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Am J Physiol Cell Physiol 293: C255-C266, 2007. First published March 28, 2007; doi:10.1152/ajpcell.00618.2006
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RECEPTORS AND SIGNAL TRANSDUCTION

Exposure to hydrogen peroxide diminishes NF-{kappa}B activation, I{kappa}B-{alpha} degradation, and proteasome activity in neutrophils

Jaroslaw W. Zmijewski,1,2 Xia Zhao,1 Zhiwei Xu,1 and Edward Abraham1

1Department of Medicine, 2Center for Free Radical Biology, University of Alabama, Birmingham, Alabama

Submitted 12 December 2006 ; accepted in final form 22 March 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Although ROS can participate in modulating the activity of the transcriptional factor NF-{kappa}B and expression of NF-{kappa}B-dependent genes, the mechanisms involved and the roles of specific ROS have not been fully determined. In particular, individual ROS appear to have differing effects on NF-{kappa}B activation dependent on the cell population studied. In the present study, we examined the ability of H2O2 to affect NF-{kappa}B activation in LPS-stimulated murine neutrophils and macrophages. Exposure of bone marrow or peritoneal neutrophils to H2O2 was associated with reduced nuclear translocation of NF-{kappa}B and decreased production of the NF-{kappa}B-dependent cytokines TNF-{alpha} and macrophage inhibitory protein-2. H2O2 treatment resulted in diminished trypsin- and chymotrypsin-like proteasome activity. The degradation of I{kappa}B-{alpha} normally found in LPS-treated neutrophils was prevented when H2O2 was added to cell cultures. In contrast to the effects found in neutrophils, H2O2 did not affect chymotrypsin-like proteasomal activity or cytokine production in LPS-stimulated macrophages, even though trypsin-like proteasomal activity was reduced. These results demonstrate that the effects of H2O2 on NF-{kappa}B and proteasomal activity are cell population specific.

reactive oxygen species; signal transduction; nuclear factor-{kappa}B

NEUTROPHILS PLAY FUNDAMENTAL ROLES in host defense responses directed toward eradication of bacteria and other microorganisms. However, neutrophils also contribute to deleterious acute inflammatory processes, including organ system dysfunction associated with sepsis, hemorrhagic shock, ischemia-reperfusion injury, and myocardial infarction (1, 11, 54). Neutrophils can be activated through the engagement of membrane-expressed Toll-like receptors (TLRs) by microbial products such as LPS. Interactions between LPS and TLR4 results in interactions between the intracellular Toll/IL-1 receptor domain with kinases and scaffolding proteins, including IL-1 receptor-associated kinase 1, TNF receptor-associated factor 6, and MyD88, leading to activation of the I{kappa}B kinase (IKK) complex, phosphorylation, ubiquitination, and degradation of I{kappa}B-{alpha} and translocation of the transcription factor NF-{kappa}B to the nucleus, where it enhances the expression of immunoregulatory molecules, including proinflammatory cytokines such as TNF-{alpha}, macrophage inhibitory protein (MIP)-2, and IL-8 (14, 42, 53, 59).

ROS (21), including superoxide and H2O2, are generated under normal metabolic conditions through cellular respiration and released in increased amounts during acute inflammatory processes (8, 15, 29). While many ROS, such as superoxide or hydroxyl radical, cross cell membranes poorly, H2O2 diffuses easily between extra- and intracellular environments. Increased release of H2O2 into the extracellular milieu, such as occurs during acute inflammatory processes in which neutrophils participate, can therefore affect oxidant-dependent intracellular processes of adjacent cellular populations.

Because H2O2 can reversibly modify intracellular targets, it is capable of modulating signaling events and cellular activation in much the same manner as traditional second messengers (19, 20, 47). H2O2 can directly participate in pathways modulating the activation of NF-{kappa}B (9, 12, 36, 44, 49, 56). While initial experiments with HeLa cells have suggested that exposure to H2O2 enhanced nuclear translocation of NF-{kappa}B, subsequent studies (24, 28, 36, 41, 55) in other cell populations found the opposite effect, with H2O2 exerting inhibitory effects on NF-{kappa}B activation. Such disparate findings suggested that the role of H2O2 in affecting pathways relating to the activation of NF-{kappa}B is likely to be cell type specific.

Proteosomal degradation of inhibitory proteins belonging to the I{kappa}B family plays a critical role in NF-{kappa}B activation (4, 6, 14, 32, 48). In particular, after phosphorylation of serine residues 32 and 36 by the activated IKK complex, I{kappa}B-{alpha} undergoes polyubiquitination and then is degraded by the 26S proteasome. Inhibition of any of these steps leads to persistence of cytoplasmic I{kappa}B-{alpha} concentrations and continued association between I{kappa}B-{alpha} and NF-{kappa}B, with consequent prevention of nuclear localization of NF-{kappa}B. While several recent studies (16, 31, 45) have indicated that ROS may be involved in the regulation of proteasomal activity, little information is available concerning the importance of interactions between ROS and proteasomal function in modulating NF-{kappa}B activation and the transcription of NF-{kappa}B-dependent genes, particularly in the clinically relevant setting of concomitant cellular stimulation, such as that induced through TLR by microbial products.

