Modulation of bone marrow-derived neutrophil signaling by H2O2: disparate effects on kinases, NF-{kappa}B, and cytokine expression

Derek Strassheim,1 Karim Asehnoune,1,2 Jong-Sung Park,1 Jae-Yeol Kim,1 Qianbin He,1 Donald Richter,1 Sanchayita Mitra,1 John Arcaroli,1 Katherine Kuhn,1 and Edward Abraham1

1Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262; and 2Service d'Anesthesie-Réanimation et Unité Propre de Recherche de l'Enseignement Superieur-Equipe d'Accueil (UPRES-EA 392), Hopital de Bicêtre, Le Kremlin Bicêtre, France 94275

Submitted 11 July 2003 ; accepted in final form 12 November 2003


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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Reactive oxygen species (ROS), including hydrogen peroxide (H2O2), are generated in increased amounts in pathological, biological processes and can play a role in signal transduction. Neutrophils often accumulate in acute inflammatory reactions, at sites where elevated concentrations of ROS are present. ROS have been demonstrated to participate in the activation of intracellular signaling pathways, including those involved in modulating nuclear accumulation and transcriptional activity of NF-{kappa}B. However, the role of ROS in affecting such events in neutrophils has not been examined. Using exposure of murine bone marrow neutrophils to H2O2 as a model of oxidative stress, we found both strong and persistent activation of ERK1/2, p38, JNK, and PKB, but not the p21-activated kinase. Stimulating the bone marrow-derived neutrophils with H2O2 did not affect nuclear translocation of NF-{kappa}B. However, production and secretion of the proinflammatory cytokine TNF-{alpha} in LPS-stimulated neutrophils were inhibited by H2O2. Exposure of LPS- or TNF-{alpha}-stimulated neutrophils to H2O2 decreased nuclear translocation of NF-{kappa}B. LPS-induced activation of the transcriptional factor AP-1 was also inhibited by H2O2. This inhibition of nuclear accumulation of NF-{kappa}B by H2O2 was not caused by an impaired capacity of LPS to stimulate the IKK pathway or to direct oxidative effects on NF-{kappa}B but rather reflected diminished degradation of I{kappa}B-{alpha}. These results indicate that oxidative stress, despite being able to selectively activate intracellular kinases in bone marrow-derived neutrophils, also inhibits NF-{kappa}B activation and associated TNF-{alpha} expression. Such inhibitory effects on neutrophil activation may limit tissue damage produced by oxidative stress.

oxidative stress; reactive oxygen species; tumor necrosis factor-{alpha}


OXIDATIVE MEDIATORS, known as reactive oxygen species (ROS), and including superoxide , hydrogen peroxide (H2O2), and hydroxyl radicals are generated in both normal and pathological, biological processes. ROS participate in the bactericidal action of phagocytic cells of the innate immune system and are produced in increased amounts in pathological conditions including ischemia-reperfusion injury (26, 29, 47, 50), chronic obstructive pulmonary disease (46), sepsis (11, 16, 34), and acute inflammatory lung injury (9, 13). ROS are generated in cells as a consequence of normal mitochondrial oxidative metabolism (14) and by neutrophils, macrophages, and other phagocytes as part of the respiratory burst that participates in microbial killing. Production of ROS in response to receptor stimulation has been demonstrated in many nonphagocytic cells, including chondrocytes, osteoblasts, fibroblasts, vascular endothelial cells, vascular smooth muscle cells, thyroid cells, neuroblastoma cells, and renal mesangial cells (18, 47).

ROS have been shown to participate in signal transduction (18, 47). Of the ROS commonly produced in tissues, H2O2 currently appears to be the most important in terms of signaling. Hydroxyl radicals are too reactive to be second messenger-like molecules, and superoxide is a very short-lived species, but H2O2 is a relatively mild oxidant, exhibiting more selective effects on biological systems (18, 47). Because of its high cell permeability, H2O2 can act as an intercellular (cell to cell) second messenger-like molecule, in addition to possessing intracellular signaling effects. Similar to classical second messengers, H2O2 is produced in response to receptor stimulation and rapidly disappears once receptor activation is terminated (47). An early description of a second messenger-like role for H2O2 was in the activation of adipocytes by insulin, where insulin stimulated H2O2 production (36) and H2O2 mimicked some of the metabolic effects of insulin (37). More recently, a role for H2O2 has been demonstrated in the activation of intracellular kinases, including MAPKs (15), PKC (27), and PKB (51), induced by interaction of growth factors and cytokines with their receptors (47). In particular, intracellular concentrations of H2O2 increase through pathways involving cellular activation by PDGF, EGF, bFGF, GM-CSF, TGF-{beta}1, IL-1, IL-3, and TNF-{alpha} (18, 47). This response has also been documented for stimulation of G protein-coupled receptors by ligands such as ANG II, thrombin, thyrotropin, lysophosphatidic acid, sphingosine 1-phosphate, serotonin, endothelin, platelet-activating factor, and bradykinin (18, 47).

