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The Journal of Immunology, 2004, 172: 2522-2529.
Copyright © 2004 by The American Association of Immunologists

Involvement of Reactive Oxygen Species in Toll-Like Receptor 4-Dependent Activation of NF-{kappa}B 1

Karim Asehnoune*,{dagger}, Derek Strassheim*, Sanchayita Mitra*, Jae Yeol Kim* and Edward Abraham2,*

* Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, Denver, CO 80262; and {dagger} Service d’ Anesthésie-Reanimation et Unité Propre de Recherche de l’ Enseignement Superieur-Equipe d’ Accueil, Hôpital de Bicêtre, Le Kremlin Bicetre, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although oxidative stress has been thought to play a general role in the activation of NF-{kappa}B, the involvement of reactive oxygen species (ROS) in facilitating nuclear translocation of NF-{kappa}B in neutrophils has not been described. In addition, the mechanisms by which ROS modulate the transcriptional activity of NF-{kappa}B in response to Toll-like receptor 4 (TLR4)-dependent signaling are not well characterized. To examine these issues, oxidant-dependent signaling events downstream of TLR4 were investigated in neutrophils stimulated with LPS. Pretreatment of neutrophils with the antioxidants N-acetylcysteine or {alpha}-tocopherol prevented LPS-induced nuclear translocation of NF-{kappa}B. Antioxidant treatment of LPS-stimulated neutrophils also inhibited the production of proinflammatory cytokines (TNF-{alpha}, macrophage inflammatory protein-2, and IL-1{beta}), as well as activation of the kinases I{kappa}B kinase {alpha}, I{kappa}B kinase {beta}, p38, Akt, and extracellular receptor-activated kinases 1 and 2. The decrease in cytoplasmic levels of I{kappa}B{alpha} produced by exposure of neutrophils to LPS was prevented by N-acetylcysteine or {alpha}-tocopherol. Activation of IL-1R-associated kinase-1 (IRAK-1) and IRAK-4 in response to LPS stimulation was inhibited by antioxidants. These results demonstrate that proximal events in TLR4 signaling, at or antecedent to IRAK-1 and IRAK-4 activation, are oxidant dependent and indicate that ROS can modulate NF-{kappa}B-dependent transcription through their involvement in early TLR4-mediated cellular responses.


   Introduction
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 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
To protect themselves from oxidative challenge, cells have developed defense mechanisms that ensure a proper balance between pro- and antioxidant molecules (1). Under normal physiological conditions, homeostatic balance exists between the formation of reactive oxygen species (ROS) 3 and their removal by endogenous antioxidant scavenging compounds (2). The intracellular redox (reduction-oxidation) state is important physiologically in terms of maintaining cellular homeostasis and is vital for proper cellular functions. Oxidative stress occurs when this balance is disrupted by excessive generation of ROS. ROS are commonly produced during inflammatory processes, are involved in signal transduction and gene activation, and can contribute to host cell and organ damage (3).

The transcriptional factor NF-{kappa}B is involved in regulating the expression of cytokines and other mediators that participate in acute inflammatory responses, many of which are associated with increased generation of ROS (4). Association of NF-{kappa}B with I{kappa}B{alpha} in the cytoplasm blocks the nuclear localization sequence of NF-{kappa}B, inhibiting its movement into the nucleus (5). Exposure of cells to inflammatory stimuli, including LPS and proinflammatory cytokines (such as TNF-{alpha} or IL-1{beta}), results in phosphorylation of I{kappa}B{alpha} on serines 32 and 36, leading to its subsequent ubiquitinylation and degradation by the 26S proteosome. Phosphorylation of I{kappa}B{alpha} is mediated by the kinases I{kappa}B kinase {alpha} (IKK{alpha}) and IKK{beta}, which are catalytically active components of the IKK complex (6).

