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Originally published online as doi:10.1189/jlb.1206735 on May 31, 2007

Published online before print May 31, 2007
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(Journal of Leukocyte Biology. 2007;82:729-741.)
© 2007 by Society for Leukocyte Biology

Role of NF-{kappa}B in transcriptional regulation of the phagocyte NADPH oxidase by tumor necrosis factor-{alpha}

Katherine A. Gauss*,1, Laura K. Nelson-Overton*, Daniel W. Siemsen*, Ying Gao*, Frank R. DeLeo{dagger} and Mark T. Quinn*

* Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana, USA; and
{dagger} Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, Montana, USA

1 Correspondence: Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717, USA. E-mail: kgauss{at}montana.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophages play an important role in the pathogenesis of chronic inflammatory disease. Activation of these phagocytes induces the production of proinflammatory cytokines, such as IL-1 and TNF-{alpha} and the generation of reactive oxygen species (ROS), such as superoxide anion (O2•–). Recently, we found that TNF-{alpha} treatment of human monocytic cells (MonoMac1) and isolated human monocytes resulted in up-regulation of the NADPH oxidase gene, neutrophil cytosolic factor 2 (NCF2). These results suggested that TNF-{alpha}, produced by activated macrophages, could serve as an autocrine/paracrine regulator of the oxidase, resulting in increased and/or prolonged production of O2•–. To gain a better understanding of the mechanisms involved in NADPH oxidase regulation by TNF-{alpha}, we evaluated transcriptional regulation of oxidase genes in MonoMac1 cells and human monocytes. We show that TNF-{alpha}-treated cells have increased levels of mRNA and up-regulated expression of NADPH oxidase subunits p47phox, p67phox, and gp91phox, as well as increased oxidase activity. Pharmacological inhibitors of NF-{kappa}B activation blocked TNF-{alpha}-induced up-regulation of NCF1, NCF2, and CYBB message, which correlated with a reduction in expression of the corresponding oxidase proteins and decreased O2•– production. These data demonstrate that the increase in and/or maintenance of O2•– production in TNF-{alpha}-treated MonoMac1 cells and monocytes are a result, in part, of transcriptional up-regulation of three essential NADPH oxidase genes via the NF-{kappa}B pathway. This novel finding supports a model, whereby TNF-{alpha}-dependent activation of NF-{kappa}B up-regulates phagocyte NADPH oxidase activity, leading to enhanced ROS production and further NF-{kappa}B activation, potentially contributing to sustained oxidant production in chronic inflammation.

Key Words: promoter • inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The phagocyte NADPH oxidase is a multiprotein enzyme complex, which plays an essential role in innate immunity (reviewed in ref. [1 ]). The NADPH oxidase is composed of a plasma membrane-associated flavocytochrome b, comprised of gp91phox and p22phox, and four cytosolic proteins (p40phox, p47phox, p67phox, and Rac2) and catalyzes the transfer of electrons from NADPH to O2, resulting in the formation of superoxide anion (O2•–) and other reactive oxygen species (ROS) important for defense against microbial pathogens (reviewed in refs. [2 3 4 ]). Chronic granulomatous disease (CGD), resulting from genetic defects in NADPH oxidase genes and characterized by recurrent infections in individuals with CGD, demonstrates the importance of the oxidase in innate host immunity [5 , 6 ]. Although the NADPH oxidase is essential to host immunity, it has also been demonstrated that excessive phagocyte ROS are involved in the tissue injury associated with a number of chronic inflammatory diseases, including rheumatoid arthritis [7 ] and atherosclerosis [8 , 9 ]. Therefore, a better understanding of the processes that regulate the formation of an active NADPH oxidase complex is essential to the development of effective treatments to control the damage associated with chronic inflammation.

Monocyte/macrophages are known to play a key role in the inflammatory processes associated with rheumatoid arthritis and atherosclerosis (reviewed in refs. [8 , 10 , 11 ]). Infiltration of stimulated T lymphocytes into target tissue is promptly followed by the appearance of monocytes, which subsequently differentiate into macrophages. Interaction between stimulated T lymphocytes and macrophages leads to macrophage activation and the production of large amounts of the proinflammatory cytokines IL-1 and TNF-{alpha} as well as ROS production via NADPH oxidase activation. ROS contribute to atherogenesis via direct lipid and lipoprotein oxidation, leading to foam cell formation [8 ], and it has been demonstrated clearly that the monocyte/macrophage NADPH oxidase is a major source of ROS in atherosclerotic lesions [12 13 14 ]. Therefore, understanding mechanisms that regulate the NADPH oxidase in monocyte/macrophages is critical for identifying therapeutic approaches to address chronic inflammatory syndromes.

Although it has been observed that the expression of various NADPH oxidase proteins is up-regulated in monocytes and/or macrophages after TNF-{alpha} treatment [15 16 17 ], little is known about the mechanisms involved in this process. Dusi et al. [16 ] reported that TNF-{alpha}-induced expression of gp91phox required the binding of PU.1 to the CYBB promoter but also demonstrated that the effect of TNF-{alpha} on O2•– production was unrelated to its effects on flavocytochrome b expression. Thus, up-regulation of NADPH oxidase activity by TNF-{alpha} is likely mediated through effects on the other oxidase proteins. Indeed, Green et al. [15 ] showed that expression of p47phox and p67phox protein was enhanced by TNF-{alpha} in murine bone marrow-derived macrophages, and we demonstrated recently that treatment of human MonoMac1 cells with TNF-{alpha} resulted in the up-regulation of neutrophil cytosolic factor 2 (NCF2), the gene encoding p67phox, required a novel TNF-{alpha}-responsive region in the NCF2 promoter [18 ]. Thus, we proposed that transcriptional regulation of the genes encoding one or more of the NADPH oxidase cytosolic components played a key role in TNF-{alpha}-induced up-regulation of monocyte/macrophage oxidase activity. As the transcription factor NF-{kappa}B can be activated by TNF-{alpha} [19 ] and ROS [20 ], these observations also raised the possibility of a positive-feedback mechanism, whereby TNF-{alpha} produced by activated macrophages could serve as an autocrine/paracrine regulator of oxidase gene expression, possibly through NF-{kappa}B, resulting in increased production of O2•– and further activation of NF-{kappa}B.