Specificity of action is a critical feature of signaling and second messenger action. Similar to nitric oxide, H2O2 lacks the chemistry that would permit specific recognition by a receptor ligand-binding site as with conventional second messengers. However, specificity may arise by alternative mechanisms. One of the principal means by which H2O2 affects signal transduction is through oxidation of susceptible cysteine residues to cysteine sulfenic acid or disulfide, a step reversed by cellular reductants (18, 26, 47). Because relatively few proteins have susceptible cysteines, with pKa lower than 8.5, few become reversibly modified by H2O2 (26). The protein tyrosine phosphatase group is susceptible to H2O2-induced inactivation because the microenvironment of the active site leads to a modifiable cysteine having a pKa of ~4.5. The inactivating effects of H2O2 on these enzymes last until cellular reductant levels return to normal (18, 26, 47). 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. ERK 1/2 (23), Src (1), PKC (27), and PKB (51) are among the kinases affected as a result of such H2O2-induced inhibition of protein tyrosine phosphatases (23, 26).

The transcription factor NF-{kappa}B has a central role in the expression of many cytokines, chemokines, and other immunoregulatory mediators involved in acute inflammatory responses (2, 19). Because oxidative stress and NF-{kappa}B activation both have important roles in inflammation (9, 13, 20), the effects of ROS on NF-{kappa}B have received considerable attention. NF-{kappa}B was one of the first transcription factors shown to be activated by ROS (48). After H2O2-induced activation of NF-{kappa}B was described in several different cell systems, this was thought to be a common mechanism involved in enhancing nuclear translocation and transcriptional activity of NF-{kappa}B (7, 12, 56). However, subsequent work indicated that ROS, in general, and H2O2, specifically, are not universally involved in NF-{kappa}B activation (7, 8, 10). In some cell types, nuclear translocation and transcriptional activity of NF-{kappa}B are actually inhibited when H2O2 is administered with NF-{kappa}B activators, such as LPS or TNF-{alpha} (25, 28, 55).

Neutrophils play a major role in pathophysiological conditions associated with ROS generation. Although production of ROS by neutrophils is essential for microbial killing, excessive generation of ROS by neutrophils and other cell populations can lead to oxidant-induced injury and organ dysfunction (9, 11, 52). Oxidative stress secondary to ischemic tissue injury as well as organ infiltration by activated neutrophils plays an important role in the pathogenesis of septic shock (16), acute inflammatory lung injury, hemorrhagic shock, and myocardial infarction (11, 13). During endotoxemia and sepsis, large numbers of neutrophils infiltrate the lungs and other organs, both producing and being exposed to high concentrations of ROS that characterize such conditions.

In the present study, we examined the effects that H2O2 exposure produced on signaling in resting bone marrow-derived neutrophils and in those activated by LPS. As noted above, neutrophils are frequently exposed to oxidative stress in acute inflammatory conditions, including that induced by endotoxemia, due to the ROS they themselves release as well as ROS released from neighboring tissues. We found that H2O2 potently and persistently activates MAPK modules (ERK1/2, p38, JNK) and PKB, but not the p21-activated kinase (PAK). In contrast, despite strong activation of these kinase cascades, H2O2 exerted a potent negative feedback effect on LPS-stimulated TNF-{alpha} production and secretion. H2O2 also inhibited LPS-stimulated nuclear accumulation of NF-{kappa}B. This inhibitory effect of H2O2 on NF-{kappa}B translocation to the nucleus was not due to an effect on the ability of LPS to stimulate the IKK pathway but rather appeared to be due to blockade of I{kappa}B-{alpha} degradation. Thus oxidative stress appears to exert negative effects on proinflammatory cytokine expression in bone marrow-derived neutrophils, primarily by inhibiting NF-{kappa}B-dependent transcription, which may thereby limit the tissue damage produced by oxidative stress, particularly in the setting of acute inflammatory conditions, such as endotoxemia associated with severe sepsis.


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

Materials. Isoflurane was obtained from Abbott Laboratories (Chicago, IL). Escherichia coli 0111:B4 endotoxin (LPS), formyl-L-methionyl-L-leucine-L-phenylalanine (fMLP), and granulocyte macrophage colony-stimulating factor (GM-CSF) were purchased from Sigma (St. Louis, MO). RPMI 1640/25 mM HEPES/L-glutamine was obtained from Bio-Whittaker Products (Walkersville, MD), and FBS and penicillin/streptomycin were purchased from Gemini Bioproducts (Calabasas, CA). Bicinchoninic acid (BCA) protein assay reagent was purchased from Pierce (Rockford, IL). Activation-specific antibodies for phospho-Thr202/Tyr204 ERK1, phospho-Thr183/Tyr185 ERK2, phospho-Thr180/Tyr182 p38, phospho-Thr183/Tyr185 JNK, phospho-Ser473 PKB, phospho-Ser199/204 PAK1/phospho-Ser192/197 PAK2, Ser180/Ser181 IKK{alpha}/{beta}, Ser32 I{kappa}B{alpha}, and total ERK1/2, p38, JNK, PKB, PAK, and I{kappa}B{alpha} were purchased from Cell Signaling Technologies (Beverly, MA). Horseradish peroxidase-labeled anti-rabbit antibodies and ECL reagents were purchased from Bio-Rad (Hercules, CA). All other reagents were purchased from Sigma unless otherwise noted in the text. Custom cocktail antibodies and columns for neutrophil isolation were purchased from Stem Cell Technologies (Vancouver, BC).