Several lines of evidence indicate that the redox status of cells participates in modulating NF-{kappa}B activation (7). For example, many of the agents that activate NF-{kappa}B result in increased intracellular formation of ROS (8). Nuclear translocation of NF-{kappa}B can be triggered by exposure to H2O2 (7, 9). A number of reports have shown that a broad range of antioxidants abolish NF-{kappa}B activation (10, 11, 12, 13, 14, 15). Such observations indicate that NF-{kappa}B activation is facilitated by conditions associated with increased intracellular oxidative stress (16). However, the target molecules subject to redox regulation that then lead to enhanced NF-{kappa}B activation have not been well characterized.

Neutrophils play a major role in causing tissue injury in acute inflammatory conditions. For example, the early stages of LPS-induced acute inflammatory lung injury are neutrophil dependent (17). In addition to producing ROS, neutrophils express proinflammatory mediators, such as TNF-{alpha} and macrophage inflammatory protein-2 (MIP-2), under the regulatory control of NF-{kappa}B (18, 19). Exposure of neutrophils to bacterial products, such as LPS, leads to enhanced nuclear translocation of NF-{kappa}B and production of proinflammatory cytokines (19). Although antioxidants have been shown to inhibit LPS-induced nuclear accumulation of NF-{kappa}B and production of proinflammatory cytokines by macrophages and other cell populations (11, 20), the role of ROS in modulating neutrophil responses initiated by exposure to LPS or other mediators capable of producing neutrophil activation has not been investigated. Additionally, intracellular signaling events that might be affected by LPS-induced production of ROS have not been delineated in neutrophils. In the present experiments we explored these issues and found that ROS were involved in enhancing nuclear translocation of NF-{kappa}B in LPS-stimulated neutrophils. IL-1R-associated kinase-1 (IRAK-1) and IRAK-4 activation, an early Toll-like receptor 4 (TLR4)-associated event, was shown to be oxidant dependent, demonstrating that ROS can affect NF-{kappa}B-dependent transcription at proximal steps in LPS-induced cellular responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

Male BALB/c mice, 8–12 wk of age, were purchased from Harlan Sprague Dawley (Indianapolis, IN). The mice were kept on a 12-h light, 12-h dark cycle with free access to food and water. All experiments were conducted in accordance with institutional review board-approved protocols.

Reagents and Abs

Isoflurane was obtained from Abbott Laboratories (Chicago, IL). Escherichia coli 0111:B4 endotoxin (LPS), myelin basic protein (MBP), {alpha}-tocopherol ({alpha}TOC), and N-acetylcysteine (NAC) were purchased from Sigma-Aldrich (St. Louis, MO). RPMI 1640/25 mM HEPES/L-glutamine was obtained from BioWhittaker (Walkersville, MD), FCS and penicillin/streptomycin were purchased from Gemini Bioproducts (Calabasas, CA). Bicinchoninic acid protein assay reagent was purchased from Pierce (Rockford, IL). Activation-specific Abs for phospho-Thr202/Tyr204 extracellular receptor-activated kinase 1 (ERK1), phospho-Thr183/Tyr185 ERK2, phospho-Thr180/Tyr182 p38, phospho-Ser473 Akt, phospho-Ser180/Ser181 IKK{alpha}/{beta}, and total ERK1/2, p38, Akt, and I{kappa}B{alpha} were purchased from Cell Signaling Technologies (Beverly, MA). Protein A/G plus agarose, polyclonal goat anti-IRAK-1 Ab, and I{kappa}B{alpha} recombinant protein were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal rabbit anti-IRAK-4 was purchased from Imgenex (San Diego, CA). HRP-labeled anti-rabbit Abs, and ECL reagents were purchased from Bio-Rad (Hercules, CA). All other reagents were purchased from Sigma-Aldrich unless otherwise noted in the text. Custom-mixed Abs and columns for neutrophil isolation were purchased from StemCell Technologies (Vancouver, British Columbia, Canada).