To explore potential mechanisms by which H2O2 might affect neutrophil function, we examined the effects of H2O2 on proteasomal activity, NF-{kappa}B activation, and expression of NF-{kappa}B-dependent cytokines after exposure to the TLR4 ligand LPS. We found that H2O2 inhibited I{kappa}B-{alpha} degradation, proteasomal activity, nuclear accumulation of NF-{kappa}B, and cytokine production in a dose- and time-dependent manner. These effects of H2O2 were not present in macrophages, indicating that they are cell population specific.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Reagents. Escherichia coli 0111:B4 endotoxin (LPS), H2O2, glucose oxidase (GO) from Aspergillus niger, dihydroethidium (DHE), and catalase were obtained from Sigma (St. Louis, MO). 2',7'-Dichlorodihydrofluorescein-diacetate (DCFH-DA) and dihydrorhodamine123 (DHR123) were purchased from Invitrogen (Carlsbad, CA), whereas Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), MG132, 7-amido-4-methyl-coumarin (AMC), and Suc-Leu-Leu-Val-Tyr-AMC were from Calbiochem (San Diego, CA). Boc-Leu-Arg-Arg-AMC was from BioMol (Plymouth Meeting, PA). Dimethoxy-naphthoquinone (DMNQ) and the RNAeasy kit were puchased from Qiagen (Valencia, CA), and ELISA kits were obtained from R&D Systems (Minneapolis, MN). Mouse alveolar macrophages (MH-S cells) were obtained from the American Type Culture Collection (No. CRL-2019, Manassas, VA).

Mice. Male BALB/c mice (8–12 wk of age) were purchased from Jackson Laboratory. Mice were kept on a 12:12-h light-dark cycle with free access to food and water. All experiments were conducted in accordance with institutional review board-approved protocols.

Cell culture. Mouse alveolar macrophages (MH-S cells) were maintained (at 37°C in 5% CO2) in RPMI-1640 growth medium (GIBCO) that contained 8% FBS (Atlanta Biologicals, Norcross, GA), L-glutamine (2 mM), penicillin (100 U/ml), and streptomycin (100 ng/ml) (Sigma). Cells were subsequently washed twice and incubated with RPMI-1640 medium (with 0.5% FBS) for 2 h and then treated as described in the figures.

Measurement of glucose/GO-generated H2O2. The concentration of H2O2 generated by glucose/GO (G/GO) in culture medium and the stability of H2O2 was determined using the xylenol orange assay (25). Briefly, GO (10 or 50 mU/ml) or H2O2 (150 µM) was incubated in 1 ml PBS or culture media (RPMI-1640) containing 20 mM glucose and 0.5% FBS for 0, 10, 20, and 30 min at 37°C. Samples (30 µl) and H2O2 standards (0–160 µM H2O2) were incubated with 270 µl methanol-xylenol orange solution [100 µM xylenol orange, 4.4 mM butylated hydroxyl toluene, 250 µM Fe(NH4)2(SO4)2, and 25 mM H2SO4] for 30 min at 25°C. Concentrations of H2O2 were determined by measurement of the ferric-xylenol orange complex using a microplate reader (wavelength: 590 nm). The specificity of the assay for H2O2 was established by coincubation of samples with catalase (200 U/ml). H2O2 concentrations were stable during 0–30 min of incubation in culture media at 37°C. The rate of H2O2 generated by G/GO in culture media was 0.35 nmol·min–1·ml–1 at 37°C.

Neutrophil isolation and culture. Bone marrow neutrophils were isolated as previously described (51, 58). Briefly, a bone marrow cell suspension was isolated from the femur and tibia of a mouse by flushing with RPMI-1640 medium containing FBS (5%). The cell suspension was passed through a glass wool column and collected by subsequent washing with PBS containing FBS (5%). Negative selection to purify neutrophils was performed by incubation of the cell suspension with primary antibodies specific for the cell surface markers F4/80, CD4, CD45R, CD5, and TER119 (StemCell Technologies) for 15 min at 4°C followed by a subsequent incubation with anti-biotin tetrameric antibody (100 µl) for 15 min and then colloidal magnetic dextran iron particles (60 µl) for an additional 15 min at 4°C. T cells, B cells, red blood cells, monocytes, and macrophages were captured in a column surrounded by a magnet, allowing the neutrophils to pass through. Neutrophil purity, as determined by Wright-Giemsa-stained cytospin preparations, was consistently >97%. Neutrophils were also isolated from the peritoneal cavity of 8- to 12-wk-old mice 4 h after an intraperitoneal injection with 2 ml of thioglycolate solution (3%). Peritoneal cell populations were consistently composed of >90% neutrophils as determined using Wright-Giemsa staining. Purified neutrophils were cultured in RPMI-1640 medium containing FBS (0.5%) and treated as described in the figures. Neutrophil viability was determined using trypan blue staining and was consistently >99%.

Imaging of DCF, DHR123, and DHE fluorescence. Intracellular levels of H2O2 were measured using the redox-sensitive probes DCFH-DA and DHR123 in conjunction with fluorescent microscopy (57, 61). Briefly, neutrophils (1.5 x 106 cells/well) were incubated in a four-well chambered coverglass (Nalge, Naperville, IL) with DCFH-DA (5 µM) or DHR123 (5 µM) for 20 min, followed by treatment with various concentrations of H2O2 or G/GO for 60 or 120 min, respectively, at 37°C. Cells were also incubated with catalase (600 U/ml) prior to the H2O2 exposure as indicated. To determine the effect of G/GO or bolus H2O2 on superoxide generation, neutrophils were loaded with DHE (10 µM) for 20 min followed by an exposure to G/GO or H2O2 as indicated. The superoxide generator DMNQ (10 µM) was used alone or with the superoxide scavenger MnTBAP (250 µM) as an additional control. Images of DCF, DHR123, or DHE were acquired by double bidirectional scans of live neutrophils using a Leica DMIRBE inverted epifluorescence/Nomarski microscope outfitted with Leica TCS NT laser confocal optics. The pinhole setting was 0.2 Airy units, and laser excitation was set for 5% to avoid dye photooxidation. The levels of fluorescence were averaged using SimplePCI software (Compix, Cranberry Township, PA). Images were processed using IPLab Spectrum and Adobe Photoshop (Adobe Systems) software.