In the present report, we evaluated the role of NF-{kappa}B as a regulator of NADPH oxidase activity in TNF-{alpha}-treated human MonoMac1 monocytes and human primary monocytes. Our data show that TNF-{alpha} treatment induced expression of the NADPH oxidase genes NCF1, NCF2, and CYBB, resulting in increased expression of p47phox, p67phox, and gp91phox (NOX2) and up-regulation of O2•– production. We also show this effect was primarily a result of activation of the NF-{kappa}B pathway but did not involve the JNK or p38 MAPK pathways. The role of NF-{kappa}B in the transcriptional regulation of three essential NADPH oxidase components in response to TNF-{alpha} is a novel finding and, given that NF-{kappa}B can be activated by TNF-{alpha} as well as ROS, supports the concept of a positive-feedback mechanism, whereby TNF-{alpha}-activated NF-{kappa}B up-regulates NADPH oxidase activity, leading to an increase in ROS levels and further activation of NF-{kappa}B.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Recombinant human TNF-{alpha} was from Fitzgerald Industries International, Inc. (Concord, MA, USA). IKK Inhibitor II (Wedelolactone), IKK Inhibitor III (BMS-345541), JNK Inhibitor I, JNK Inhibitor II, p38 MAPK inhibitor, and p38 MAPK Inhibitor III were from Calbiochem/EMD Biosciences (San Diego, CA, USA). Diogenes cellular luminescence enhancement system was from National Diagnostics (Atlanta, GA, USA). Taqman probes and primer sets were purchased from Applied Biosystems (Foster City, CA, USA). mAb 7D5 was a kind gift of Dr. Michio Nakamura (Nagasaki University, Nagasaki, Japan).

Cell culture
The human MonoMac1 cell line (DSM ACC252, German Microorganism and Cell Culture Collection, Braunschweig, Germany) was used for these studies due to their morphological, histochemical, phenotypic, and functional property similarities to mature monocytes (e.g., phagocytosis, FcR, and specific surface marker expression) [21 ]. MonoMac1 cells were grown in RPMI-1640 media supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 50 µg/ml streptomycin, nonessential amino acids, and sodium pyruvate. Cells were seeded at 3–5 x 105 cells/mL, 12–24 h prior to treatment.

Monocyte purification
Human blood monocytes were purified in accordance with a protocol approved by the Institutional Review Board at Montana State University (Bozeman, MT, USA). Briefly, mononuclear cells were isolated from peripheral blood of healthy donors using dextran sedimentation for 45 min., followed by Histopaque 1077 gradient separation, as described by Schulert and Allen [22 ]. The monocyte/lymphocyte layer was collected, washed twice with cold RPMI, and plated on FBS-coated, 60 mm Petri dishes at 107 cells/plate in RPMI-1640 media supplemented with 10% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 50 µg/ml streptomycin, nonessential amino acids, and sodium pyruvate. Petri dishes were coated with FBS overnight and washed twice with PBS immediately before use. After incubation of the cells for 4 h at 37°C and 5% CO2, the media containing nonadherent cells were removed and replaced with fresh media ± TNF-{alpha} and/or inhibitor, as indicated. At the indicated times, monocytes were harvested by removing the media, washing with PBS, and incubating for 15–20 min in cold PBS containing 5% FBS and 0.02% EDTA to lift the cells. Monocytes were collected, washed with HBSS containing 10 mM Hepes, pH 7.4, and resuspended in the desired buffer for further analyses. Monocyte preparations contained ~10% lymphocytes and no granulocytes, as determined by microscopic analysis.

Measurement of O2•– production
MonoMac1 cells and monocytes were plated for 12 h prior to treatment with media (control) or 20 ng/mL TNF-{alpha}. At the indicated times, cells were harvested and washed three times in HBSS. Cells were resuspended in HBSS containing CaCl2 and MgCl2 (HBSS+), and O2•– production was determined using Diogenes, a cellular chemiluminescence system specific for O2•– detection [23 ]. Assays were performed in Corning half-area chemiluminescence plates, and each well contained 50 µL Diogenes reagent, 2 x 105 cells, and HBSS+ added to a final volume of 100 µL, with each sample run in triplicate. The cells were activated by addition of 50 ng/mL PMA in the presence or absence of 5 U/mL bovine erythrocyte superoxide dismutase (SOD; Sigma Chemical Co., St. Louis, MO, USA), and the reactions were monitored for 2 h at 37°C using a Fluoroskan Ascent FL microtiter plate reader (Thermo Electron Corp., Waltham, MA, USA).

For inhibitor experiments, cells were plated as above but treated for 30–60 min with inhibitor prior to TNF-{alpha} treatment and activation. O2•– production was determined, and the data were analyzed as described above.

Quantitative (q)RT-PCR
MonoMac1 cells were seeded at 4 x 105 cells/mL, 24 h prior to TNF-{alpha} treatment. At the indicated times, total RNA was isolated from untreated and 20 ng/mL TNF-{alpha}-treated cells using a RNeasy mini kit and subjected to qRT-PCR analysis using gene-specific Taqman probes and primers (Table 1 ). ß-actin was used as the endogenous control, and IL-8 and TNF-{alpha} were used as positive controls for inhibitor assays. Predesigned probe and primer sets for ß-actin, IL-8, and TNF-{alpha} were obtained from Applied Biosystems (Foster City, CA, USA). Samples were analyzed on a 7500 real-time PCR system (Applied Biosystems), and data are presented as relative mRNA levels with Time 0 and no TNF-{alpha} treatment set to 1.


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  		Table 1. Established and Putative PRRs That Contain Tyrosine-Based Signaling Motifs

 
For inhibitor assays, cells were treated with or without inhibitor for 30–60 min prior to TNF-{alpha} treatment and subsequent qRT-PCR analysis. Data were analyzed as above.

Immunoblot analysis
Cells were treated with inhibitor, as indicated, prior to treatment with media (control) or 20 ng/mL TNF-{alpha} for 12 h. Cells were pelleted by centrifugation and resuspended in 5 mL Dulbecco’s PBS (DPBS; Invitrogen, Carlsbad, CA, USA) and treated with diisopropylfluorophosphate (0.5 µL/mL) for 15 min on ice. Subsequently, cells were washed with DPBS and resuspended in 1 mL relaxation buffer (10 mM NaCl, 100 mM KCl, 10 mM Hepes, pH 7.4) containing protease inhibitor cocktail (Sigma Chemical Co.), 625 µM PMSF, and 1% octyl-ß-D-glucopyranoside. Detergent extracts were stirred for 30 min on ice and centrifuged for 30 min at 100,000 g, and supernatants were collected. Samples (25, 50, or 100 µg) were separated by SDS-PAGE on gradient gels and transferred to a nitrocellulose membrane as described [24 ]. Prestained molecular weight standards (Amersham Biosciences, Piscataway, NJ, USA) were included on all gels for reference. Previously characterized anti-p67phox mAb 81.1 [25 ], anti-gp91phox mAb 54.1 [26 ], p22phox mAb 44.1 [26 ], anti-p40phox mAb 1.9 [27 ], and anti-p47phox polyclonal antibody R360 [25 ] were used to probe Western blots, followed by HRP-conjugated goat anti-mouse or anti-rabbit secondary antibodies (BioRad, Richmond, CA, USA) and chemiluminescence development. Developed blots were analyzed using an IS-1000 Alpha Imager digital imaging system (Alpha Innotech, San Leandro, CA, USA).