Isolation and culture of bone marrow-derived mouse neutrophils. Bone marrow neutrophils were isolated as described previously (58). 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, 1% FCS and then incubated with primary antibodies specific for cell surface markers F4/80, CD4, CD45R, CD5, and TER119 for 15 min at 4°C. This custom mixture (StemCell Technologies) is specific for T and B cells, RBC, monocytes, and macrophages. After a 15-min incubation, 100 µl of antibiotin tetrameric antibody 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. Neutrophil purity, as determined by Wright's stained cytospin preparations, was greater than 97%. Bone marrow neutrophils (2 x 106/0.5 ml) were cultured in RPMI 1640, 0.2% FCS, with or without drugs as described in figure legends.

Quantitative PCR. Quantitative PCR was performed as described previously (58). RNA was isolated using the RNAeasy kit (Qiagen, Valencia, CA) following the manufacturer's protocol. Primers and probes for TNF-{alpha} were designed using Primer Express software supplied by Perkin Elmer (Foster City, CA). The TNF-{alpha} primer and probe consisted of the following: forward primer, 5'-CTGTAGCCCACGTCGTAGTCAA-3'; reverse primer, 5'-CTCCTGGTATGAGATAGCAAATCG-3'; probe, 5'-TGCCCCGACTACGTGCTCCTCAC-3'. To optimize the primer sets, a primer optimization experiment was performed as described in the manufacturer's protocol. On the basis of the primer optimization, the concentration of primers and probe for TNF-{alpha} contained 200 nM for the probe, forward primer, and reverse primer. In each experiment, a ribosomal RNA control probe, a forward primer, and a reverse primer (Perkin Elmer), at concentrations of 50 nM, were used to normalize the amount of RNA in each sample. All reagents used in the one-step RT-PCR were purchased from Perkin Elmer. Each one-step RT-PCR contained a total volume of 50 µl. The reverse transcription reaction was performed for 30 min at 48°C using MultiScribe Reverse Transcriptase with a final concentration of 0.25 U/µl. After the reverse transcription step, AmpliTaq Gold polymerase, with a final concentration of 0.025 U/µl, was activated by an increase in temperature to 95°C for 10 min followed by 40 cycles of amplification (95°C for 15 s and 60°C for 1 min) with a Gene Amp 5700 Sequence Detection System (Applied Biosystems, Foster City, CA). The quantity of cytokine mRNA was determined from a standard curve with 10-fold dilutions of known amounts of target RNA with each primer and probe set. RNA amounts were determined using software provided with the Gene Amp 5700 Sequence Detection System. Quantification was determined by dividing the amount of 18S ribosomal RNA by the target amount for each cytokine sample.

Cytokine ELISA. Immunoreactive TNF-{alpha} was quantitated using commercially available ELISA kits (R&D Systems, Minneapolis, MN), according to manufacturer's instructions and as described previously (58).

EMSA. Nuclear extracts were prepared and assayed by EMSA as previously described (58). For the analysis of NF-{kappa}B, the {kappa}B-DNA sequence of the Ig gene was used. Synthetic double-strand 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'. For AP-1, the consensus oligonucleotide used was 5'-CGC TTG ATG AGT CAG CCG GAA-3' and 3'-GCG AAC TAC TCA GTC GGC CTT-5', from Promega (Madison, WI).

Western blot analysis. Western blots for phosphorylated and total kinases were performed as described previously (58). Parallel samples for total protein kinase were run along with samples for activation-specific phosphorylation analysis. Densitometry was performed using the chemiluminescence system and analysis software (Bio-Rad) to determine the ratio between phosphorylated and total kinase.

Cell viability/lactate dehydrogenase assay. Lactate dehydrogenase (LDH) activity was measured to determine cell viability using a kit and techniques per the manufacturer's (Roche, Indianapolis, IN) instructions with a calorimetric assay system. Culture supernatants obtained for secreted cytokine analysis were used for the determination of LDH activity. Levels of LDH are an indicator of cell necrosis, because only damaged or dead cells release LDH.

Superoxide production. The respiratory burst of murine bone marrow-derived neutrophils was determined by the cytochrome c reduction method (32). Two million cells were resuspended in 1 ml of Dulbecco's PBS containing calcium, magnesium and glucose, and 80 nM cytochrome c (Sigma). The neutrophils were cultured with 1 µg/ml LPS or buffer for 30 min and then stimulated with either 1 µg/ml LPS or 1 µM fMLP combinations for 10 min, followed by centrifugation to pellet the cells. Supernatants were immediately measured for absorbance at 550 nm.