Purification of nuclear proteins. Nuclear proteins were purified from 7.5 x 106 neutrophils lysed in 200 µl buffer containing 10 mM Tris (pH 7.5), 10 mM NaCl, 3 mM MgCl2, 0.02% Nonidet P-40 (NP-40), 1 mM EGTA, 1 mM sodium orthovanadate, 50 mM sodium fluoride, and protease inhibitors (100 µM PMSF, 10 µg/µl leupeptin, 10 µg/µl aprotinin, 5 µg/µl pepstatin A, and 1 nM okadaic acid). Lysed cells were collected in microcentrifuge tubes and centrifuged at 2,700 g for 10 min at 4°C. The supernatant containing the cytosol was centrifuged at 20,800 g for 15 min at 4°C to obtain the cytosolic fraction. The pellet containing nuclei was washed three times by a gentle resuspension in 200 µl wash buffer [10 mM PIPES (pH 6.8), 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 25 mM NaCl, 1 mM sodium orthovanadate, 50 mM sodium fluoride, and protease inhibitors] and centrifuged at 2,700 g for 5 min at 4°C. Nuclear proteins were extracted using buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 0.5% (vol/vol) NP-40, 1 mM EDTA, 1 mM EGTA, 1 nM okadaic acid, and protease inhibitors. Nuclear lysates were sonicated and centrifuged at 10,000 g for 15 min at 4°C. The protein concentration of the supernatant was determined using Bradford reagent (Bio-Rad, Hercules, CA) with BSA as a the standard.

Western blot analysis of I{kappa}B-{alpha} and p65. Briefly, neutrophils (3.6 x 106 cells/well) or isolated nuclei were lysed using buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 0.5% (vol/vol) NP-40, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 50 mM NaF, 0.5 mM PMSF, 2 µg/ml leupeptin, 2 µg/ml pepstatin A, 2 µg/ml aprotinin, and 1 nM okadaic acid. Cell lysates were sonicated and centrifuged at 10,000 g for 15 min at 4°C. The protein concentration in the supernatant was determined using Bradford reagent (Bio-Rad) with BSA as the standard. Samples were mixed with Laemmli sample buffer and boiled for 5 min. Equal amounts of proteins were resolved by 10% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Immobilon P, Millipore, Billerica, MA). Membranes were probed with specific antibody to I{kappa}B-{alpha} (Cell Signaling), p65 (Santa Cruz Biotechnology), or actin (Sigma), followed by detection with horseradish peroxidase-conjugated anti-mouse or goat anti-rabbit IgG. Bands were visualized by enhanced chemiluminescence (ECL plus, Amersham) and quantified by AlphaEaseFC software (Alpha Innotech). Each experiment was carried out two or more times using cell populations obtained from separate groups of mice.

RT-PCR. Total RNA was purified with TRI reagent (Sigma), and cDNA was synthesized using Taqman reverse transcription reagents (Roche). RT-PCR was performed using a Lightcycler 480 SYBR Green I Master system (Roche) according to the manufacturer's instructions. The following primers (Roche) were used to amplify the mouse TNF-{alpha} transcript: forward, 5'- CCTCCCTCTCATCAGTTCTA-3'; and reverse, 5'-CTTTGAGATCCATGCCG-3'. The mouse GAPDH transcript was used as an internal control (forward, 5'-TCACTGGCATGGCCTTCC- 3'; and reverse, 5'-GGCGGCACGTCAGATCC-3').

Cytokine ELISA. ELISA was used to measure proinflammatory cytokine release from neutrophils or MH-S cells into culture media. Levels of TNF-{alpha} or MIP-2 were determined using commercially available ELISA kits (R&D Systems) according to the manufacturer's instructions and as previously described (51, 58).

Measurement of proteasome activity. Briefly, cell lysates (100 µl/sample) were obtained from neutrophil or MH-S cultures (7.5 x 106 cells/well) using buffer containing 10 mM Tris (pH 7.5), 1 mM EDTA, 20% glycerol, 2 mM ATP, 0.5% Triton X-100, and protease inhibitors (50 µM PMSF, 50 µM tosyl-phenylalanyl-chloromethyl-ketone, 2 µg/ml aprotinin, and 2 µg/ml leupeptin). Cell extracts (15 µg/100 µl) were then incubated with the fluorogenic peptide substrate Suc-Leu-Leu-Val-Tyr-AMC (100 µM) or Boc-Leu-Arg-Arg-AMC (100 µM) to determine proteasome chymotrypsin-like or trypsin-like activity, respectively (7, 17, 40, 43). Fluorescence was measured in a microtiter plate fluorometer (Bio-Rad) at 2-min intervals over a 60-min period at 37°C with an excitation filter of 380 nm and an emission filter of 460 nm. Proteasomal independent activity was determined by performing the assay in the presence of the proteasome inhibitor MG132 (10 µM). Proteasomal activity was determined using rate (fluorescence units/min) and standard AMC concentration curve, with calculated values expressed as picomoles per milliliter per milligram. The rates of chymotrypsin-like and trypsin-like activity (fluorescence units/min) were derived by subtracting the rate obtained in the presence of the proteasome inhibitor (MG132) from the values obtained in its absence. Assays were performed at least in triplicate, and results are presented from three or more independent experiments using neutrophils purified from separate groups of mice (n = 3–4 in each group).