Isolation and culture of bone marrow-derived mouse neutrophils

Bone marrow neutrophils were isolated as described previously (21). To obtain the bone marrow cell suspension, the femur and tibia of a mouse were flushed with RPMI 1640. Tissue fragments were removed by rapid filtration through a glass-wool column, and cells were collected by centrifugation. The cell pellets were resuspended in RPMI 1640/2% FCS and then incubated with primary Abs specific for cell surface markers F4/80, CD4, CD45R, CD5, and TER119 for 15 min at 4°C. This custom mixture (Stem Cell Technologies) is specific for T and B cells, RBC, monocytes, and macrophages. After 15-min incubation, 100 µl of anti-biotin tetrameric Ab complexes were added, and the cells were incubated for 15 min at 4°C. After this, 60 µl of colloidal magnetic dextran iron particles were added to the suspension and incubated for 15 min at 4°C. The entire cell suspension was then placed into a column surrounded by a magnet. The T cells, B cells, RBC, monocytes, and macrophages were captured in the column, allowing the neutrophils to pass through by negative selection methods. Viability, as determined by trypan blue exclusion, was consistently >98%. Neutrophil purity, as determined by Wright’s-stained cytospin preparations, was >97%. Less than 0.3% of the purified cell population consisted of mononuclear cells. Bone marrow neutrophils (2 x 106/0.5 ml) were cultured in RPMI 1640/0.1% FCS with or without drugs as described in figure legends. LPS, NAC, and {alpha}TOC were made fresh for each experiment. In each experiment {alpha}TOC and NAC were added to the cell cultures 15 min before stimulation with LPS.

Cytokine ELISA

Immunoreactive TNF-{alpha}, IL-1{beta}, and MIP-2 were quantitated using commercially available ELISA kits (R&D Systems, Minneapolis, MN), according to the manufacturer’s instructions and as described previously (21).

EMSA

Nuclear extracts were prepared and assayed by EMSA as previously described (21). For analysis of NF-{kappa}B, the {kappa}B DNA sequence of the Ig gene was used. Synthetic double-stranded sequences (with enhancer motifs underlined) were filled in and labeled with [{alpha}-32P]dATP using Sequenase DNA polymerase as follows: {kappa}B, 5'-TTTTCGAGCTCGGGACTTTCCGAGC-3'and 3'-GCTCGAGCCCTGAAAGGCTCGTTTT-5'.

Western blot analysis

Western blots for phosphorylated and total kinases were performed as described previously (21). Parallel samples for total protein kinase were run with samples for activation-specific phosphorylation analysis. Densitometry was performed using chemiluminescence system and analysis software (Bio-Rad) to determine the ratio between phosphorylated and total kinase. Densitometry data for each kinase are shown in graphic form, with baseline phosphorylation levels before LPS stimulation (i.e., at 0 min) assigned a value of 100%.

Kinase assays

IKK. Cells were washed in PBS, then placed in lysis buffer (Cell Signaling Technologies), and rocked for 30 min on ice. After cell extracts were centrifuged at 14,000 x g for 15 min at 4°C, the protein concentration of the supernatant was determined by bicinchoninic acid protein assay, and 500 µg of protein was incubated overnight at 4°C in 300 µl of cell lysis buffer and protease inhibitor mixture from Sigma-Aldrich (P-83-40) with 1.5 µg of mouse anti-IKK{alpha} or anti-IKK{beta} mAbs (Cell Signaling Technologies). Fifty microliters of 50% protein A-Sepharose was then added to each sample and incubated for 2 h at 4°C. The samples were centrifuged, and the beads were washed twice with lysis buffer (Cell Signaling Technologies) and twice with kinase buffer (Cell Signaling Technologies). After this, the beads were incubated at 30°C in a final volume of 20 µl of kinase buffer with 1 µg of I{kappa}B{alpha} expressed in E. coli as a 70-kDa tagged fusion protein (Santa Cruz Biotechnology), 100 µM ATP, and 0.5 µl of [{gamma}-32P]ATP (5 µCi/sample). SDS sample buffer was then added to the protein A beads. The samples were boiled for 5 min and resolved on 8% SDS-PAGE. The gels were dried, and the intensity of the radioactive signal was quantified using a phosphorimager and ImageQuant (Bio-Rad). To determine the total amount of IKK{alpha} and IKK{beta} in each sample, 30 µg of cytoplasmic protein was resolved on 8% SDS-PAGE, then electrotransferred to a nitrocellulose membrane that was probed with either anti-IKK{alpha} or anti-IKK{beta} Abs.

