH oxidase complex (7, 14, 15), a tempting explanation for the selective action of DPI could be that DPI interacts with and incapacitates p40phox. To test whether they could interact directly, we mixed DPI with recombinant p40phox and afterward analyzed the protein by reversed-phase LC and tandem mass spectrometry (MS/MS). We also tested the interaction of DPI and the recombinant p40phox in the presence of a strong redox agent (10 mM DTT) to determine if DTT enhances binding. The data processing on the corresponding LC-MS/MS data was performed using the Mascot database search engine in several stages to identify the p40phox peptides carrying common modifications and/or putative DPI modification. Up to 97% of the protein sequence was characterized using the LC-MS/MS method. However, no covalent modification derived from DPI was identified on any peptide in the samples (with or without DTT). We also performed the Wildcard open-modification search using the Byonic database search engine to identify unexpected modifications on the p40phox peptides but did not find any potential modifications corresponding to theoretical DPI-induced peptide modifications (based on the m.w. of DPI) (Supplemental Table I). Thus, it is safe to say, we did not find any evidence of direct interaction between DPI and p40phox.

Neutrophils are phagocytic cells with an unprecedented ability to generate ROS by the NADPH oxidase. One part of the cellular cytochrome b558 is present in the plasma membrane of neutrophils, and assembly and activation of the surface-located NADPH oxidase results in the release of ROS to the extracellular milieu. However, most of the cytochrome b558 is not present on the cell surface but rather in the membranes of intracellular granules, with the specific granule membrane being the main pool (5). Activation of the granule-localized NADPH oxidase results in the generation of ROS at intracellular sites, which can be mature phagolysosomes after ingestion of particles or so far rather poorly characterized (granule-containing) vesicles (6, 9). NADPH oxidase–derived ROS are critical components of the antimicrobial arsenal of neutrophils; CGD cells are hampered in terms of microbial killing, and CGD is a severe immune deficiency (10). Phagocyte ROS are also of importance for regulation of inflammatory signaling; CGD phagocytes produce elevated levels of proinflammatory cytokines, and CGD is a hyperinflammatory (or even autoinflammatory) disease (10). Additionally, we recently showed that intracellular ROS generated at locations distinct from phagosomes are essential for the ability to form neutrophil extracellular traps (22). Such neutrophil extracellular traps are fibrillary webs based on cellular DNA and chromatin that are covered with granule proteins and are cast out by cells under certain circumstances (23). Thus, neutrophil ROS clearly have additional functions other than merely to kill microbes, and although the mechanistic details of how they affect cell signaling are vague, it is likely that the effects depend on where in the cell the ROS are generated (7). To further our understanding of these matters, pharmacological inhibitors that specifically block the phagocyte NADPH oxidase at different cellular sites would be a great benefit.

Novel inhibitors of the phagocyte NADPH oxidase are continuously discovered, and one interesting recent compound is GSK, which is a specific small molecule inhibitor of gp91phox (NOX2) (16). Part of our data demonstrate that GSK effectively inhibits ROS production of primary human neutrophils and, importantly, that it is equally effective at blocking enzyme activity at the plasma membrane and at intracellular membranes. We also show that the effect of GSK is very rapid and that the inhibitory effect was fully reversible, which fits well with in vivo data from rodent models (16). This is, to our knowledge, the first characterization of this compound in primary human neutrophils with distinction of cellular localization of ROS production.

Also, the classic NADPH oxidase inhibitor DPI blocks enzyme activity (and thus ROS production) at the plasma membrane as well as in internal membranes. However, for DPI, the inhibitory effect was selective, and significantly lower doses were needed to inhibit ROS production at intracellular sites as compared to those needed to inhibit extracellular ROS release. In addition, the inhibition of the intracellular NADPH oxidase displayed a faster onset and was irreversible; after repeated washing of DPI-treated neutrophils, the ability to produce extracellular, but not intracellular, ROS was restored. Even though the exact details are not clear, DPI is agreed to interact with the FAD part of the NADPH oxidase (24), which is localized on the cytosolic part of the membrane harboring the gp91phox/p22phox heterodimer. This would mean that DPI interferes with enzymatic activity from the cytoplasm, regardless of whether the NADPH oxidase is present in the plasma membrane or in internal membranes. Thus, simple differences in membrane permeability or subcellular localization cannot explain our data on selectivity of DPI for NADPH oxidase in internal membranes.