    RESULTS
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Direct effects of H2O2 on kinase activation in neutrophils. The p38 kinase is important in neutrophil activation, particularly after exposure to LPS (40, 41), and has been shown to be subject to redox regulation in other cell types (47, 57). Rapid and transient activation of protein kinases is characteristic after engagement of the fMLP (30), and other G protein-coupled receptors (39), reflecting rapid turn on and off of these types of receptors especially compared with the more prolonged temporal profile of LPS or cytokine receptor-activated events (39).

Addition of H2O2 to neutrophils induced dose-dependent increases in p38 activation (Fig. 1A). Activation was apparent in the low-to-mid millimolar range, similar to concentrations used in studies of other investigators (15, 27, 36, 51) and present in vivo during pathophysiological conditions associated with oxidative stress (35). As expected, the G protein-coupled receptor agonist fMLP stimulated p38 very rapidly, within 30 s, with subsequent exponential decay (Fig. 1B). H2O2 activated p38 equally rapidly, but in contrast to fMLP, where there was a decrement in p38 activation starting at 3 min after stimulation, H2O2-induced p38 activation was more persistent, only beginning to decrease at 40 min. Exposure of neutrophils to LPS resulted in persistence of p38 activation that was similar to that produced by H2O2, but the increase in phosphorylated p38 was less rapid, rising above baseline values more than 5 min after LPS was added to the neutrophils. To determine whether the effects of LPS or fMLP on p38 phosphorylation were due to production of reactive oxygen intermediates by the bone marrow-derived neutrophils, we determined superoxide production after exposure to LPS or fMLP alone and compared that to superoxide production in neutrophils primed with LPS and then stimulated with fMLP, a condition known to result in enhanced superoxide release by bone marrow-derived neutrophils (22). As shown in Fig. 1C, although neutrophils primed with LPS and then stimulated with fMLP released large amounts of superoxide, such effects were not seen when fMLP or LPS alone was used (22).



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Fig. 1. Activation of p38 by H2O2. Bone marrow-derived neutrophils were cultured with increasing concentrations of H2O2 for 10 min (A) or for increasing times with 1 µM formyl-L-methionyl-L-leucine-L-phenylalanine (fMLP) or 1 µg/ml LPS, with or without 0.5 mM H2O2 (B). The reactions were terminated with SDS-PAGE sample buffer. Cell lysates were subjected to SDS-PAGE and Western blot analysis with activation-specific antibodies for phospho-Thr180/Tyr182 p38 and total p38. Agonist treatments did not change the levels of total p38. The fold increase in p38 activation is plotted vs. log10 [H2O2] (A) or time (min) (B). Superoxide production by neutrophils primed with LPS and then stimulated with fMLP for 10 min was increased, but such increases in superoxide production were not found in unprimed neutrophils cultured with LPS or fMLP alone (C). The data shown are means ± SE and are representative of 3 independent experiments.

 

As was the case for p38, ERK1/2 was both rapidly and persistently activated in neutrophils exposed to H2O2 (Fig. 2A). fMLP-induced activation of ERK1/2, while rapid, was of brief duration, with return to baseline values after 10 min. Phosphorylation of ERK1/2 after exposure of neutrophils with LPS was only apparent after 20-min incubation and was less intense than that produced by stimulation of neutrophils with H2O2.



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Fig. 2. Activation of ERK1/2 and JNK by H2O2. Neutrophils were cultured for increasing times with 1 µM fMLP or 1 µg/ml LPS, with or without 0.5 mM H2O2. The reactions were terminated with SDS-PAGE sample buffer. Cell lysates were subjected to SDS-PAGE and Western blot analysis with activation-specific antibodies for phospho-Thr202/Tyr204 ERK1, phospho-Thr183/Tyr185 ERK2, and phospho-Thr183/Tyr185 JNK, as well as for total ERK, and total JNK. Agonist treatments did not alter the levels of total ERK1/2 or JNK. The fold increase in activation is plotted with respect to time. The data shown are representative of 3 independent experiments

 

The JNK signaling module is involved in neutrophil activation (52) and has been described to be redox sensitive in other cell types (47, 57). Similar to the patterns seen with ERK1/2 and p38, JNK activation in neutrophils exposed to H2O2 was both rapid and persistent (Fig. 2B). In contrast, fMLP-induced phosphorylation of JNK, while rapid, occurring within 30 s of cell exposure, was transient and no longer apparent at 10 min of culture. JNK activation induced by LPS only became apparent 20 min after LPS was added to the cells and was of a lesser degree than that induced by H2O2 at all time points. Thus, as with both p38 and ERK1/2, the unique feature of H2O2-stimulated JNK activation is that it is both rapid and long lived, showing kinetics and intensity that are distinct from those observed after neutrophil exposure to fMLP or LPS.

PKB is essential for superoxide generation by neutrophils in response to fMLP and other chemokines (32), whereas PAK is involved in chemotaxis and superoxide generation (4). As with p38, ERK1/2, and JNK, fMLP activation led to rapid activation of both PKB and PAK (Fig. 3, A and B). However, although H2O2 rapidly activated PKB in a sustained manner, it failed to activate PAK.