IRAK-1 and IRAK-4. Equal amounts of whole cell extracts were incubated with polyclonal goat anti-IRAK-1 or with polyclonal rabbit anti-IRAK-4 Ab for 2 h at 4°C on a rotor, after which 50 µl of 50% protein G plus agarose was added to each sample and incubated for an additional 2 h at 4°C. The samples were precipitated in a microcentrifuge, and the beads were washed twice with lysis buffer (Cell Signaling Technologies) and twice with kinase buffer (Cell Signaling Technologies). The beads were incubated at 30°C in a final volume of 20 µl of kinase buffer in the presence of MBP as a substrate (1 µg/sample), 100 µM ATP, and 0.5 µl of [{gamma}-32P]ATP (5 µCi/sample). After SDS sample buffer was added to the protein A beads, the samples were boiled for 5 min and then subjected to SDS-PAGE analysis. The gels were dried, and the intensity of the radioactive signal was quantified using a phosphorimager and ImageQuant (Bio-Rad). To determine the total and phosphorylated amounts of IRAK-1 and the total amount of IRAK-4 in each sample, equal amounts of whole cell extracts were incubated with polyclonal goat anti-IRAK-1 Ab or polyclonal rabbit anti IRAK-4 for 2 h at 4°C on a rotor, after which 50 µl of 50% protein G plus agarose was added to each sample and incubated for an additional 2 h at 4°C. The samples were precipitated in a microcentrifuge, and the beads were washed four times with lysis buffer (Cell Signaling Technologies). The samples were boiled for 5 min, resolved on 8% SDS-PAGE, and electrotransferred to a nitrocellulose membrane that was then probed with anti-IRAK-1 or with anti-IRAK-4 Abs.

Statistical analysis

Values are expressed as the mean ± SEM. Data were analyzed by ANOVA, and differences between means were determined using the Bonferroni multiple comparison test. Significance is defined as p < 0.05.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Nuclear translocation of NF-{kappa}B is oxidant dependent in LPS-stimulated neutrophils

The time course for LPS-mediated nuclear accumulation of NF-{kappa}B in neutrophils is shown in Fig. 1A. Increased concentrations of NF-{kappa}B in the nucleus were present within 20 min of LPS exposure, and nuclear translocation of NF-{kappa}B was maximal 40 min after addition of LPS to the cultures. Pretreatment of neutrophils with NAC or {alpha}TOC inhibited LPS induced NF-{kappa}B activation (Fig. 1, B and C).



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  		FIGURE 1. Antioxidants inhibit nuclear translocation of NF-{kappa}B in LPS-stimulated neutrophils. Culture of neutrophils with LPS (1 µg/ml) induces nuclear accumulation of NF-{kappa}B (A), that is inhibited by pretreatment with the antioxidants {alpha}TOC (50 µM) and NAC (30 mM; B). B, Neutrophils were incubated with {alpha}TOC or NAC or were left untreated (LPS) for 15 min before LPS was added to the cultures. Neutrophils not treated with antioxidants or LPS were included as controls (C). Nuclear extracts were obtained after 40 min of LPS stimulation. A representative experiment is shown in B. Two additional experiments produced similar results. Phosphorimaging data from the three independent studies are shown in C, presented as the mean ± SE. **, p < 0.01 vs neutrophils cultured with LPS alone.

 
Antioxidants inhibit LPS-induced proinflammatory cytokine secretion by neutrophils

To assess the role of ROS in LPS-induced cellular activation, neutrophils were incubated with LPS and with varying concentrations of NAC or {alpha}TOC. As expected, concentrations of TNF-{alpha}, IL-1{beta}, and MIP-2 were increased in supernatants of neutrophils stimulated with LPS (Fig. 2). The release of TNF-{alpha} and MIP-2 from LPS-stimulated neutrophils was inhibited in a dose-dependent manner by NAC and {alpha}TOC (Fig. 2A). Pretreatment of neutrophils with {alpha}TOC, but not NAC, also prevented LPS-induced secretion of IL-1{beta} (Fig. 2B).