The fact that the inhibitory effect of DPI on extracellular ROS production was reversible and could be easily washed off is in contrast to the generally accepted view that DPI is an irreversible inhibitor of flavin (25), a view that, in large part, is based on early biochemical work and the fact that radiolabeled DPI was found to bind one or more proteins of the NADPH oxidase (26, 27). To our knowledge, however, all such characterizations were done in cell-free systems with detergent-solubilized neutrophil membranes (often from nonhuman species) and not on intact and viable cells. Results from our simple and straightforward experiments were, however, very clear in that extracellular ROS production was recovered after washing.

Given the promiscuous nature of DPI (28), it is not unlikely that this drug interacts with cellular components critical for intracellular, but not extracellular, NADPH oxidase activation, but we can presently only speculate as to the nature and identity of such components. One obvious candidate in this respect is the cytoplasmic subunit p40phox, which is dispensable for ROS production in cell-free systems (29) and for extracellular ROS release from cell lines and murine neutrophils (1113). Interestingly, the reported phenotype of neutrophils from CGD patients deficient in the NCF4 gene that encodes p40phox is perfectly in line with this, and these cells produce normal levels of extracellular ROS but lack the ability to generate ROS in intracellular compartments (14, 15). We tested whether DPI was able to interact directly with (recombinant) p40phox in a minimalistic cell-free assay using mass spectrometry (MS)–based proteomic data to identify modifications on the p40phox protein. The manual inspection of the lists of potential candidates did not yield any plausible candidate for a DPI-induced peptide modification. Furthermore, we inspected the list of the relative abundance of the precursor ions in the LC-MS files to determine mass signals that are absent in the control samples but abundant in the DPI-treated samples and that could potentially arise from the DPI reaction with the peptide. None of the 400 most-abundant MS signals that were inspected corresponded to plausible modified p40phox peptides. Thus, in our simplistic cell-free system, we could not find any evidence of direct interaction between DPI and p40phox. We cannot rule out the possibility that these two molecules indeed interact in the much more complex environment of a living cell.

Another possibility that is worth considering is that the selectivity of DPI for internal NADPH oxidase activity depends on p40phox indirectly. The p40phox subunit binds with high affinity to the phospholipid phosphatidylinositol-3-phosphate [PI(3)P] by its PX domain (30, 31) which is critical for optimal NADPH oxidase activity (11). This lipid, PI(3)P, is generated from phosphatidylinositol on the cytosolic side of primarily intracellular membranes (3133). Because the PX-mediated interaction of PI(3)P and p40phox on internal membranes facilitates assembly and activation of the NADPH oxidase by conformational effects (8), it could be that the p40phox-containing enzyme complex exhibits additional interaction sites for DPI on the FAD part of gp91phox, as compared with the plasma membrane–associated complex lacking p40phox.

Additionally, the detection of intracellular ROS is critically determined by the presence and activity of MPO (22), and although we ascertained that MPO activity was not affected by DPI, it is possible that signals regulating the granule fusion events necessary for the delivery of MPO to the ROS-generating compartments (34) are inhibited by DPI.

Taken together, our data show that GSK is equally potent in inhibiting NADPH oxidase activity at extracellular and intracellular sites and that it, thus, should be a useful tool to pharmacologically mimic neutrophils of classic CGD. Additionally, we show that DPI displays a selective inhibition of the intracellular neutrophil NADPH oxidase. Even though we can presently not describe the molecular mechanisms that underlie this selectivity, our data suggest that DPI at low doses could be used as a tool to obtain neutrophils specifically lacking intracellular oxidase activity, i.e., quite similar to primary neutrophils from p40phox-deficient patients.

MS proteomic analysis was performed at the Proteomics Core Facility of Sahlgrenska Academy, University of Gothenburg. The Proteomics Core Facility is grateful to the Inga-Britt and Arne Lundbergs Research Foundation for the donation of the Orbitrap Fusion Tribrid MS instrument.