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Fig. 3. Activation of PKB but not p21-activated kinase (PAK) by H2O2. Neutrophils were cultured for increasing times with 1 µM fMLP or 1 µg/ml LPS, with or without 0.5 mM H2O2. The reactions were terminated with SDS-PAGE sample buffer. Cells lysates were subjected to SDS-PAGE and Western blot analysis. PKB or PAK analysis was performed using activation-specific antibodies for phospho-Ser473 PKB and phospho-Ser199/204 PAK1, phospho-Ser192/197 PAK2, as well as for total PKB and PAK. Agonist treatments did not change levels of total PKB or PAK. The fold increase in activation is plotted with respect to time. The data shown are representative of 3 independent experiments.

 

Effects of H2O2 on transcriptional factor activation in neutrophils. In acute inflammatory conditions, such as endotoxemia, neutrophils express proinflammatory mediators, such as cytokines, under the regulatory control of the transcriptional factors NF-{kappa}B and AP-1. Because the kinases affected by H2O2 in the above experiments, including p38, JNK, and PKB, have been demonstrated to be involved in activation of NF-{kappa}B or AP-1 in neutrophils (5, 40, 58), we hypothesized that exposure to H2O2 would modulate these transcriptional factors.

No alteration in nuclear translocation of NF-{kappa}B was found in murine bone marrow neutrophils incubated with H2O2 (Fig. 4A). As expected, marked increase in nuclear levels of NF-{kappa}B was present in neutrophils exposed to LPS. H2O2 administered concurrently with LPS inhibited the activation of NF-{kappa}B to levels below those produced by LPS alone (Fig. 4A). Exposure of murine bone marrow neutrophils to fMLP did not result in any alterations in nuclear levels of NF-{kappa}B (data not shown).



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Fig. 4. H2O2 inhibits LPS- and TNF-{alpha}-induced NF-{kappa}B activation. Neutrophils were cultured for increasing times with 0.5 mM H2O2 alone, 1 µg/ml LPS alone, 1 µg/ml LPS and 0.5 mM H2O2, 2 nM TNF-{alpha} alone, or a combination of 2 nM TNF-{alpha} and 0.5 mM H2O2, and then nuclear extracts were obtained (A and B). C: effects of increasing concentrations of H2O2 on LPS-induced nuclear translocation of NF-{kappa}B. Nuclear extracts were subjected to EMSA and analyzed by autoradiography. The data shown are representative of 3 independent experiments.

 

Given that H2O2 inhibited LPS-induced nuclear translocation of NF-{kappa}B, we examined the possibility that H2O2 would have similar effects in neutrophils stimulated by a Toll-like receptor (TLR)-independent mechanism relevant to acute inflammation, such as exposure to proinflammatory cytokines. Stimulation of neutrophils with TNF-{alpha} resulted in increased nuclear levels of NF-{kappa}B. Addition of H2O2 to neutrophil cultures containing TNF-{alpha} potently inhibited NF-{kappa}B activation compared with that seen in neutrophils incubated with TNF-{alpha} alone (Fig. 4B). H2O2 dose dependently inhibited NF-{kappa}B translocation (Fig. 4C); the IC50 for this response was calculated by nonlinear regression to be 175 µM.

The AP-1 transcription factor is involved in proinflammatory cytokine production by neutrophils (21) and has been described as being sensitive to redox regulation (49). Similar to the lack of effect of H2O2 on nuclear accumulation of NF-{kappa}B, exposure of neutrophils to H2O2 did not result in increased nuclear accumulation of AP-1 (data not shown). Although stimulation of neutrophils with LPS enhanced nuclear translocation of AP-1, inhibition of this response was found in neutrophils exposed to H2O2 concurrently with LPS (Fig. 5A).



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Fig. 5. Effects of H2O2 on LPS-induced activation of CREB and AP-1. For AP-1, neutrophils were stimulated for increasing times with 1 µg/ml LPS alone or the combination of LPS and 0.5 mM H2O2. A: nuclear extracts were prepared and subjected to EMSA. For CREB, neutrophils were stimulated with either 2 nM TNF-{alpha} or 2 nM TNF-{alpha} plus 0.5 mM H2O2, and then the cells were lysed by addition of SDS-PAGE sample buffer. B: lysates were subjected to SDS-PAGE and Western blot analysis with an activation-specific antibody recognizing phospho-Ser133 CREB.

 

Inhibitory effects of H2O2 on LPS- or cytokine-induced activation of transcriptional factors were not found to be a universal response. For example, whereas exposure of neutrophils to LPS or TNF-{alpha} resulted in increased levels of the transcriptionally active serine 133-phosphorylated form of CREB, no modification of this response was found when neutrophils were treated with H2O2 in conjunction with LPS or TNF-{alpha} (Fig. 5B).