Statistical analyses. For each experiment, neutrophils were isolated and pooled from groups of mice (n = 3–4) and all conditions were studied at the same time. Data are presented as means ± SD for each experimental group. One-way ANOVA, the Tukey-Kramer multiple-comparison test (for multiple groups), or Student's t-test (for comparisons between two groups) were used. P < 0.05 was considered significant.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
H2O2 inhibits LPS-mediated activation of NF-{kappa}B. The effects of H2O2 on LPS-dependent activation of NF-{kappa}B were determined in isolated neutrophils exposed either to bolus H2O2 or to H2O2 generated by the G/GO system. Unlike bolus H2O2, which is consumed relatively rapidly by cells, GO produces H2O2 at a constant rate, resulting in increases in H2O2 steady-state levels (39). The rate of H2O2 production by G/GO in PBS and culture media (RPMI-1640 and 0.5% FBS) was determined using the xylenol orange assay (25). The rate of H2O2 generation by 10 and 50 mU G/GO in culture media was 3.5 and 17.5 nmol·min–1·ml–1 at 37°C, respectively. The concentration of H2O2 (150 µM) in media or in PBS did not decrease over the 30-min period of incubation at 37°C.

In initial experiments, isolated neutrophils were exposed to varying concentrations of H2O2 or G/GO, and the level of intracellular H2O2 was determined by using the redox-sensitive probes DCFH-DA or DHR123 and confocal microscopy, an effective and reproducible measure of fluorescence (57, 60, 61). As shown in Fig. 1A, exposure of neutrophils to either bolus H2O2 or to G/GO increased DCF fluorescence, whereas elimination of H2O2 from culture media by the addition of catalase abolished this effect. Similar, H2O2-dependent increases in the level of fluorescence were seen for DHR123 (data not shown).


Figure 1
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  		Fig. 1. Effects of H2O2 on 2',7'-dichlorodihydrofluorescein-diacetate (DCFH-DA) and dihydroethidium (DHE) oxidation in neutrophils. A: neutrophils loaded with DCFH-DA were exposed to H2O2 (0 or 250 µM) or glucose/glucose oxidase (G/GO; 0 or 10 mU/ml) for 60 min. Catalase (CAT; 600 U/ml) was added to the cell cultures 10 min prior to H2O2 treatment as indicated. DCF, dichlorofluorescein. B: neutrophils loaded with DHE were exposed to G/GO (0 or 10 mU/ml), bolus H2O2 (250 µM), or dimethoxy-naphthoquinone (DMNQ; 10 µM) for 1 h. C: neutrophils were pretreated with or without Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP; 250 µM) and then exposed to DMNQ for 1 h. Mean DCF or DHE fluorescence intensities were obtained from 3 or more randomly chosen fields of cells and are expressed as pixel intensities per cell; n = 3–4 repetitions for each experiment. CTL, control. *P < 0.05 compared with untreated cells; **P < 0.05 compared with G/GO- or H2O2-treated cells.

 
No increase in superoxide generation occurred in neutrophils cultured with H2O2 or G/GO (Fig. 1B). In these experiments, neutrophils were loaded with the superoxide-sensitive probe DHE followed by exposure to G/GO (10 mU/ml), H2O2 (250 µM), or the redox cycling agent DMNQ, a generator of intracellular superoxide. Because DHE is directly oxidized by superoxide, specificity of the assay for superoxide was shown by diminished DHE fluorescence when DMNQ-exposed neutrophils were cocultured with the superoxide dismutase mimetic MnTBAP (Fig. 1C).

Culture of neutrophils with LPS resulted in nuclear translocation of NF-{kappa}B that was inhibited in a dose-dependent manner by H2O2 (Fig. 2A). Similarly, LPS-induced increases in mRNA levels for TNF-{alpha}, a cytokine whose transcription is dependent on NF-{kappa}B (18, 22), were diminished by the addition of H2O2 to LPS-stimulated neutrophils (Fig. 2B). The specificity of the inhibitory properties of H2O2 on LPS-mediated TNF-{alpha} expression was demonstrated by the reversal of this effect when catalase was added to neutrophils cocultured with LPS and H2O2 or G/GO (Fig. 2, C and D). Exposure of LPS-stimulated neutrophils to H2O2 or G/GO also resulted in decreased secretion of TNF-{alpha} protein as well as that for MIP-2, a CXC chemokine whose transcription is also regulated by NF-{kappa}B (22) (Fig. 2, E and F). These inhibitory effects of H2O2 or G/GO on NF-{kappa}B-dependent cytokine secretion were reversed by the addition of catalase to the cultures, showing the specificity of the effect for H2O2. Of note, we (37) have previously shown that incubation of neutrophils with compounds that increase intracellular superoxide, such as paraquat, increase LPS-mediated cytokine production. However, neither H2O2 bolus nor G/GO alone had any effect on intracellular superoxide concentrations (Fig. 1B).