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  		FIGURE 2. Treatment of LPS-stimulated neutrophils with {alpha}TOC or NAC inhibits proinflammatory cytokine secretion. Neutrophils were incubated with control medium or with the indicated concentrations of {alpha}TOC (A) or NAC (B) for 15 min, then cultured with or without LPS (1 µg/ml). After 90 min the supernatants were collected and assayed for concentrations of TNF-{alpha}, MIP-2, and IL-1{beta}. Combined data are presented from three independent experiments. *, p < 0.05; **, p < 0.01 (vs neutrophils cultured with LPS alone).

 
NAC and {alpha}TOC inhibit IKK activation and stabilize I{kappa}B{alpha} in LPS-stimulated neutrophils

A potential mechanism for the inhibitory effect that antioxidants exert on LPS-induced nuclear accumulation of NF-{kappa}B could be through blocking activation of the IKK complex. IKK-mediated phosphorylation of I{kappa}B-{alpha} on serines 32 and 36 leads to subsequent ubiquitinylation and degradation of I{kappa}B-{alpha}, with removal of its tonic inhibitory effects on nuclear translocation of NF-{kappa}B (6).

Activation of IKK was initially assessed by determining phosphorylation of serine 180 in IKK{alpha}, and serine 181 in IKK{beta}. As shown in Fig. 3A, stimulation of neutrophils with LPS resulted in activation of IKK that peaked between 15 and 30 min. Neutrophil pretreatment with NAC and {alpha}TOC inhibited LPS-induced IKK phosphorylation. To confirm that antioxidants also affected functional, downstream effects of IKK activation, we examined cytoplasmic levels of I{kappa}B{alpha} in neutrophils treated with LPS alone or with LPS plus NAC or {alpha}TOC. In neutrophils challenged with LPS, I{kappa}B{alpha} underwent time-dependent degradation (Fig. 3B). However, in neutrophils incubated with LPS and either antioxidant, no degradation of I{kappa}B{alpha} was apparent.



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  		FIGURE 3. Effects of antioxidants on LPS-induced IKK phosphorylation and I{kappa}B{alpha} degradation. Neutrophils were pretreated with {alpha}TOC (50 µM) or NAC (30 mM) or were left untreated (LPS) for 15 min, then cultured with LPS (1 µg/ml). At the indicated times after addition of LPS, the reactions were terminated by addition of SDS-PAGE sample buffer. Cell lysates were subjected to Western blotting using Abs specific for Ser180/Ser181 IKK{alpha}/{beta} (A) or total I{kappa}B{alpha} (B). Densitometry data are shown in graphic form, with baseline phosphorylation of Ser180/Ser181 IKK and total I{kappa}B{alpha} levels before LPS stimulation (i.e., 0 min) assigned a value of 100%. The data shown are representative of three independent experiments.

 
To more completely determine the effects of antioxidants on IKK activation, we directly measured the kinase activity of IKK{alpha} and IKK{beta}. Stimulation of neutrophils with LPS primarily resulted in increased activation of IKK{beta} (Fig. 4A), although late increases in IKK{alpha} activity also appeared to be present (Fig. 4B). Pretreatment of neutrophils with antioxidants abolished LPS-induced activation of IKK{beta} (Fig. 4A) and prevented the increased activity of IKK{alpha} found in LPS-stimulated neutrophils (Fig. 4B).



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  		FIGURE 4. Antioxidants prevent LPS-induced increases in IKK{alpha} and IKK{beta} kinase activities. Neutrophils were pretreated with {alpha}TOC (50 µM) or NAC (30 mM) or were left untreated for 15 min, then cultured with LPS (1 µg/ml). At the indicated times after addition of LPS, cellular extracts were obtained, and IKK{alpha} or IKK{beta} was immunoprecipitated using specific Abs. IKK{alpha} and IKK{beta} kinase activities were determined in vitro in the presence of [{gamma}-32P]ATP using I{kappa}B{alpha} as a substrate. Total protein levels of IKK{alpha} and IKK{beta} were analyzed by Western blotting. Radioactive I{kappa}B{alpha} was quantified using a phosphorimager and ImageQuant (Bio-Rad). Representative experiments are shown for IKK{beta} (A) and IKK{alpha} (B) kinase activities. In the figures, densitometry data are shown in graphic form, with the kinase activity of IKK{alpha} or IKK{beta} at baseline, before LPS addition to cultures (i.e., 0 min) given a value of 100%. The data shown are representative of three independent experiments.