Involvement of H2O2 on LPS-induced TNF-{alpha} expression by neutrophils. Because oxidative stress, as modeled by H2O2 exposure, diminished activation of NF-{kappa}B and AP-1 in LPS-stimulated neutrophils, we examined whether this effect would translate into an effect on TNF-{alpha} expression, a proinflammatory cytokine that occupies an important role in endotoxemia-induced acute inflammatory responses (3). LPS-stimulated neutrophils secrete the proinflammatory cytokine TNF-{alpha} in response to increased transcriptional activation of NF-{kappa}B (20, 21). As expected, neutrophils stimulated with LPS demonstrated increased TNF-{alpha} secretion (Fig. 6A). The response was significantly attenuated by concurrent incubation of neutrophils with H2O2. This inhibitory effect of H2O2 on TNF-{alpha} secretion was dose dependent (Fig. 6C). The IC50 determined by nonlinear regression was 142 µM (Fig. 6C), very close to the 175 µM for the inhibition of NF-{kappa}B activation by H2O2 in LPS-stimulated neutrophils (Fig. 4C).



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Fig. 6. Inhibition of LPS-stimulated TNF-{alpha} secretion and mRNA accumulation by H2O2. A: neutrophils were cultured for the times shown with LPS (1 µg/ml) alone, LPS plus catalase (650 IU/ml), LPS plus catalase (650 IU/ml) and H2O2 (0.5 mM), or LPS plus H2O2 (0.5 mM). The reactions were terminated by centrifugation and the supernatants were used for determination of TNF-{alpha} levels by ELISA. B: viability of the neutrophils exposed to LPS or to LPS and H2O2 was determined by lactate dehydrogenase assay. C: IC50 for H2O2 inhibition of LPS-induced TNF-{alpha} secretion was determined. D: cell pellets were used for analysis of TNF-{alpha} mRNA levels. The results show means ± SE and are representative of 3 similar independent experiments.

 

The inhibitory effects of H2O2 on cytokine production by neutrophils were not due to any effect on viability. In particular, the viability of neutrophils exposed to H2O2 relative to that of cells exposed only to LPS was determined by LDH assay and found to be essentially unchanged (Fig. 6B). These results were confirmed by trypan blue exclusion (data not shown).

To demonstrate that the inhibitory effects of H2O2 on LPS-induced secretion were specific, catalase, which facilitates the degradation of H2O2 to H2O and O2, was added to the neutrophil cultures. Coincubation with LPS, H2O2, and catalase eliminated the suppression in TNF-{alpha} secretion produced by H2O2 (Fig. 6A).

The inhibition of proinflammatory cytokine release by H2O2 was profound enough so as to suggest that it might be mirrored by an equivalent decrease in transcription of the TNF-{alpha} gene. To explore this issue, we examined mRNA levels in neutrophils stimulated with LPS alone or with LPS plus H2O2. Amounts of mRNA for TNF-{alpha} were increased by LPS but inhibited by concurrent incubation with H2O2 and LPS (Fig. 6D).

Mechanisms of H2O2-induced inhibition of NF-{kappa}B activation. There are several possible mechanisms by which oxidative stress might inhibit nuclear translocation of NF-{kappa}B and transcription of NF-{kappa}B genes. Direct inhibition of NF-{kappa}B itself is possible by virtue of H2O2 directly oxidizing susceptible cysteine or other residues in the NF-{kappa}B complex. To test this mechanism, we incubated nuclear extracts from LPS-stimulated neutrophils with H2O2 and then determined whether binding to the oligonucleotides used in EMSA was modified by such exposure. As shown in Fig. 7, no alteration in NF-{kappa}B binding was found after incubation with H2O2. Such results demonstrate that the inhibitory effects of H2O2 are not due to direct actions on the NF-{kappa}B complex.



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Fig. 7. H2O2 does not affect NF-{kappa}B interaction with its DNA binding sites. Neutrophils were stimulated for increasing times with 1 µg/ml LPS, or LPS and 0.5 mM H2O2, and nuclear extracts were prepared. The nuclear extracts were then incubated with either buffer or 0.5 mM H2O2 for 15 min at 30°C and subjected to EMSA. The data shown are representative of 3 independent experiments.

 

A second mechanism for the inhibitory effect that H2O2 exerts on the activation of NF-{kappa}B by LPS could be through inhibition of the IKK pathway, responsible for the phosphorylation of I{kappa}B-{alpha}, which then leads to subsequent degradation of I{kappa}B-{alpha} and removal of its tonic inhibitory effect on nuclear translocation of NF-{kappa}B. H2O2 has previously (28) been shown to inhibit IKK in alveolar epithelial type II cell lines.

In initial experiments, we examined the effects of H2O2 on LPS-induced activation of the IKK complex in neutrophils. Activation of IKK was assessed by determining phosphorylation of Ser180 in IKK{alpha}, and Ser181 in IKK{beta}, both of which are essential for IKK activity (33). As shown in Fig. 8A, stimulation of neutrophils with LPS resulted in a time-dependent biphasic activation of IKK. Coincubation with LPS and H2O2 did not have any effect on IKK phosphorylation.