This work was supported by grants from the Swedish Research Council (2016-00982), the Swedish Heart-Lung Foundation (20180218), the King Gustaf V Memorial Foundation (FAI-2017-0368), the Patent Revenue Fund for Research in Preventive Odontology, and the Swedish state under the TUA-agreement (TUAGBG-628751).

The online version of this article contains supplemental material.

Abbreviations used in this article:

     
  • CGD

    chronic granulomatous disease

  •  
  • CL

    chemiluminescence

  •  
  • DPI

    diphenyleneiodonium

  •  
  • GSK

    GSK2795039

  •  
  • KRG

    Krebs–Ringer phosphate buffer

  •  
  • LC

    liquid chromatography

  •  
  • MPO

    myeloperoxidase

  •  
  • MS

    mass spectrometry

  •  
  • MS/MS

    tandem mass spectrometry

  •  
  • PI(3)P

    phosphatidylinositol-3-phosphate

  •  
  • ROS

    reactive oxygen species

  •  
  • SOD

    superoxide dismutase.

1
Björkman
L.
,
C.
Dahlgren
,
A.
Karlsson
,
K. L.
Brown
,
J.
Bylund
.
2008
.
Phagocyte-derived reactive oxygen species as suppressors of inflammatory disease.
Arthritis Rheum.
58
:
2931
2935
.
2
Cachat
J.
,
C.
Deffert
,
S.
Hugues
,
K. H.
Krause
.
2015
.
Phagocyte NADPH oxidase and specific immunity.
Clin. Sci. (Lond.)
128
:
635
648
.
3
Hoffmann
M. H.
,
H. R.
Griffiths
.
2018
.
The dual role of reactive oxygen species in autoimmune and inflammatory diseases: evidence from preclinical models.
Free Radic. Biol. Med.
125
:
62
71
.
4
Belambri
S. A.
,
L.
Rolas
,
H.
Raad
,
M.
Hurtado-Nedelec
,
P. M.
Dang
,
J.
El-Benna
.
2018
.
NADPH oxidase activation in neutrophils: role of the phosphorylation of its subunits.
Eur. J. Clin. Invest.
48
(
Suppl. 2
):
e12951
.
5
Borregaard
N.
,
J. M.
Heiple
,
E. R.
Simons
,
R. A.
Clark
.
1983
.
Subcellular localization of the b-cytochrome component of the human neutrophil microbicidal oxidase: translocation during activation.
J. Cell Biol.
97
:
52
61
.
6
Bylund
J.
,
K. L.
Brown
,
C.
Movitz
,
C.
Dahlgren
,
A.
Karlsson
.
2010
.
Intracellular generation of superoxide by the phagocyte NADPH oxidase: how, where, and what for?
Free Radic. Biol. Med.
49
:
1834
1845
.
7
Dahlgren
C.
,
A.
Karlsson
,
J.
Bylund
.
2019
.
Intracellular neutrophil oxidants: from laboratory curiosity to clinical reality.
J. Immunol.
202
:
3127
3134
.
8
Nunes
P.
,
N.
Demaurex
,
M. C.
Dinauer
.
2013
.
Regulation of the NADPH oxidase and associated ion fluxes during phagocytosis.
Traffic
14
:
1118
1131
.
9
Björnsdottir
H.
,
A.
Welin
,
C.
Dahlgren
,
A.
Karlsson
,
J.
Bylund
.
2015
.
Quantification of heterotypic granule fusion in human neutrophils by imaging flow cytometry.
Data Brief
6
:
386
393
.
10
Seger
R. A.
,
D.
Roos
,
B. H.
Segal
,
T. W.
Kuijpers
; Immunology And Immune System Disorders.
2017
.
Chronic Granulomatous Disease: Genetics, Biology and Clinical Management.
Nova Science Publishers, Inc.
,
Hauppauge, NY
.
11
Ellson
C.
,
K.
Davidson
,
K.
Anderson
,
L. R.
Stephens
,
P. T.
Hawkins
.
2006
.