 
Role of oxidants on LPS-induced activation of p38, ERK, and Akt in neutrophils

Exposure of neutrophils to LPS has been shown to activate mitogen-activated protein kinases (MAPK), including p38 (22), ERK1/2 (23), and Akt (21), all of which are implicated in enhancing nuclear translocation of NF-{kappa}B (4). To examine the role of ROS in modulating the activation of these kinases, we determined levels of phosphorylated kinases in LPS-stimulated neutrophils and in those pretreated with NAC or {alpha}TOC. Antioxidant treatment prevented LPS-induced activation of p38 and Akt, and strongly inhibited that of ERK1/2 (Fig. 5).



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  		FIGURE 5. Effects of {alpha}TOC and NAC on phosphorylation of p38, Akt, and ERK in LPS-stimulated neutrophils. Neutrophils were pretreated with {alpha}TOC (50 µM) or NAC (30 mM) or were left untreated (LPS) for 15 min, then cultured with LPS (1 µg/ml). At the indicated times after the addition of LPS, cellular extracts were obtained, and levels of phosphorylated (p-p38, p-Akt, p-ERK) and total kinases were determined. Representative gels are shown. Densitometry data are presented, with the baseline kinase phosphorylation before LPS addition to cultures (i.e., 0 min) given a value of 100%. The data shown are representative of three independent experiments.

 
LPS-induced ROS increase IRAK-1 and IRAK-4 activity

The above experiments showed that ROS were involved in TLR4-associated nuclear translocation of NF-{kappa}B and activation of kinases, including IKK, p38, ERK 1/2, and Akt. Because IKK, p38, ERK1/2, and Akt have previously been shown to be activated in neutrophils cultured with LPS as well as other stimuli (6, 21, 23, 24, 25), our experiments did not identify an oxidant-dependent step that could be specifically affected through TLR4-associated mechanisms. To address this issue, we examined the effects of antioxidant treatment on LPS-induced IRAK-1 and IRAK-4 activation. IRAK-1 and IRAK-4 are rapidly recruited to the TLR/IL-1R complex after ligand occupancy (26, 27).

Kinase activity of IRAK-1 (Fig. 6A) and IRAK-4 (Fig. 6B) increased in neutrophils stimulated with LPS. Both phosphorylation and activation of IRAK-1 were diminished in neutrophils treated with antioxidants before LPS exposure (Fig. 6A). Similarly, as shown in Fig. 6B, activation of IRAK-4 was diminished in neutrophils exposed to antioxidants before stimulation with LPS.



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  		FIGURE 6. Antioxidants inhibit LPS-induced IRAK-1 and IRAK-4 kinase activities in LPS-stimulated neutrophils. Neutrophils were pretreated with {alpha}TOC (50 µM) or NAC (30 mM) or were left untreated (LPS) for 15 min, then cultured with LPS (1 µg/ml). At the indicated times after addition of LPS, cellular extracts were obtained. Phosphorylated and total protein levels of IRAK-1 (A) as well as total protein levels of IRAK-4 (B) were analyzed by Western blotting. The kinase activities of IRAK-1 and IRAK-4 were determined after immunoprecipitation from the cellular extracts using IRAK-1- or IRAK-4-specific Abs, followed by in vitro incubation in the presence of [{gamma}-32P]ATP using MBP as substrate. Representative experiments are shown for IRAK-1 (A) and IRAK-4 (B) kinase activities. Quantitation of {gamma}-32P-labeled MBP is presented, with the baseline IRAK-1 or IRAK-4 kinase activity before LPS addition to cultures (i.e., 0 min) given a value of 100%. The data shown are representative of three independent experiments.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
A number of studies have suggested that oxidative stress is involved in the activation of NF-{kappa}B (7, 9, 28). Evidence linking ROS to NF-{kappa}B comes from complementary observations demonstrating that nuclear translocation of NF-{kappa}B can be enhanced by exposure of cells to ROS, and cytokine-induced activation of NF-{kappa}B can be prevented by antioxidants (7, 9). However, despite these findings, the assumption that oxidants play a general role in the activation of NF-{kappa}B has been the subject of considerable debate (9, 29). In part, this controversy arises from data showing that exogenous ROS do not activate NF-{kappa}B in all cell types (7). Even in a single cell type, variability in the ability of ROS to modulate accumulation of NF-{kappa}B in the nucleus has been demonstrated (30). Neutrophils have only recently been shown to be transcriptionally active (31), and the role of ROS in affecting NF-{kappa}B activity in this cell population has not previously been examined.