Flow cytometric analysis
MonoMac1 cells were treated with media (control) or 20 ng/mL TNF-{alpha} for 12 h, diluted by addition of 4 mL DPBS containing 5% heat-inactivated FCS and 7.5 mM sodium azide (FACS buffer), and incubated for 5 min. The cells were separated into three groups. One group received no treatment (cells only); one group was treated with Alexa 488-conjugated goat anti-mouse IgG secondary antibody (2° antibody) for 1 h on ice; and one group was treated for 1 h on ice with mAb 7D5, which recognizes an extracellular epitope on gp91phox [28 ], followed by washing and addition of secondary antibody for 1 h. Following a wash with FACS buffer, all samples were resuspended in 2 mL FACS buffer and analyzed using a FACScan flow cytometer (BD Biosciences, San Jose, CA, USA). The data are presented as mean fluorescence intensity.

Analysis of inhibitor efficacy
IKK Inhibitor II (Wedelolactone) and IKK Inhibitor III (BMS-345541) inhibit the NF-{kappa}B pathway. Likewise, IKK Inhibitor II is reported to have no effects on p38 MAPK or Akt and IKK. Inhibitor III shows no activity toward IKK{epsilon} or more than 15 unrelated protein kinases. The reported IC50 values for IKK Inhibitors II and III were reported to be ~35 µM [29 ] and 4 µM [30 ], respectively. JNK Inhibitor I (cell-permeable peptide) and JNK Inhibitor II (SP600125) inhibit the JNK pathway. JNK Inhibitor I has no effect on ERK1/2 or p38 activities with an IC50 of ~1 µM [31 ], and JNK Inhibitor II exhibits over 300-fold greater selectivity for JNK, as compared with ERK1 and p38 MAPKs, and has a reported IC50 of 40–90 nM [32 ]. The inhibitors used for the p38 MAPK pathway were p38 MAPK Inhibitor III (ML3403) with a reported IC50 of 0.38 µM [33 ] and p38 MAPK inhibitor with a reported IC50 of 35 nM [34 ].

MonoMac1 cells were seeded at 2 x 105 cells/well in 96-well microtiter plates for 24 h, treated with buffer (control) or the indicated inhibitor for 30–60 min, and then treated with media (control) or 20 ng/mL TNF-{alpha} treatment for 30 min. Cells were fixed and analyzed for total and phosphorylated forms of NF-{kappa}B p65 (Ser 536), JNK, and p38 MAPK using specific colorimetric assay FACE kits (Active Motif, Carlsbad, CA, USA), according to the manufacturer’s protocol. Absorbance was measured on a SpectraMax PLUS microtiter plate spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).

Statistical analysis
One-way ANOVA was performed on the indicated sets of data, followed by Tukey’s post test for pair-wise comparisons (GraphPad Prism Software, San Diego, CA, USA). Differences at P < 0.05 were considered to be statistically significant and are indicated.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
TNF-{alpha} treatment enhances PMA-stimulated O2•– production by MonoMac1 cells
In previous studies investigating the transcriptional regulation of NCF2, we found that TNF-{alpha} treatment of various myeloid cell lines and primary monocytes resulted in an increase in NCF2 mRNA and p67phox protein. We hypothesized that an increase in p67phox expression may lead to an increased and/or prolonged production of O2•–. To further characterize the effects of TNF-{alpha} on NADPH oxidase activity in monocyte/macrophages, we evaluated PMA-activated O2•– production in MonoMac1 cells pretreated for 10 h with or without TNF-{alpha}. As seen in Figure 1A , PMA-activated MonoMac1 cells produced significant amounts of O2•– compared to cells with no PMA activation. Furthermore, treatment with TNF-{alpha} prior to PMA activation resulted in a statistically significant increase in O2•– production of two- to threefold relative to the non-TNF-{alpha}, PMA-activated cells. Note that the assay used here was specific for the measurement of O2•–, as addition of SOD to the assay completely eliminated the signal, indicating the entire response was a result of O2•– and not some other ROS (Fig. 1A) . Analysis of O2•– production over time demonstrated that the increase in O2•– production was evident after ~6 h of TNF-{alpha} treatment and statistically significant by 10 h (Fig. 1B) . These data demonstrate that TNF-{alpha} pretreatment results in enhanced PMA-stimulated NADPH oxidase activity in MonoMac1 cells.


Figure 1
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  		Figure 1. TNF-{alpha} enhances O2•– production by activated MonoMac1 cells (A), which were treated with media (control) or 20 ng/mL TNF-{alpha} for 10 h and then activated with PMA (50 ng/mL) in the presence or absence of 100 µM SOD. O2•– production was determined using a chemiluminescence assay, as described. The presence or absence of TNF-{alpha}, PMA, and/or SOD is indicated, and the data are presented relative to the no TNF-{alpha}/no PMA control (Control), which was set to 1. (B) MonoMac1 cells were treated with media (control) or 20 ng/mL TNF-{alpha} for the indicated lengths of time and then activated with PMA. O2•– production is shown relative to the no TNF-{alpha}/no PMA control (set to 1; not shown) at each time-point. The data are presented as the mean ± SEM of triplicate samples from one experiment, which is representative of at least three independent experiments. Statistically significant differences (a, P<0.05; b, P<0.01; c, P<0.001) are indicated.

 
TNF-{alpha} treatment up-regulates NCF1, NCF2, and CYBB message in MonoMac1 cells
To determine if transcription of other NADPH oxidase genes, in addition to NCF2, was altered by TNF-{alpha} treatment, we analyzed the change in message for CYBA, CYBB, NCF1, NCF2, and NCF4 using qRT-PCR with TaqMan probes and primer sets specific for the individual oxidase genes (see Table 1 for sequences). As with NCF2, mRNA levels for NCF1 and CYBB also increased with TNF-{alpha} treatment (Fig. 2 ). The increase in mRNA levels for NCF1 was statically significant after 3 h of TNF-{alpha} treatment and after 6 h for NCF2 and CYBB, relative to the corresponding samples with no TNF-{alpha} treatment. The largest fold change was seen for NCF1 message, with a ten- to 20-fold increase. The increase in NCF2 mRNA was three- to fivefold, and there was a smaller, yet consistent, approximate twofold change observed for CYBB mRNA. In contrast, no increase in message was apparent for CYBA or NCF4 (Fig. 2) , even after 24 h of TNF-{alpha} treatment (data not shown).