Figure 2
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  		Fig. 2. H2O2 inhibits LPS-dependent cytokine expression and nuclear translocation of NF-{kappa}B in neutrophils. A: representative Western blot show H2O2-dependent inhibition of LPS-mediated p65 nuclear accumulation. In this experiment, neutrophils were left untreated or incubated with LPS (1 µg/ml) or H2O2 (250 µM) alone or H2O2 and LPS (top) or LPS alone and increasing concentrations of H2O2 (0–250 µM) for 1 h (bottom). CAT (600 U/ml) was added to the cultures 10 min before H2O2 and LPS treatment. Nuclear extracts were subjected to SDS-PAGE and Western blot analysis with antibodies specific for p65. B: neutrophils were exposed to LPS (1 µg/ml) and increasing concentrations of H2O2 (0–500 µM) for 2 h. TNF-{alpha} mRNA levels were determined using RT-PCR. C–F: neutrophils were left untreated or cultured with LPS (1 µg/ml), bolus H2O2 (250 µM), or G/GO (10 mU/ml) alone or in the indicated combinations with or without CAT (600 U/ml). TNF-{alpha} mRNA levels (C and D) as well as TNF-{alpha} and macrophage inhibitory protein-2 (MIP-2) protein concentrations in the culture media (E and F) were then determined. Three additional experiments demonstrated similar results. *P < 0.05 compared with untreated cells; **P < 0.05 compared with cells treated with LPS alone.

 
Culture of neutrophils with H2O2 or catalase alone had no effect on the production of MIP-2 or TNF-{alpha} (data not shown). Levels of cell death in LPS-stimulated neutrophils cultured with or without G/GO or H2O2 for 120 min, as in the experiments shown in Fig. 2, were consistently <1%.

H2O2 increases I{kappa}B-{alpha} protein levels in neutrophils. As shown in Fig. 2, exposure of neutrophils to H2O2 significantly inhibited LPS-mediated p65 nuclear accumulation. These results suggested a possible role for H2O2 in cytosolic retention of NF-{kappa}B. Because of the involvement of I{kappa}B-{alpha} in maintaining cytoplasmic sequestration of NF-{kappa}B (3, 6), we examined the effect of H2O2 on I{kappa}B-{alpha} levels in neutrophils. As shown in Fig. 3A, exposure of neutrophils to H2O2 or G/GO resulted in a dose-dependent increase in I{kappa}B-{alpha} protein levels. Similar to findings previously demonstrated in cell populations other than neutrophils (34, 52), stabilization of I{kappa}B-{alpha} was found when the proteasome inhibitor MG132 was added to neutrophils (Fig. 3A). The specificity of the effects of H2O2 on maintaining I{kappa}B-{alpha} levels was demonstrated by its ablation when catalase was added to the neutrophil cultures (Fig. 3, B and C). Treatment with catalase alone had no significant effect on I{kappa}B-{alpha} levels.


Figure 3
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  		Fig. 3. I{kappa}B-{alpha} levels are increased in neutrophils cultured with H2O2. A: neutrophils were left unstimulated or were cultured for 60 min with increasing concentrations of H2O2, G/GO, or MG132 as indicated. Cell lysates were prepared and subjected to SDS-PAGE and Western blot analysis with antibodies specific for total I{kappa}B-{alpha}. Amounts of protein loaded were confirmed by probing membranes with actin-specific antibodies. B and C: neutrophils were incubated with H2O2 (250 µM) or G/GO (10 mU/ml) with or without CAT (600 mU/ml) for 60 min. A representative Western blot is shown (B) as well as means ± SD of optical densitometry from 3 independent experiments (C). *P < 0.05 compared with untreated cells; **P < 0.05 compared with LPS-treated cells. The Western blot shown was obtained using a brief exposure after the addition of ECL reagents to prevent overexposure of I{kappa}B-{alpha} bands after H2O2 treatment.

 
In a recent study (51), we found that the decrease in I{kappa}B-{alpha} levels in neutrophils normally produced by culture with LPS was inhibited by H2O2. To further explore this effect, neutrophils were exposed to LPS in the presence of H2O2 (500 µM) for 0, 10, 20, 40, or 60 min, and the amounts of I{kappa}B-{alpha} were determined. In LPS-stimulated neutrophils, levels of I{kappa}B-{alpha} were decreased within 10 min of culture initiation and remained lower than baseline through the 60-min time point (Fig. 4). In contrast, coincubation of neutrophils with LPS and H2O2 resulted in robust (nearly 4-fold) increases of I{kappa}B-{alpha} levels. Of note, this effect of H2O2 was ablated in the presence of catalase, demonstrating its specificity for H2O2.


Figure 4
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  		Fig. 4. Neutrophil exposure to H2O2 prevents LPS-mediated I{kappa}B-{alpha} degradation. Neutrophils were cultured without or with LPS (1 µg/ml), H2O2 (500 µM), and CAT (600 U/ml) for the indicated time periods. I{kappa}B-{alpha} and actin were determined using SDS-PAGE and Western blot analysis (A) and optical densitometry (B). A second experiment provided similar results.

 
H2O2 inhibits proteasomal activity in neutrophils. Degradation of I{kappa}B-{alpha} depends on IKK-induced phosphorylation of Ser32 and Ser36, leading to ubiquitination and subsequent proteosomal degradation (4, 14). Although H2O2 inhibited LPS-induced nuclear accumulation of NF-{kappa}B and increased intracellular concentrations of I{kappa}B-{alpha} (Figs. 3 and 4), our previous experiments showed that the addition of H2O2 to neutrophils with or without LPS stimulation had no effect on IKK activity (51). Since recent studies (16, 31, 46) have shown that proteasomal activity can be modulated by the intracellular redox balance, a possible mechanism for H2O2-mediated inhibition of I{kappa}B-{alpha} degradation may be through inhibition of proteosomal activity, an issue not previously explored in neutrophils.