The antioxidants used in these experiments have differing mechanisms of action, strengthening conclusions that ROS are involved in TLR4 mediated NF-{kappa}B activation in neutrophils. NAC exerts antioxidant activity through two mechanisms, by preventing the formation of ROS and also through scavenging ROS once formed (20). Direct effects of NAC in blocking ROS generation were shown by its ability to decrease PMA-induced H2O2 production in human neutrophils (32). NAC also acts by increasing intracellular content of glutathione, altering redox balance and inhibiting the formation of ROS (33). In contrast, {alpha}TOC, being lipid soluble, is associated with the hydrophobic lipid interior of cellular membranes and reacts with ROS that are formed in or gain access to this environment (20). {alpha}TOC prevents membrane polyunsaturated fatty acids from undergoing lipid peroxidation, which leads to loss of membrane integrity (34). Of note, the doses of antioxidants that were effective in inhibiting LPS-induced cytokine production in the present experiments are similar to those previously reported to be effective in other cell types (11, 35, 36).

Although ROS can activate signaling pathways leading to NF-{kappa}B activation (8), the specific intracellular events affected by oxidant stress have not been completely defined. Kinases shown to be activated by H2O2 include Syk, Akt, p38, ERK, and the IKK complex (37, 38, 39). It was recently demonstrated in a leukemia cell line that H2O2 induces NF-{kappa}B activation through Syk-mediated phosphorylation of I{kappa}B{alpha} (39). Although H2O2 has been shown to activate p38 MAPK (40, 41) and ERK1/2 (42, 43), its effect on these kinases is controversial, as some reports have found inhibition (44, 45). Akt has been demonstrated to participate in NF-{kappa}B activation at least in part through its effects in phosphorylating IKK{alpha} (46). H2O2 activates Akt in several cell types (47, 48, 49). It has been hypothesized that these effects of H2O2 on Akt were indirect, being mediated through oxidant-induced activation of p38 MAPK, which then subsequently phosphorylated and activated Akt (50).

Although the presumption has been that oxidants directly activate kinases, it is also possible their effects may be indirect and result from inactivation of pathways that are normally inhibitory for kinase activity. One of the principle means by which ROS affect signal transduction is through oxidation of susceptible cysteine residues to cysteine sulfonic acid or disulfide, a step reversed by cellular reductants and antioxidants (51). The protein tyrosine phosphatase group is particularly susceptible to H2O2-induced inactivation (52). Such inhibition of tyrosine phosphatases by H2O2 results in transient activation of a number of protein kinase pathways that are normally tonically inhibited by protein tyrosine phosphatase activity. In particular, ERK1/2 (53) and Akt (49) are among the kinases whose activity has been shown to be increased as a result of such H2O2-induced inhibition of protein tyrosine phosphatases (53).