Figure 2
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  		Figure 2. NCF1, NCF2, and CYBB mRNA is increased in TNF-{alpha}-treated MonoMac1 cells. Total RNA was isolated from MonoMac1 cells after treatment with media (open bars) or TNF-{alpha} (solid bars) for the indicated times. RNA was subjected to qRT-PCR analysis using gene-specific Taqman probes and primers for CYBA, CYBB, NCF1, NCF2, and NCF4, and ß-actin was used as the endogenous control. The data are shown as relative mRNA levels, and the Time 0/no TNF-{alpha} sample was set to 1. The data are presented as the mean ± SEM of triplicate samples from one experiment, which is representative of at least two independent experiments. Statistically significant differences (a, P<0.05; b, P<0.01; c, P<0.001) between TNF-{alpha}-treated and corresponding untreated samples at each time-point are indicated.

 
To determine if the changes in message levels for NCF1, NCF2, and CYBB corresponded to changes in protein expression, Western blot analysis was performed to compare oxidase protein expression in untreated and TNF-{alpha}-treated MonoMac1 cells (Fig. 3 ). Consistent with the increased mRNA levels observed for NCF1, NCF2, and CYBB, expression of these oxidase components also increased after TNF-{alpha} treatment (Fig. 3A) , and the relative changes in protein expression were confirmed by densitometric analysis of the blots (Fig. 3B) . The lack of change in mRNA levels for NCF4 and CYBA was consistent with the absence of change in their protein levels seen after TNF-{alpha} treatment (Fig. 3A) . As a result of the relatively small change in CYBB mRNA and protein levels, we also analyzed the amount of cell surface flavocytochrome b protein expression on cells treated with and without TNF-{alpha} using flow cytometry. Consistent with the degree of change observed for CYBB mRNA and gp91phox protein, an increase in the amount of gp91phox protein expressed on the cell surface with TNF-{alpha} treatment was observed (1.5- to twofold increase; Fig. 3C ). As monocyte/macrophages do not contain an intracellular pool of flavocytochrome b [35 ], flow cytometry provides an accurate measure of the total flavocytochrome b in these cells. Overall, the correlation between the delayed timing of the increase in O2•– production and the increase in NCF1, NCF2, and CYBB mRNA suggested that enhanced NADPH oxidase activity in TNF-{alpha}-treated MonoMac1 cells was a result, in part, of increased concentrations of the NADPH oxidase components p47phox, p67phox, and gp91phox via transcriptional up-regulation.


Figure 3
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  		Figure 3. TNF-{alpha} treatment up-regulates p67phox, p47phox, and gp91phox protein expression in MonoMac1 cells (A), which were incubated for 12 h without (–) or with (+) 20 ng/mL TNF-{alpha}, and proteins were separated by SDS-PAGE and immunoblotted with antibodies against p22phox, p40phox, p47phox, p67phox, and gp91phox, as described. Representative blots are shown from two independent experiments. (B) Immunoblots were analyzed by densitometry, and the data are presented as the fold change of TNF-{alpha}-induced protein expression versus the corresponding non-TNF-{alpha} control sample. (C) Cells were incubated for 12 h with media (open histogram) or with TNF-{alpha} (solid histogram), labeled with no antibody (Cells Only, dotted line), secondary antibody only (2° Control, dashed line), or anti-gp91phox antibody 7D5 plus secondary antibody (7D5+2° antibody) and analyzed by flow cytometry. For each assay, 10,000 cells were collected, and the data are plotted as mean fluorescence intensity. The data are representative of at least two independent experiments.

 
Inhibition of NF-{kappa}B activation blocks TNF-{alpha}-induced up-regulation of NCF1, NCF2, and CYBB and p47phox, p67phox, and gp91phox expression
To identify the TNF-{alpha}-induced signal transduction pathway(s) required for the up-regulation of p47phox, p67phox, and gp91phox in MonoMac1 cells, we used small molecule, pharmacological inhibitors to block specific signaling pathways. We concentrated on the NF-{kappa}B, JNK, and p38 MAPK pathways, as these are three of the major pathways induced by TNF-{alpha} [36 , 37 ]. As effective chemical inhibitor concentrations can vary between cell types, a cell-based ELISA was used to determine inhibitor concentrations required to inhibit phosphorylation and therefore, activation of these kinases in MonoMac1 cells. Two inhibitors were used to analyze each pathway and when possible, were chosen based on their reported target specificity (see Materials and Methods).

MonoMac1 cells were treated without or with increasing concentrations of inhibitor prior to TNF-{alpha} treatment, and the phosphorylated forms of NF-{kappa}B p65, JNK, and p38 MAPK as well as total NF-{kappa}B p65, JNK, and p38 MAPK were measured. As seen in Figure 4 , there was a statistically significant increase in the levels of phosphorylated NF-{kappa}B p65 in TNF-{alpha}-treated cells versus untreated cells, whereas the levels of total NF-{kappa}B p65 remained fairly constant in treated and untreated cells. NF-{kappa}B p65 phosphorylation was inhibited effectively in TNF-{alpha}-treated MonoMac1 cells by IKK Inhibitor II and IKK Inhibitor III at concentrations of 20–50 µM and 10–20 µM, respectively (Fig. 4) . The levels of phosphorylated JNK and p38 MAPK, but not total levels of these kinases, also increased after TNF-{alpha} treatment; however, these increases were not statistically significant, suggesting that TNF-{alpha} does not activate the JNK and p38 MAPK pathways as effectively as the NF-{kappa}B pathway in MonoMac1 cells. Nevertheless, JNK phosphorylation was inhibited by JNK Inhibitors I and II in TNF-{alpha}-treated MonoMac1 cells at concentrations of 1–2 µM and 100–150 nM, respectively (Fig. 4) , and p38 MAPK phosphorylation was inhibited effectively by p38 MAPK Inhibitor III and p38 MAPK inhibitor at concentrations of 0.6–1 µM and 100–150 nM, respectively (Fig. 4) . Note that these concentrations were within the range of published IC50 concentrations for all of these inhibitors.