PtdIns3P binding to the PX domain of p40phox is a physiological signal in NADPH oxidase activation.
EMBO J.
25
:
4468
4478
.
12
Ellson
C. D.
,
K.
Davidson
,
G. J.
Ferguson
,
R.
O’Connor
,
L. R.
Stephens
,
P. T.
Hawkins
.
2006
.
Neutrophils from p40phox-/- mice exhibit severe defects in NADPH oxidase regulation and oxidant-dependent bacterial killing.
J. Exp. Med.
203
:
1927
1937
.
13
Tian
W.
,
X. J.
Li
,
N. D.
Stull
,
W.
Ming
,
C. I.
Suh
,
S. A.
Bissonnette
,
M. B.
Yaffe
,
S.
Grinstein
,
S. J.
Atkinson
,
M. C.
Dinauer
.
2008
.
Fc gamma R-stimulated activation of the NADPH oxidase: phosphoinositide-binding protein p40phox regulates NADPH oxidase activity after enzyme assembly on the phagosome.
Blood
112
:
3867
3877
.
14
Matute
J. D.
,
A. A.
Arias
,
N. A.
Wright
,
I.
Wrobel
,
C. C.
Waterhouse
,
X. J.
Li
,
C. C.
Marchal
,
N. D.
Stull
,
D. B.
Lewis
,
M.
Steele
, et al
.
2009
.
A new genetic subgroup of chronic granulomatous disease with autosomal recessive mutations in p40 phox and selective defects in neutrophil NADPH oxidase activity.
Blood
114
:
3309
3315
.
15
van de Geer
A.
,
A.
Nieto-Patlán
,
D. B.
Kuhns
,
A. T.
Tool
,
A. A.
Arias
,
M.
Bouaziz
,
M.
de Boer
,
J. L.
Franco
,
R. P.
Gazendam
,
J. L.
van Hamme
, et al
.
2018
.
Inherited p40phox deficiency differs from classic chronic granulomatous disease.
J. Clin. Invest.
128
:
3957
3975
.
16
Hirano
K.
,
W. S.
Chen
,
A. L.
Chueng
,
A. A.
Dunne
,
T.
Seredenina
,
A.
Filippova
,
S.
Ramachandran
,
A.
Bridges
,
L.
Chaudry
,
G.
Pettman
, et al
.
2015
.
Discovery of GSK2795039, a novel small molecule NADPH oxidase 2 inhibitor.
Antioxid. Redox Signal.
23
:
358
374
.
17
Holland
P. C.
,
H. S.
Sherratt
.
1972
.
Biochemical effects of the hypoglycaemic compound diphenyleneiodonnium. Catalysis of anion-hydroxyl ion exchange across the inner membrane of rat liver mitochondria and effects on oxygen uptake.
Biochem. J.
129
:
39
54
.
18
Bøyum
A.
,
D.
Løvhaug
,
L.
Tresland
,
E. M.
Nordlie
.
1991
.
Separation of leucocytes: improved cell purity by fine adjustments of gradient medium density and osmolality.
Scand. J. Immunol.
34
:
697
712
.
19
Bylund
J.
,
H.
Björnsdottir
,
M.
Sundqvist
,
A.
Karlsson
,
C.
Dahlgren
.
2014
.
Measurement of respiratory burst products, released or retained, during activation of professional phagocytes.
Methods Mol. Biol.
1124
:
321
338
.
20
Welin
A.
,
H.
Björnsdottir
,
M.
Winther
,
K.
Christenson
,
T.
Oprea
,
A.
Karlsson
,
H.
Forsman
,
C.
Dahlgren
,
J.
Bylund
.
2015
.
CFP-10 from Mycobacterium tuberculosis selectively activates human neutrophils through a pertussis toxin-sensitive chemotactic receptor.
Infect. Immun.
83
:
205
213
.
21
Wiśniewski
J. R.
,
A.
Zougman
,
N.
Nagaraj
,
M.
Mann
.
2009
.
Universal sample preparation method for proteome analysis.
Nat. Methods
6
:
359
362
.
22
Björnsdottir
H.
,
A.
Welin
,
E.
Michaëlsson
,
V.
Osla
,
S.
Berg
,
K.
Christenson
,
M.
Sundqvist
,
C.
Dahlgren
,
A.
Karlsson
,
J.
Bylund
.
2015
.
Neutrophil NET formation is regulated from the inside by myeloperoxidase-processed reactive oxygen species.