As shown in Fig. 5, both chymotrypsin- and trypsin-like proteasomal activity were inhibited in a dose- and time-dependent manner by the addition of H2O2 to neutrophils. Statistically significant inhibition of chymotrypsin-like activity was evident starting at concentrations of 50 µM H2O2 (Fig. 5, A and B). While trypsin-like proteasomal activity was also inhibited by H2O2, the effects of H2O2 only became statistically significant at concentrations of 100 µM or above. Inhibition of both chymotrypsin- and trypsin-like proteasomal activity by H2O2 was rapid, occurring within 30 min after the addition of H2O2 to neutrophils (Fig. 5, C and D). The inclusion of catalase in neutrophil cultures blocked the effects of H2O2 on proteasomal activity, demonstrating that this effect was specific for H2O2. As shown in Fig. 5E, there was no effect of ATP on chymotrypsin-like proteasomal activity. While trypsin-like activity decreased with increasing ATP concentrations, there was a consistent inhibitory effect of H2O2 (Fig. 5F).


Figure 5
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  		Fig. 5. H2O2 inhibits proteasomal activity in neutrophils. Neutrophils were exposed to increasing concentrations of H2O2 (0–250 µM) for 60 min (A and B) or with H2O2 (250 µM) for the indicated time periods (C and D). Proteasomal chymotrypsin-like (A and C) or trypsin-like (B and D) activity was measured in cell lysates. E and F: proteasomal activity in cell lysates obtained form control and H2O2-treated neutrophils was determined in the presence of ATP (0–1 mM). Values are expressed as picomoles per milliliter per milligram; n = 3–6 repetitions for each experiment. *P < 0.05 compared with untreated cells; **P < 0.05 compared with H2O2-treated cells.

 
Similar to bolus H2O2, the inclusion of GO in neutrophil cultures resulted in dose- and time-dependent inhibition of chymotrypsin- and trypsin-like proteasomal activity (Fig. 6, A and B). Exposure of neutrophils to moderate, nontoxic concentrations of G/GO (10 mU/ml) led to progressively decreased chymotrypsin- or trypsin-like activity, with nearly 80% inhibition of trypsin-like activity being present after 120 min of culture (Fig. 6D). As was the case with bolus H2O2, the inhibitory effects of G/GO on proteasomal activity were abrogated by coculture of neutrophils with catalase.


Figure 6
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  		Fig. 6. GO-generated H2O2 inhibits proteasome activity. Proteasomal activity was determined in cell lysates obtained from neutrophils treated with GO at 0–10 mU/ml for 60 min (A and B) or 10 mU/ml for 0–120 min (C and D). Chymotrypsin-like (A and C) and trypsin-like (B and D) activity are shown. n = 3–6 repetitions for each experiment. *P < 0.05 compared with untreated cells; **P < 0.05 compared with GO-treated cells.

 
H2O2 inhibits proteasomal trypsin-like but not chymotrypsin-like activity in macrophages. Previous studies (10, 44) have indicated that the effects of reactive oxygen intermediates on NF-{kappa}B activation are cell type specific. For example, exposure of macrophages to H2O2 has been shown to induce nuclear translocation of NF-{kappa}B and to enhance LPS-mediated production of NF-{kappa}B-dependent cytokines, such as TNF-{alpha} (26, 56). In contrast, as shown in Fig. 2, exposure of neutrophils to H2O2 diminished LPS-induced nuclear accumulation of NF-{kappa}B and LPS-associated production of TNF-{alpha} and MIP-2. The apparently divergent responses of macrophages and neutrophils to H2O2 may be a result of a differing intracellular antioxidant balance in the two cell types, relatively different sensitivity of proteasomal activity to H2O2, or both factors. To explore these issues, we examined the effects of H2O2 exposure on LPS-induced cytokine production and proteasomal activity in the murine macrophage cell line MH-S.

As shown in Fig. 7, neither bolus H2O2 nor H2O2 generated by G/GO induced production of TNF-{alpha} or MIP-2 by macrophages. Similarly, H2O2 appeared to have no effects on LPS-associated cytokine production by MH-S cells. Of note, the culture conditions for macrophage exposure to H2O2 utilized the same cell and media concentrations as for neutrophils, where blockade of LPS-induced cytokine production by H2O2 was found.


Figure 7
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  		Fig. 7. Effects of H2O2 on LPS-mediated cytokine release from MH-S macrophages. Macrophages were left unstimulated or were cultured with LPS (1 µg/ml) and increasing concentrations of H2O2 (0–250 µM; A and C) or G/GO (0–10 mU/ml; B and D) for 4 h. Concentrations (means ± SD) for MIP-2 (A and B) and TNF-{alpha} (C and D) in culture media are shown. Four independent experiments were performed.

 
When macrophages were cultured with increasing concentrations of H2O2 (0–250 µM) or G/GO (0–20 mU/ml), there was significant inhibition of trypsin-like but not chymotrypsin-like proteasomal activity (Fig. 8). The addition of catalase to macrophage cultures containing H2O2 (250 µM) or G/GO (20 mU/ml) prevented any decrease in trypsin-like proteasomal activity but had no effects on chymotrypsin-like activity.