Figure 4
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  		Figure 4. Determination of effective inhibitor concentrations in TNF-{alpha}-treated MonoMac1 cells, which were treated with the indicated concentrations of inhibitor prior to TNF-{alpha} treatment for 30 min and then fixed. Control samples received no TNF-{alpha}. NF-{kappa}B p65, JNK, and p38 MAPK total (open bars) and phosphorylated (solid bars) protein were analyzed as described. The data are presented as the mean ± SEM of triplicate samples from one experiment, which is representative of at least two independent experiments. Statistically significant differences (a, P<0.05; b, P<0.01) between the levels of phosphorylated proteins in control (No TNF-{alpha}) and TNF-{alpha}-treated/no inhibitor samples are indicated. Statistically significant differences (c, P<0.05; d, P<0.01) between the TNF-{alpha}-treated/no inhibitor and the TNF-{alpha}-treated/plus inhibitor samples are also indicated.

 
Using the effective inhibitor concentrations determined above, we evaluated which pathway(s) were involved in TNF-{alpha}-induced up-regulation of NCF2, NCF1, and CYBB transcription. Cells were treated with inhibitor prior to 6 h of TNF-{alpha} treatment, and total RNA was analyzed by qRT-PCR. As seen in Figure 5 , IKK Inhibitor II and IKK Inhibitor III effectively blocked TNF-{alpha}-induced up-regulation of NCF2 mRNA at or below concentrations determined to be effective at blocking the NF-{kappa}B pathway in these cells. In contrast, inhibitors of the JNK and p38 MAPK pathways did not inhibit TNF-{alpha}-induced up-regulation of NCF2 mRNA, even at concentrations higher than those determined to be effective at blocking these respective pathways (data not shown).


Figure 5
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  		Figure 5. Inhibitors of NF-{kappa}B activation block up-regulation of NCF2, NCF1, and CYBB in TNF-{alpha}-treated MonoMac1 cells, which were treated with buffer or the indicated concentrations of inhibitor for 1 h prior to incubation with control media (open bars) or TNF-{alpha} (solid bars) for 9 h. Total RNA was isolated and subjected to qRT-PCR analysis for NCF2, NCF1, and CYBB, as indicated, and ß-actin was used as the endogenous control. The data are shown as relative mRNA levels with the no inhibitor/no TNF-{alpha} sample set to 1 and are presented as the mean ± SEM of triplicate samples from one experiment, which is representative of at least two independent experiments. Statistically significant differences between controls and the corresponding TNF-{alpha}-treated samples (a, P<0.05; b. P<0.01; c, P<0.001) and between the TNF-{alpha}-treated samples without and with inhibitor (d, P<0.05; e, P<0.01; f, P<0.001) are indicated.

 
We next evaluated the effects of IKK Inhibitors II and III on the changes in NCF1 and CYBB mRNA induced by TNF-{alpha} treatment using inhibitor concentrations determined to be effective at blocking TNF-{alpha}-induced up-regulation of NCF2 mRNA. Similar to NCF2, both IKK inhibitors completely blocked TNF-{alpha}-induced up-regulation of NCF1 and CYBB message (Fig. 5) . In contrast, neither of the JNK or p38 MAPK pathway inhibitors affected the TNF-{alpha}-induced up-regulation of NCF1 or CYBB mRNA at or above concentrations determined to be effective in blocking these respective pathways (data not shown).

Immunoblotting was performed to determine if the decrease in oxidase mRNAs after IKK inhibitor treatment led to a corresponding decrease in oxidase protein expression. Cells were treated with inhibitor prior to TNF-{alpha} treatment, and total cell lysates were analyzed. As shown in Figure 6 , treatment with both IKK inhibitors inhibited TNF-{alpha}-induced up-regulation of p67phox, p47phox, and gp91phox expression. Although inhibitor-treated samples showed basal p47phox protein expression was similar to that of control cells (no inhibitor, no TNF-{alpha}), basal p67phox and gp91phox protein expression in inhibitor-treated samples was below that of untreated cells, suggesting that NCF2 and CYBB basal expression may also be regulated, in part, via the NF-{kappa}B pathway. This result is consistent with recent studies demonstrating that NF-{kappa}B was required for basal as well as LPS/IFN-{gamma}-stimulated CYBB expression in murine J774.A1 cells [38 ]. Overall, the ability of NF-{kappa}B pathway inhibitors to block TNF-{alpha}-induced up-regulation of NCF2, NCF1, and CYBB mRNA and corresponding protein in MonoMac1 cells clearly demonstrates that this response is mediated through the NF-{kappa}B pathway.


Figure 6
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  		Figure 6. Inhibitors of NF-{kappa}B activation block up-regulation of p67phox, p47phox, and gp91phox in TNF-{alpha}-treated MonoMac1 cells, which were treated as described in the Figure 5 legend with 25 µM IKK II and 5 µM IKK III, as indicated, for 24 h. The cells were lysed and immunoblotted with antibodies against p47phox, p67phox, and gp91phox. Representative blots are shown from two independent experiments.

 
To verify the results observed with the small-molecule inhibitors of the NF-{kappa}B pathway, we attempted to block the NF-{kappa}B pathway by transiently transfecting MonoMac1 cells with a dominant-negative I{kappa}B{alpha} expression plasmid. However, the low transfection efficiency in MonoMac1 cells did not allow for equal or greater levels of expression of dominant-negative I{kappa}B{alpha} protein relative to endogenous I{kappa}B{alpha}, as determined by Western blot analysis (data not shown). Therefore, to determine if the IKK inhibitors were specific for the NF-{kappa}B pathway, we analyzed changes in expression of the gene encoding TNF-{alpha}, which is known to be regulated via the NF-{kappa}B pathway in response to TNF-{alpha} (reviewed in ref. [36 ]). MonoMac1 cells were treated with the IKK inhibitor prior to TNF-{alpha} treatment for 6 h, and relative levels of TNF-{alpha} mRNA were determined using qRT-PCR. As seen in Figure 7 (upper panels), the levels of TNF-{alpha} mRNA increased approximately sevenfold in response to TNF-{alpha} treatment, and this increase was inhibited completely by IKK Inhibitor III. Similar inhibition was seen after treatment with IKK Inhibitor II (data not shown). In contrast, treatment with p38 Inhibitor III did not affect the TNF-{alpha}-dependent increase in TNF-{alpha} mRNA (Fig. 7 , upper right panel). IL-8 is also known to be up-regulated by TNF-{alpha}, and studies have shown that this response can occur via p38 MAPK, NF-{kappa}B, or both [39 40 41 ]. Therefore, the effects of IKK and p38 inhibitors on TNF-{alpha}-induced increases in IL-8 mRNA were also analyzed (Fig. 7 , lower panels). Although p38 Inhibitor III inhibited the TNF-{alpha}-induced increase significantly in IL-8 mRNA, it did not ablate the response. In comparison, the TNF-{alpha}-dependent increase in IL-8 mRNA was inhibited only slightly by IKK Inhibitor III (Fig. 7 , lower left panel) and IKK II Inhibitor (data not shown). Thus, these results are consistent with reports that IL-8 can be regulated via the p38 MAPK and NF-{kappa}B pathways in response to TNF-{alpha}. Altogether, these data confirm specificity of the pharmacological inhibitors to their respective pathways.