In the present experiments we found that exposure of neutrophils to LPS resulted in activation of the IKK{beta} to a greater extent than IKK{alpha}. Gene targeting studies have demonstrated that IKK{beta} is the IKK subunit required for NF-{kappa}B activation induced by proinflammatory stimuli, such as LPS (54). IKK{alpha} deletion has no effect on NF-{kappa}B activation by LPS and most other agents, with the exception of lymphotoxin-{beta} (39). In these experiments antioxidants inhibited LPS-induced activation of both IKK{beta} and IKK{alpha}. A previous study using HeLa cells showed that NAC suppressed NF-{kappa}B activation through inhibition of IKK, but did not examine upstream kinases (55). As expected, we found that inhibition of IKK activation by antioxidants resulted in preservation of levels of I{kappa}B{alpha}, providing a mechanism for the observed reduction in translocation of NF-{kappa}B to the nucleus and suppression of the expression of NF-{kappa}B-dependent cytokines under such conditions.

We (21, 24) and others (22, 23, 25) have demonstrated that stimulation of neutrophils with LPS results in activation of ERK1/2, Akt, and p38. As noted above, previous studies (40, 41, 42, 43, 44, 45) have shown varying effects of ROS on the specific activity of p38 and ERK1/2. In the present experiments antioxidants blocked phosphorylation of ERK1/2, Akt, and p38, as well as activation of IKK{beta}, suggesting that the oxidant-responsive step in LPS-induced signaling was upstream of these kinases.

The ability of antioxidants to block IRAK-1 and IRAK-4 activation after exposure of neutrophils to LPS suggests a common oxidant-dependent mechanism for the activation of p38, ERK1/2, Akt, and IKK{beta}. IRAKs play a crucial role in the signaling pathways initiated by members of the TLR/IL-1R family (27, 56). Neither IL-1R nor TLR has intrinsic kinase activity, so cellular activation is dependent on the assembly of kinases and scaffolding proteins about the receptor. Interaction of TLR4 with LPS results in the rapid recruitment of IRAK-1 to the receptor complex, which includes upstream components, including MyD88 and IRAK-4. MyD88 facilitates the interaction between IRAK-1 and IRAK-4, resulting in the phosphorylation and activation of IRAK-1 by IRAK-4 (57, 58, 59). Of note, one previous study showed that recruitment of IRAK-1 to the IL-1R in a T cell line was regulated by intracellular oxidant balance (60). Once activated, IRAK-1 can recruit and activate TNFR-associated factor 6, producing activation of the TGF-{beta}-activated kinase 1 (TAK1). TAK1 is a MAPK kinase kinase able to phosphorylate IKK{alpha} and IKK{beta} as well as c-Jun N-terminal kinase and p38 MAPKs. In addition to its effects on TAK1, TNFR-associated factor 6 participates in additional downstream pathways, leading to the activation of ERK1/2 and protein kinase C{zeta}. Therefore, because of its proximal position in TLR4-dependent signaling, activation of IRAK-4 appears to be an appropriate candidate for the oxidant-dependent step that the present experiments identified as being present in LPS-dependent neutrophil signaling.


   Footnotes
 
1 This work was supported by grants from Ministère Français des Affaires Étrangères (Program LAVOISIER), Société Française d’Anesthésie-Réanimation (to K.A.), and National Institutes of Health Grants HL62221 and PO1HL68743 to (E.A.). Back

2 Address correspondence and reprint requests to Dr. Edward Abraham, Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, Box C272, 4200 East 9th Ave, Denver, CO 80262. E-mail address: edward.abraham{at}uchsc.edu Back

3 Abbreviations used in this paper: ROS, reactive oxygen species; ERK1/2, extracellular receptor-activated kinases 1 and 2; IKK, I{kappa}B kinase; IRAK, IL-1R-associated kinase; MAPK, mitogen-activated protein kinase; MBP, myelin basic protein; MIP-2, macrophage inflammatory protein-2; NAC, N-acetylcysteine; p38, p38 mitogen-activated protein kinase; PI3K/Akt, phosphatidylinositol 3-kinase/Akt; TAK, TGF-{beta}-activated kinase; TLR, Toll-like receptor; {alpha}TOC, {alpha}-tocopherol. Back

Received for publication August 7, 2003. Accepted for publication November 26, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Forman, H. J., M. Torres. 2001. Redox signaling in macrophages. Mol Aspects Med. 22:189.[Medline]
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