Free Radic. Biol. Med.
89
:
1024
1035
.
23
Boeltz
S.
,
P.
Amini
,
H. J.
Anders
,
F.
Andrade
,
R.
Bilyy
,
S.
Chatfield
,
I.
Cichon
,
D. M.
Clancy
,
J.
Desai
,
T.
Dumych
, et al
.
2019
.
To NET or not to NET:current opinions and state of the science regarding the formation of neutrophil extracellular traps.
Cell Death Differ.
26
:
395
408
.
24
Jaquet
V.
,
L.
Scapozza
,
R. A.
Clark
,
K. H.
Krause
,
J. D.
Lambeth
.
2009
.
Small-molecule NOX inhibitors: ROS-generating NADPH oxidases as therapeutic targets.
Antioxid. Redox Signal.
11
:
2535
2552
.
25
Altenhöfer
S.
,
K. A.
Radermacher
,
P. W.
Kleikers
,
K.
Wingler
,
H. H.
Schmidt
.
2015
.
Evolution of NADPH oxidase inhibitors: selectivity and mechanisms for target engagement.
Antioxid. Redox Signal.
23
:
406
427
.
26
Cross
A. R.
,
O. T.
Jones
.
1986
.
The effect of the inhibitor diphenylene iodonium on the superoxide-generating system of neutrophils. Specific labelling of a component polypeptide of the oxidase.
Biochem. J.
237
:
111
116
.
27
O’Donnell
B. V.
,
D. G.
Tew
,
O. T.
Jones
,
P. J.
England
.
1993
.
Studies on the inhibitory mechanism of iodonium compounds with special reference to neutrophil NADPH oxidase.
Biochem. J.
290
:
41
49
.
28
Aldieri
E.
,
C.
Riganti
,
M.
Polimeni
,
E.
Gazzano
,
C.
Lussiana
,
I.
Campia
,
D.
Ghigo
.
2008
.
Classical inhibitors of NOX NAD(P)H oxidases are not specific.
Curr. Drug Metab.
9
:
686
696
.
29
Abo
A.
,
A.
Boyhan
,
I.
West
,
A. J.
Thrasher
,
A. W.
Segal
.
1992
.
Reconstitution of neutrophil NADPH oxidase activity in the cell-free system by four components: p67-phox, p47-phox, p21rac1, and cytochrome b-245.
J. Biol. Chem.
267
:
16767
16770
.
30
Ellson
C. D.
,
S.
Gobert-Gosse
,
K. E.
Anderson
,
K.
Davidson
,
H.
Erdjument-Bromage
,
P.
Tempst
,
J. W.
Thuring
,
M. A.
Cooper
,
Z. Y.
Lim
,
A. B.
Holmes
, et al
.
2001
.
PtdIns(3)P regulates the neutrophil oxidase complex by binding to the PX domain of p40(phox).
Nat. Cell Biol.
3
:
679
682
.
31
Kanai
F.
,
H.
Liu
,
S. J.
Field
,
H.
Akbary
,
T.
Matsuo
,
G. E.
Brown
,
L. C.
Cantley
,
M. B.
Yaffe
.
2001
.
The PX domains of p47phox and p40phox bind to lipid products of PI(3)K.
Nat. Cell Biol.
3
:
675
678
.
32
Nicot
A. S.
,
J.
Laporte
.
2008
.
Endosomal phosphoinositides and human diseases.
Traffic
9
:
1240
1249
.
33
Vieira
O. V.
,
R. J.
Botelho
,
L.
Rameh
,
S. M.
Brachmann
,
T.
Matsuo
,
H. W.
Davidson
,
A.
Schreiber
,
J. M.
Backer
,
L. C.
Cantley
,
S.
Grinstein
.
2001
.
Distinct roles of class I and class III phosphatidylinositol 3-kinases in phagosome formation and maturation.
J. Cell Biol.
155
:
19
25
.
34
Björnsdottir
H.
,
D.
Granfeldt
,
A.
Welin
,
J.
Bylund
,
A.
Karlsson
.
2013
.
Inhibition of phospholipase A(2) abrogates intracellular processing of NADPH oxidase derived reactive oxygen species in human neutrophils.
Exp. Cell Res.
319
:
761
774
.

The authors have no financial conflicts of interest.

This article is distributed under the terms of the CC BY 4.0 Unported license.

Supplementary data