Figure 7
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  		Figure 7. Demonstration of inhibitor specificity. MonoMac1 cells were treated as described in the Figure 5 legend, and total RNA was subjected to qRT-PCR analysis for TNF-{alpha} and IL-8, and ß-actin was the endogenous control. The data are shown as relative mRNA levels, and the no inhibitor/no TNF-{alpha} sample was set to 1 and is presented as the mean ± SEM of triplicate samples from one experiment, which is representative of at least two independent experiments. Statistically significant differences between controls and the corresponding TNF-{alpha}-treated samples (a, P<0.01; b, P<0.001) and between the TNF-{alpha}-treated samples without and with inhibitor (c, P<0.05; d, P<0.01; e, P<0.001) are indicated.

 
As a final verification of inhibitor specificity, we evaluated an inhibitor from each group in HeLa cells containing a NF-{kappa}B-luciferase reporter [42 ]. IKK Inhibitor II completely blocked TNF-{alpha}-induced up-regulation of the NF-{kappa}B-luciferase reporter, whereas the JNK and p38 MAPK inhibitors had no effect on the luciferase reporter activity (Supplemental Fig. S1). Thus, these data provide additional confirmation of the specificity of these inhibitors. Taken together, our results provide convincing evidence that the TNF-{alpha}-induced transcriptional up-regulation of NCF1, NCF2, and CYBB and subsequent protein expression in MonoMac1 cells are mediated through the NF-{kappa}B pathway.

Pharmacological inhibition of NF-{kappa}B activation blocks TNF-{alpha}-induced enhancement of NADPH oxidase activity in MonoMac1 cells
To determine if the inhibition of TNF-{alpha}-induced up-regulation of p47phox, p67phox, and gp91phox correlated with a reduction in TNF-{alpha}-induced O2•– production in MonoMac1 cells, cells were treated with inhibitor prior to TNF-{alpha} treatment (12 h) and measurement of O2•– production. Consistent with our oxidase gene and protein expression data, inhibitors of the NF-{kappa}B pathway (Fig. 8 ) but not the JNK or p38 MAPK pathways (data not shown) blocked the TNF-{alpha}-induced increase completely in O2•– production in MonoMac1 cells. The concentrations of IKKs II and III inhibitor (25 µM and 5 µM, respectively) required to block the TNF-{alpha}-induced increase in O2•– production were within the range of the IKK inhibitor concentrations (50 µM and 3 µM, respectively) required to block the TNF-{alpha}-induced increases in NF-{kappa}B p65 phosphorylation, NCF1, NCF2, and CYBB mRNA, and corresponding proteins. The ablation of basal as well as TNF-{alpha}-stimulated ROS production at 50 and 10 µM IKK Inhibitors II and III, respectively, can be explained by the significant reduction in p67phox and gp91phox protein expression in the basal and TNF-{alpha}-treated samples observed after 24 h of inhibitor treatment (Fig. 6) . The highest concentration (150 nM) of the p38 MAPK inhibitor caused a slight decrease in O2•– production when compared with control TNF-{alpha}-treated samples, although the decrease was not as dramatic as the complete return to basal O2•– levels observed with the IKKs II and III inhibitor treatment at 25 and 5 µM, respectively. High concentrations of p38 MAPK Inhibitor III (600 nM) also caused a slight decrease in O2•– production; however, this effect was not dose-responsive or consistent between experiments (data not shown). Overall, these data show that the increase in O2•– production induced by TNF-{alpha} treatment of MonoMac1 cells is regulated primarily via the NF-{kappa}B pathway.


Figure 8
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  		Figure 8. Inhibitors of the NF-{kappa}B pathway block TNF-{alpha}-dependent up-regulation of NADPH oxidase activity in MonoMac1 cells, which were treated with buffer, IKK Inhibitor II, or IKK Inhibitor III, as indicated, for 1 h prior to treatment with media (open bar) or 20 ng/mL TNF-{alpha} (solid bar) for 12 h. Cells were activated with PMA (50 ng/mL), and O2•– production was monitored. SOD-inhibitable O2•– production is shown relative to the no inhibitor/no TNF-{alpha} sample (set to 1). The data are presented as the mean ± SEM of triplicate samples from one experiment, which is representative of two independent experiments. Statistically significant differences between control and corresponding TNF-{alpha}-treated samples (a, P<0.001) and between TNF-{alpha}-treated samples without and with inhibitor (b, P<0.001) are indicated.

 
Inhibition of NF-{kappa}B activation blocks TNF-{alpha}-induced up-regulation of NADPH oxidase proteins and activity in human monocytes
In previous studies, we and others [16 , 18 ] demonstrated that TNF-{alpha} treatment of human blood monocytes resulted in a prolonged maintenance of stimulated O2•– levels over time, and untreated cells showed a decrease in O2•– levels when compared with freshly plated cells. Based on our observations in MonoMac1 cells, we considered whether the response to TNF-{alpha} was also mediated by the NF-{kappa}B pathway in human primary monocytes. Isolated blood monocytes were treated with IKK Inhibitor III prior to TNF-{alpha} treatment, and at the indicated times, cells were stimulated with PMA, and O2•– production was monitored. As seen in Figure 9A , TNF-{alpha}-treated monocytes maintained their ability to generate O2•– over time in culture ({blacksquare}), and there was a time-dependent loss of oxidase activity in the control, non-TNF-{alpha}-treated cells ({square}), and major differences were present at times >6 h. It is notable that treatment of monocytes with IKK Inhibitor III prior to TNF-{alpha} treatment (•) also resulted in a time-dependent loss in oxidase activity, which was similar in magnitude and kinetics to that observed in control, non-TNF-{alpha}-treated cells ({square}), demonstrating that maintenance of NADPH oxidase activity in cultured monocytes required the NF-{kappa}B pathway.