Figure 8
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  		Fig. 8. H2O2 inhibits proteasomal trypsin-like but not chymotrypsin-like activity in MH-S macrophages. MH-S cells were incubated with H2O2 (0, 50, 100, or 250 µM; A and B) or G/GO (0, 5, 10 or 20 mU/ml; C and D) for 60 min, and levels of chymotrypsin-like (A and C) or trypsin-like (B and D) activity in cell lysates were measured. Means ± SD from 4 independent experiments are shown. *P < 0.05 compared with untreated cells; **P < 0.05 compared with G/GO- and H2O2-treated cells.

 
H2O2 inhibits proteasomal activity, LPS-mediated I{kappa}B-{alpha} degradation, and TNF-{alpha} production by peritoneal neutrophils. To determine if the inhibitory effects of H2O2 were limited to bone marrow neutrophils, we examined I{kappa}B-{alpha} degradation, TNF-{alpha} production, and proteasomal activity in mature neutrophils. In these experiments, peritoneal neutrophils were acquired from mice injected intraperitoneally with 3% thioglycolate. As shown in Fig. 9, A and B, exposure of peritoneal neutrophils to LPS resulted in decreased levels of I{kappa}B-{alpha}, whereas coincubation with H2O2 (300 µM) inhibited this effect. LPS-induced secretion of TNF-{alpha} was also significantly reduced when peritoneal neutrophils were cocultured with H2O2 (Fig. 9C). The addition of catalase to peritoneal neutrophils incubated with both LPS and H2O2 resulted in increased TNF-{alpha} release to levels that were not significantly different from those produced by cells stimulated with LPS alone. As was the case with bone marrow neutrophils, treatment with H2O2 significantly inhibited both chymotrypsin- and trypsin-like proteasomal activity in peritoneal neutrophils (Fig. 9, D and E).


Figure 9
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  		Fig. 9. H2O2 inhibits LPS-mediated I{kappa}B-{alpha} degradation, TNF-{alpha} production, and chymotrypsin- and trypsin-like proteasomal activity in peritoneal neutrophils. Amounts of I{kappa}B-{alpha} and proteasomal activity were determined in untreated neutrophils (control) or in neutrophils incubated with LPS (1 µg/ml) or H2O2 (300 µM) alone or H2O2 with LPS for 60 min, whereas TNF-{alpha} concentrations in the media were measured after 4 h treatment. A: representative Western blot showing I{kappa}B-{alpha} and actin concentrations in control and LPS-treated peritoneal neutrophils with or without the addition of H2O2. B: I{kappa}B-{alpha} density (means ± SD) calculated as the ratio of I{kappa}B-{alpha} and actin. n = 3 independent experiments with neutrophils isolated from different groups of mice. *P < 0.05 compared with control cells; **P < 0.05 compared with LPS-treated cells. C: TNF-{alpha} protein concentrations (means ± SD) in culture media. CAT (600 U/ml) was added to the cultures 10 min before LPS or H2O2. n = 3. *P < 0.05 compared with control cells; **P < 0.05 compared with LPS-treated cells; ***P < 0.05 compared with LPS + H2O2-treated cells. D and E: proteasomal chymotrypsin-like (D) or trypsin-like (E) activity (means ± SD) in cell lysates obtained from untreated neutrophils (control) or exposed to H2O2. n = 3 separate experiments with neutrophils isolated from different groups of mice. *P < 0.05 compared with control cells.

 

   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, H2O2 potently inhibited nuclear translocation of NF-{kappa}B and production of proinflammatory cytokines in LPS-stimulated neutrophils. These findings are consistent with previous results (33, 51) demonstrating a reduction in the expression of TNF-{alpha} and IL-8 after exposure of neutrophils to H2O2. A potential mechanism for these effects of H2O2 on NF-{kappa}B activation is through inhibition of proteasomal activity, a finding that was demonstrated in the present study in both resting and LPS-stimulated neutrophils. The inhibitory actions of H2O2 on the expression of NF-{kappa}B-dependent cytokines and on proteasomal activity were not generalizable to all cell types, since similar effects were not seen in macrophages in this study or in previous reports (26, 35, 56), indicating that the ability of H2O2 to inhibit NF-{kappa}B activation through reducing proteasome activity is cell population specific.

In previous experiments, we found that exposure of bone marrow neutrophils to H2O2 decreased LPS-induced production of TNF-{alpha}, a NF-{kappa}B-dependent cytokine, but did not affect the activation of IKK or phosphorylation of I{kappa}B-{alpha} (51). Those results suggested that the inhibitory effects of H2O2 were downstream of IKK. The present study, by showing that H2O2 inhibits proteasome activity in both bone marrow and peritoneal neutrophils, provides an important mechanism for the ability of H2O2 to suppress NF-{kappa}B activity. In particular, exposure of neutrophils to H2O2 resulted in diminished proteasomal activity and increased cytoplasmic levels of I{kappa}B-{alpha}. Even in resting bone marrow neutrophils not stimulated with LPS, there were increased I{kappa}B-{alpha} concentrations after cellular exposure to H2O2, indicating that a baseline level of proteasomal activity, which is modifiable by H2O2, is involved in maintaining steady-state concentrations of I{kappa}B-{alpha} and preventing the accumulation of NF-{kappa}B in the nucleus. After TLR4 engagement, the inhibitory effects of H2O2 on proteasomal function were sufficiently potent to prevent the degradation of I{kappa}B-{alpha} that normally occurs in this setting, thereby inhibiting nuclear translocation of NF-{kappa}B and expression of NF-{kappa}B-dependent cytokines. A similar effect of H2O2 was observed in LPS-treated mature peritoneal neutrophils. As was the case in bone marrow neutrophils, H2O2-dependent inhibition of proteasomeal activity was also associated with loss of LPS-mediated I{kappa}B-{alpha} degradation and cytokine production by peritoneal neutrophils.