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Fig. 8. Effects of H2O2 on IKK signaling and I{kappa}B-{alpha} degradation. Neutrophils were stimulated for increasing times with 1 µg/ml LPS or LPS plus 0.5 mM H2O2. The reactions were terminated by addition of SDS-PAGE sample buffer. Cell lysates were subjected to SDS-PAGE and Western blotting using activation-specific antibodies for Ser180/Ser181 IKK{alpha}/{beta} (A), for Ser32 I{kappa}B{alpha} (B), and antibodies specific for total I{kappa}B{alpha} (C). The data shown are representative of 3 independent experiments.

 

To confirm the finding that H2O2 did not affect IKK activation in neutrophils, we determined phosphorylation of serine 32 in I{kappa}B-{alpha}, an event that is directly regulated by the IKK complex. Stimulation of neutrophils by LPS resulted in rapid phosphorylation of I{kappa}B-{alpha} (Fig. 8B). The addition of H2O2 to neutrophils cultured with LPS did not alter levels of I{kappa}B-{alpha} phosphorylation. These findings are consistent with those shown in Fig. 8A and demonstrate that the IKK pathway is not subject to inhibition by H2O2 in LPS-stimulated neutrophils.

A third mechanism by which H2O2 could inhibit nuclear translocation of NF-{kappa}B is by diminishing LPS-induced degradation of I{kappa}B-{alpha}, thereby enhancing cytoplasmic retention of NF-{kappa}B. To explore this possibility, we examined cytoplasmic levels of I{kappa}B-{alpha} in neutrophils treated with LPS alone or with LPS and H2O2. In neutrophils challenged with LPS, I{kappa}B-{alpha} underwent time-dependent degradation (Fig. 8C). However, in neutrophils incubated with both LPS and H2O2, no degradation of I{kappa}B-{alpha} was apparent. Such results indicate that the mechanism by which H2O2 inhibits LPS-induced nuclear translocation of NF-{kappa}B is through preservation of cytoplasmic I{kappa}B-{alpha} levels.


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 DISCUSSION
 REFERENCES
 
In this study, we investigated the effects of oxidative stress on signaling pathways in neutrophils purified from bone marrow. Previous studies showed that both ROS and antioxidants could affect intracellular signaling cascades, including the activation of transcriptional regulatory factors, such as NF-{kappa}B and AP-1 (7, 8, 10, 18, 47, 48). However, this is the first report to demonstrate that exogenous ROS directly modulate such pathways in neutrophils. The concentrations of H2O2 in our studies are similar to those used in nonneutrophil systems examining oxidative stress (1, 15, 23, 27, 36, 37, 51, 55) and have been demonstrated to be present in vivo during pathophysiological states, such as sepsis and acute lung injury (35), where activated neutrophils play an important role. In the present experiments, modeling the situation where neutrophils are exposed to increased extracellular concentrations of ROS, culture with H2O2 resulted in activation of p38, ERK1/2, JNK, and PKB, but not PAK.

The effects of H2O2 on intracellular signaling pathways occurred as rapidly as those observed following incubation of bone marrow-derived neutrophils with the G protein-coupled receptor agonist fMLP and were present within 30 s. Many G protein-coupled receptors, including the fMLP receptor, activate responses such as stimulation of phospholipases within seconds, and this rapidity extends to activation of MAPKs (31). Such kinetics after fMLP exposure reflect the rapid activation of heterotrimeric G proteins, such as Gi, through which fMLP receptors signal. Gi is reportedly directly activated in cardiac myocytes by H2O2 via modification of Cys287 (42, 43). Our data, showing similar kinetics of kinase phosphorylation after neutrophil exposure to fMLP or H2O2, are consistent with a similar mechanism for the two stimuli that may involve Gi activation. It is also possible that the temporal profile of MAPK activation induced by H2O2 reflects the rapidity by which protein tyrosine phosphatase inhibition results in activation of MAPK modules (6, 17). However, unlike responses to fMLP that diminished to near baseline levels within 10 min, those induced by H2O2 continued at high levels for more than 30 min, similar to the time course of kinase activation after neutrophil incubation with LPS. The persistence of MAPK activation by H2O2 is not inconsistent with direct activation of Gi (39, 40), because Gi is not subjected to the same rapid desensitization as the fMLP receptor (54). The rapid termination of fMLP-stimulated events, such as MAPK activation, is believed to reflect phosphorylation and termination of fMLP receptor signaling (54).

Given the magnitude and duration in activation of the specific MAP kinases induced by H2O2, one would predict that exposure to H2O2 would result in downstream effects, such as enhanced cytokine production. In particular, p38 MAP kinase has been linked to secretion of TNF-{alpha} in LPS-exposed neutrophils and macrophages, primarily through NF-{kappa}B-dependent mechanisms (40, 41). Direct exposure of macrophages to H2O2 results in increased secretion of TNF-{alpha} (24, 30), and addition of antioxidants to LPS-stimulated macrophages decreases LPS-induced production of TNF-{alpha} protein in a time- and dose-dependent manner (38).