Figure 9
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  		Figure 9. TNF-{alpha}-induced maintenance of O2•– levels in monocytes is regulated via the NF-{kappa}B pathway. (A) Human blood monocytes were isolated and treated with buffer ({blacksquare}, {square}) or 3 µM IKK Inhibitor III (•) for 1 h prior to treatment with media ({square}) or 20 ng/mL TNF-{alpha} ({blacksquare}, •). At the indicated times, cells were stimulated with 50 ng/mL PMA, and O2•– production was determined. The data are presented as percent of the response measured at time = 0 for each sample and represent the mean ± SEM of triplicate samples. The PMA-activated responses at time = 0 ranged from 300 to 400 chemiluminescent units. All data points represent three independent experiments, except for the 3-h point, which represents two independent experiments. (B) Monocytes were treated as in A, and proteins were separated by SDS-PAGE and immunoblotted with antibodies against p47phox, p67phox, and gp91phox. Representative blots are shown from two independent experiments.

 
To confirm the loss in monocyte oxidase activity was a result of diminished oxidase protein expression, as demonstrated above in MonoMac1 cells, we evaluated oxidase protein expression in monocytes treated identically as in the oxidase assays described above. Consistent with oxidase activity, p67phox, p47phox, and gp91phox expression was similar between samples out to ~6 h, regardless of treatment (Fig. 9B) . However, at 24 and 48 h, there were major differences in oxidase protein expression between control and TNF-{alpha}-treated samples, which correlated directly with the relative levels of O2•– produced by these cells (Fig. 9A) . Likewise, inhibition of the NF-{kappa}B pathway inhibited the ability of TNF-{alpha} to maintain oxidase protein expression in cultured primary monocytes, and protein expression in IKK Inhibitor III-treated cells was similar to or even below that of control cells at similar time-points. Furthermore, the reduced expression of oxidase protein was also consistent with the diminished levels of O2•– produced by these cells. It should be noted that the basal levels of p67phox and gp91phox expression were also reduced by IKK inhibitor treatment (Fig. 9B) . This result is similar to our observations in MonoMac1 cells (Fig. 6) , again suggesting that basal and TNF-{alpha}-induced NCF2 and CYBB gene expression is regulated via the NF-{kappa}B pathway. Overall, these results clearly demonstrate the physiological relevance of the NF-{kappa}B pathway in TNF-{alpha}-induced regulation of oxidase gene expression and O2•– production in human monocytes.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Although monocytes and neutrophils express the same phagocyte NADPH oxidase components, there are some notable differences in the regulation of the NADPH oxidase activity between these cell types [35 , 43 , 44 ]. While monocytes show a gradual increase in O2•– production, which peaks ~1 h after stimulation with soluble agonists [13 ], the response in neutrophils peaks in 2–10 min [45 ]. In addition, after sufficient recovery, monocytes are capable of mounting an additional response, which is typically not the case for neutrophils [46 ]. Lastly, differences in regulation through alternative signal transduction pathways are likely, given the fact that activating agents of the monocyte/macrophage NADPH oxidase do not necessarily activate the neutrophil NADPH oxidase (reviewed in refs. [8 , 43 ]). These differences support the hypothesis that regulation of the NADPH oxidase contributes to the distinct roles of monocyte/macrophages and neutrophils in chronic versus acute inflammation, respectively [45 , 47 , 48 ].

It is well established that the macrophage respiratory burst can be enhanced markedly by pre-exposure to priming agents, such as LPS, TNF-{alpha}, or IFN-{gamma} [49 50 51 ]. Although the mechanisms of priming are not understood completely, it has become increasingly clear that NADPH oxidase subunits can be transcriptionally up-regulated in response to certain cytokines (TNF-{alpha}, IFN-{gamma}, IL-15), leading to an increase in or prolonged production of O2•– by mature monocytes [15 16 17 18 , 38 ]. In previous studies, we demonstrated that NCF2 was transcriptionally regulated in TNF-{alpha}-treated MonoMac1 cells. Here, we extend this observation by further evaluating the effects of TNF-{alpha} on NADPH oxidase activity and investigating the signaling mechanisms underlying this response in these cells and in primary monocytes. After ~10 h of TNF-{alpha} treatment, there was a significant increase in the levels of O2•– produced upon PMA activation relative to untreated, PMA-activated cells. The length of time required to observe a significant increase in TNF-{alpha}-induced O2•– levels suggested that regulation of oxidase activity was, in part, at the transcriptional level. Indeed, analysis of changes in message and protein for NADPH oxidase subunits indicated significant changes in NCF1, NCF2, and CYBB mRNA were evident by 3–6 h and were followed by corresponding increases in p47phox, p67phox, and gp91phox protein. Furthermore, the kinetics of up-regulation of these three NADPH oxidase subunits correlated directly with the enhancement of O2•– production after TNF-{alpha} treatment. The significantly larger increase in p47phox and p67phox protein levels (ten- to 20- and three- to fivefold, respectively) versus the relatively smaller change observed for gp91phox protein (1.5- to threefold) after TNF-{alpha} treatment suggests that changes in expression of the two cytosolic components versus changes in flavocytochrome b expression may have a greater impact on the increase in O2•– production in TNF-{alpha}-treated cells. This observation is consistent with other reports demonstrating that the amount of gp91phox did not correlate with the functional capacity of the enzyme [52 ] and that oligonucleotide inhibition of gp91phox expression induced by INF-{gamma} and TNF-{alpha} failed to contribute to changes in NADPH oxidase activity [16 ].

To further elucidate the signal transduction pathway(s) involved in the transcriptional regulation of NCF1, NCF2, and CYBB in TNF-{alpha}-treated MonoMac1 cells, we used pharmacological inhibitors to specifically block the NF-{kappa}B, JNK, and p38 MAPK pathways. We found that TNF-{alpha}-mediated up-regulation of NCF1, NCF2, and CYBB gene expression and subsequent protein expression could be blocked by NF-{kappa}B pathway inhibitors but not by inhibitors of the JNK or p38 MAPK pathways. In addition, inhibitor treatment resulted in a decrease in basal levels of p67phox and gp91phox protein, suggesting that basal expression of these proteins may also be regulated via the NF-{kappa}B pathway. These results are consistent with a recent report demonstrating a role for NF-{kappa}B in the basal, as well as LPS/IFN-{gamma}-induced regulation of CYBB expression in murine J774.A1 monocyte/macrophages [38 ]. In their report, two NF-{kappa}B-binding sites were identified in the mouse CYBB promoter, and putative, corresponding elements were noted in the human CYBB promoter [38 ]. However, functionality of the putative NF-{kappa}B elements in the human CYBB promoter has not been determined. We searched the NCF1 and NCF2 promoters for NF-{kappa}B elements similar in sequence to those identified in the murine CYBB promoter using the TFSEARCH program [53 ] and identified one putative NF-{kappa}B element corresponding in sequence to the murine CYBB NF-{kappa}B1 element in each of the NCF1 and NCF2 promoters (Fig. 10 ), both containing two nucleotide differences when compared with the mouse CYBB {kappa}B1 promoter element. In any case, further studies are required to determine if NF-{kappa}B binds to the putative {kappa}B elements in the human NCF1, NCF2, and CYBB promoters and if these sites are functionally regulated by NF-{kappa}B.