A previous study (31) using cultured endothelial cells found that exposure to H2O2 resulted in diminished trypsin- and chymotrypsin-like proteasomal activity. The present experiments demonstrate similar inhibitory effects of H2O2 on proteasomal function in neutrophils and also suggest that the effects of H2O2 on proteasomal chymotrysin-like activity, rather than on trypsin-like activity, appear to be of primary importance in modulating the expression of NF-{kappa}B-dependent genes, such as proinflammatory cytokines. In particular, exposure of neutrophils to H2O2 induced inhibition of both chymotrypsin- and trypsin-like activity and was accompanied by decreased LPS-mediated production of TNF-{alpha} and MIP-2, whereas the addition of H2O2 to cultures of LPS-stimulated MH-S macrophages resulted in inhibition of trypsin-like but not chymotrypsin-like proteasomal activity and had no effects on LPS-induced cytokine secretion. The mechanisms responsible for the differences in sensitivity to H2O2 demonstrated by macrophages versus neutrophils may relate either to differing basal intracellular levels of antioxidants in the two cell populations, which may mitigate effects of cellular exposure to H2O2, or to structural alterations in proteasomal assembly that may make the proteasomes in neutrophils more sensitive to the effects of H2O2 than are those in macrophages.

Recent data from our laboratory have shown that H2O2 and superoxide have distinct effects in neutrophils, with H2O2 being anti-inflammatory while superoxide is proinflammatory (37). In particular, incubation of neutrophils with paraquat, an intracellular generator of superoxide, resulted in increased nuclear translocation of NF-{kappa}B and enhanced production of NF-{kappa}B-dependent cytokines, such as TNF-{alpha} and MIP-2. The addition of paraquat to neutrophils cultured with LPS produced an even greater nuclear accumulation of NF-{kappa}B and production of TNF-{alpha} and MIP-2 than found after exposure of neutrophils to LPS alone. Generation of superoxide in H2O2-exposed neutrophils appears to play no role in mediating the observed inhibitory effects on proteasomal function and NF-{kappa}B activation. In particular, in the present experiments, we found no increase in intracellular superoxide concentrations in neutrophils exposed either to bolus H2O2 or to GO.

There are several possible direct and indirect mechanisms through which H2O2 may inhibit proteasomal function in neutrophils. Previous studies (31, 45, 46) have shown that exposure to H2O2 diminished 26S proteasomal activity in cell populations of endothelial or acute myelogenous leukemia origin. Those experiments found a differential effect of H2O2 on the 20S and 26S proteasome, with 26S proteasomal activity being much more sensitive to H2O2 than was that of the 20S proteasome (45, 46). The inhibitory effects of H2O2 on the 26S proteasome were not due to fragmentation or degradation of its subunits but were accompanied by reversible oxidative modification of amino acid residues that may participate in proteasomal activation.

Because the expression of inducible nitric oxide (NO) synthase (iNOS) is NF-{kappa}B dependent, intracellular concentrations of NO increase after TLR4 engagement (27). NO and H2O2 appear to have counterbalancing effects on intracellular antioxidant concentrations, oxidant-induced iron signaling, and 26S proteasome activity, with recent studies (30, 31) demonstrating that NO enhances both trypsin- and chymotrypsin-like functions. Inhibition of such potentiating effects of NO on proteasomal activity through affecting transferrin receptor-related events or diminishing NF-{kappa}B-dependent transcription of iNOS would result in a decrease in 26S proteasome activity in H2O2-treated cells compared with those found when elevated concentrations of H2O2 are absent.

Alterations in intracellular ATP concentrations induced by H2O2 may also affect 26S proteasome activity. In particular, ATP levels are diminished after cellular exposure to H2O2 (23, 50). As ATP is required for optimal 26S proteasomal activity (4), H2O2-induced reductions in ATP concentrations may prevent attainment of maximum LPS-induced increases in proteasomal function.

Neutrophils appear to play a major contributory role in organ dysfunction associated with severe infection, such as sepsis or acute lung injury (1, 2). Because activated neutrophils accumulate in the lungs and other tissues during such pathophysiological processes, and both produce and are exposed to high levels of ROS, the present findings suggest that modulation of the levels of various oxidant species may have therapeutic benefit. In particular, interventions that increase H2O2 concentrations may diminish NF-{kappa}B activation and the generation of NF-{kappa}B-dependent cytokines and other immunoregulatory mediators that contribute to organ failure and death in the setting of overwhelming infection. Previous clinical trials using nonspecific antioxidants have yielded disappointing results in improving outcome for patients with sepsis or acute lung injury (5, 13, 38). However, a tailored approach that results in weighting the oxidant balance toward H2O2 while decreasing concentrations of more proinflammatory ROS, such as superoxide, may yet prove to be beneficial in such clinical situations.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Heart, Lung, and Blood Institute Grants HL-62221, HL-76206, and HL-068743 (to E. Abraham).


   ACKNOWLEDGMENTS
 
The authors thank Dr. Jack Lancaster, Jr., for helpful advice and Dr. Youhong Zhang for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. Abraham, Dept. of Medicine, School of Medicine, Univ. of Alabama, BDB 420, 1530 3rd Ave. S, Birmingham, AL 35294-0012 (e-mail: eabraham{at}uab.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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