In the present studies, we found that H2O2 had inhibitory effects on LPS-stimulated secretion of TNF-{alpha} by bone marrow-derived neutrophils. The ability of LPS to enhance TNF-{alpha} mRNA accumulation was diminished by H2O2, suggesting that H2O2 might reduce LPS-induced gene transcription. We found that H2O2 itself did not activate NF-{kappa}B in bone marrow-derived neutrophils but did inhibit LPS- or TNF-{alpha}-induced nuclear translocation of this transcriptional factor. Such findings contrast with those reported in previous studies that demonstrated activation of NF-{kappa}B by ROS (48, 56) but are consistent with reports that H2O2 can inhibit LPS-stimulated activation of NF-{kappa}B (28, 55). There is also evidence that the ability of ROS to activate NF-{kappa}B is cell-type specific (7, 28, 55). The effects of ROS on NF-{kappa}B regulation in neutrophils have not previously been examined, so the present experiments represent the first report that ROS, particularly H2O2, are able to modulate nuclear translocation of NF-{kappa}B in this cell population. It should be noted, however, that these experiments used bone marrow-derived neutrophils, and other neutrophil populations may respond in a different manner to ROS exposure.

NF-{kappa}B is normally retained in the cytoplasm through its association with inhibitory molecules of the I{kappa}B family (20). The present experiments examined various mechanisms that could be induced by H2O2 to inhibit LPS-associated nuclear accumulation of NF-{kappa}B and subsequent transcription of NF-{kappa}B-dependent genes, such as TNF-{alpha} (20). Direct oxidative effects of H2O2 on NF-{kappa}B that prevent binding to its nucleotide consensus sequence were examined and found to be absent. Other investigators reported that H2O2 can inhibit activation of the IKK complex induced by either LPS (55) or TNF-{alpha} (28). However, we did not find evidence that H2O2 blocked LPS-induced activation of IKK in neutrophils. In particular, both phosphorylation of IKK subunits and phosphorylation of I{kappa}B-{alpha} occurred equally well in the presence of H2O2. In contrast, we found that LPS-induced degradation of I{kappa}B-{alpha} was inhibited by the presence of H2O2, indicating that the mechanism by which H2O2 inhibited activation of NF-{kappa}B was through preserving cytoplasmic levels of I{kappa}B-{alpha}. Other investigators reported that oxidative stress decreases activity of the proteasome pathway for intracellular proteolysis (44, 45). Because this pathway is responsible for degradation of phosphorylated I{kappa}B-{alpha}, an essential step for activation of NF-{kappa}B (20), blockade of proteosomal degradation of I{kappa}B-{alpha} may represent the mechanism by which H2O2 inhibits nuclear translocation of NF-{kappa}B. An additional possible mechanism for the inhibitory effects of H2O2 on I{kappa}B-{alpha} degradation would involve interference with ubiquitination of phosphorylated I{kappa}B-{alpha}. The ubiquitination of I{kappa}B proteins identifies them for degradation by proteosomes. Interference of I{kappa}B-{alpha} polyubiquitination by H2O2 would preserve cytoplasmic levels of I{kappa}B-{alpha} after LPS stimulation of neutrophils, as seen in the present experiments, and would also inhibit nuclear translocation of NF-{kappa}B.

The potential distinctions between the role of H2O2 as a mediator of oxidative stress and as a participant in intracellular signaling are important. Our study models the signaling effects of extracellular oxidative stress that would be expected to result in globally increased intracellular H2O2 concentrations. By contrast, in normal redox conditions where pathological extracellular redox imbalance does not exist, receptor-stimulated H2O2 is localized within the cell to the plasma membrane and certain granule species (53).

The present findings have potentially important physiological implications, because they indicate that oxidative stress exerts negative regulatory effects on proinflammatory neutrophil pathways activated by LPS and cytokines, including TNF-{alpha}. The negative feedback effect of H2O2 on the production of proinflammatory mediators and cytokines, such as TNF-{alpha}, would be expected to retard neutrophil-driven inflammatory cascades. These inhibitory actions of H2O2, and potentially of other ROS, may thereby limit the extent of tissue damage due to the pathological accumulation of activated neutrophils in conditions, such as sepsis, where increased release of ROS is associated with elevated tissue concentrations of bacterial products, including LPS, and proinflammatory cytokines.


    ACKNOWLEDGMENTS
 
GRANTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-62221 and P01 HL-68743 (to E. Abraham), French Ministry of Foreign Affair (LAVOISIER program), French Society of Anesthesiology and Critical Care Medicine: Societe Francaise d'Anesthesie et de Reanimation (to K. Asehnoune).


    FOOTNOTES
 

Address for reprint requests and other correspondence: D. Strassheim, Division of Pulmonary Sciences and Critical Care Medicine, Univ. of Colorado Health Sciences Center, Box C272, 4200 East 9th Ave., Denver, CO 80262 (E-mail: derek.strassheim{at}uchsc.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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