Figure 10
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  		Figure 10. Alignment of murine CYBB NF-{kappa}B1 element with putative human CYBB, NCF1, and NCF2 {kappa}B elements. Bold characters indicate identical nucleotides in all four sequences; underlined characters indicate three out of four nucleotides with identical nucleotides; and A/G indicates two out of four nucleotides are A or G. Numbers in parentheses denote the number of base pairs upstream of the ATG start site corresponding to the 5' end of the sequence.

 
The correlation between the timing of increased p47phox, p67phox, and gp91phox protein expression and enhanced O2•– production in TNF-{alpha}-treated cells, as well as the complete inhibition of both with IKK inhibitors, suggested that the increase in O2•– production was primarily a result of transcriptional up-regulation of NADPH oxidase genes via the NF-{kappa}B pathway. The concentration of IKK Inhibitor II needed to sufficiently block TNF-{alpha}-induced increases in O2•– production was, however, somewhat lower than that required to block TNF-{alpha}-induced NF-{kappa}B p65 phosphorylation and up-regulation of NCF1, NCF2, and CYBB gene expression. This finding suggests that there are possibly other TNF-{alpha}-induced mechanisms, in addition to transcriptional up-regulation of the NADPH oxidase components, which may contribute to the overall enhancement of NADPH oxidase activity and are regulated via the NF-{kappa}B signaling pathway. We did observe a relatively small decrease in O2•– production in cells treated with the highest concentrations of p38 MAPK inhibitor, suggesting the possibility that the TNF-{alpha}-induced increase in NADPH oxidase activity may also be regulated, to a slight degree, by the p38 MAPK pathway, a pathway shown to be important in regulating the NADPH oxidase activity in neutrophils via priming [54 55 56 57 58 ]. Although this study does not completely rule out the possibility that some of the TNF-{alpha}-induced ROS production in MonoMac1 cells is generated by other mechanisms (i.e., other NOX proteins or a mitochondrial source), our data clearly show that the majority of TNF-{alpha}-induced increase in NADPH oxidase activity in MonoMac1 cells is regulated via the NF-{kappa}B pathway and correlates well with the timing of transcriptional up-regulation of NCF1, NCF2, and CYBB gene expression.

Previously, we found that TNF-{alpha} treatment of human blood monocytes induced an increase in NCF2 message and subsequent p67phox protein expression [18 ], which is consistent with our present findings and the conclusion that the enhancing effects of TNF-{alpha} on O2•– production are a result of regulation of NADPH oxidase gene expression. These results are generally consistent with previous reports demonstrating an increase in p67phox and p47phox protein in murine bone marrow-derived macrophages treated with TNF-{alpha} [15 ], an increase in gp91phox in THP-1 cells treated with IFN-{gamma}/TNF-{alpha} [17 ], and an increase in p47phox and gp91phox in monocytes treated with TNF-{alpha} [16 ]. It should be noted, however, that some differences were observed when comparing our p67phox expression data in monocytes with studies published previously. For example, Dusi et al. [16 ] reported similar up-regulation of p47phox and gp91phox expression in TNF-{alpha}-treated monocytes at 48 h; whereas, they observed no differences in p67phox protein levels compared with control cells at 48 h. In contrast, we found that p67phox expression is maintained by TNF-{alpha}, which would be required for maintenance of oxidase activity. The reason for this difference is not clear, but may simply be a result of differences in sample preparation. Another possibility may be variability between cell preparations, as we observed donor-to-donor variability in the timing and/or changes of oxidase protein expression and activity. However, regardless of this variability between donors, TNF-{alpha} treatment consistently resulted in the maintenance of p67phox, p47phox, and gp91phox protein levels and oxidase activity, and this response was blocked completely by NF-{kappa}B pathway inhibitors. Thus, the maintenance of p67phox, p47phox, and gp91phox protein levels in TNF-{alpha}-treated monocytes is mediated through the NF-{kappa}B pathway, demonstrating the physiological relevance of this pathway in transcriptional regulation of oxidase gene expression and subsequent enhanced O2•– production.

Chronic inflammatory diseases, such as atherosclerosis and rheumatoid arthritis, are characterized by the migration of blood-derived inflammatory cells into the target tissue, where they generate ROS and other inflammatory products [7 ]. It is also well established that NF-{kappa}B can be activated by NADPH oxidase through ROS intermediates [20 , 59 60 61 ]. This study provides evidence that transcriptional regulation of three essential NADPH oxidase components in response to TNF-{alpha} is mediated through the NF-{kappa}B pathway. This novel finding, as well as our previous study [18 ], provides further evidence to support a positive-feedback model, whereby NF-{kappa}B activation, as a result of TNF-{alpha} produced by activated macrophages, could lead to up-regulation of NADPH oxidase expression and subsequent O2•– production, which in turn, could further activate NF-{kappa}B in the same cells (autocrine) and neighboring phagocytes (paracrine). As a consequence, this positive-feedback loop could result in sustained production of O2•– and contribute to the pathogenesis of chronic inflammatory diseases. Given the intricate relationship between NF-{kappa}B and ROS in chronic inflammation, establishing the pathways that regulate the NADPH oxidase activity may eventually help to identify critical events associated with the pathogenesis of chronic inflammation.


   ACKNOWLEDGEMENTS
 
This work was supported in part by National Institutes of Health grants AR42426 and RR020185, U.S. Department of Agriculture National Research Initiative/Competitive Grant Proposal grant 2006-01690, an Equipment grant from the M. J. Murdock Charitable Trust, and the Montana State University Agricultural Experimental Station. K. A. G. is the recipient of an American Heart Association Scientist Development grant. We thank Dr. Michio Nakamura (Nagasaki University, Nagasaki, Japan) for the kind gift of mAb 7D5; Drs. Lee-Ann Allen and Grant Schulert (University of Iowa and VA Medical Center, Iowa City, IA, USA) for advice on monocyte preparation; and Dr. Richard Ye (Department of Pharmacology, University of Illinois, Chicago, IL, USA) for advice in using dominant-negative expression plasmids.

Received December 15, 2006; revised May 2, 2007; accepted May 4, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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