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Rationale: Although reactive oxygen species (ROS) are generally considered to be proinflammatory and to contribute to cellular and organ dysfunction when present in excessive amounts, there is evidence that specific ROS, particularly hydrogen peroxide (H2O2), may have antiinflammatory properties.
Objectives: To address the role that increases in intracellular H2O2 may play in acute inflammatory processes, we examined the effects of catalase inhibition or the absence of catalase on LPS-induced inflammatory responses.
Methods: Neutrophils from control or acatalasemic mice, or control neutrophils incubated with the catalase inhibitor aminotriazole, were treated with LPS, and levels of reactive oxygen species, proteasomal activity, NF-κB activation, and proinflammatory cytokine expression were measured. Acute lung injury (ALI) was produced by intratracheal injection of LPS into control, acatalasemic-, or aminotriazole-treated mice.
Measurements and Main Results: Intracellular levels of H2O2 were increased in acatalasemic neutrophils and in neutrophils exposed to aminotriazole. Compared with LPS-stimulated neutrophils from control mice, neutrophils from acatalasemic mice or neutrophils treated with aminotriazole demonstrated reduced 20S and 26S proteasomal activity, IκB-α degradation, NF-κB nuclear accumulation, and production of the proinflammatory cytokines TNF-α and macrophage inhibitory protein (MIP)-2. The severity of LPS-induced ALI was less in acatalasemic mice and in mice treated with aminotriazole as compared with that found in control mice.
Conclusions: These results indicate that H2O2 has antiinflammatory effects on neutrophil activation and inflammatory processes, such as ALI, in which activated neutrophils play a major role.
Reactive oxygen species are generally thought to have proinflammatory effects. However, recent studies suggest that different reactive oxygen intermediates may have distinct effects on cellular activation.
These results indicate that H2O2 has antiinflammatory effects on neutrophil activation and inflammatory processes, such as ALI, in which activated neutrophils play a major role.
Reactive oxygen species (ROS) are generated during normal and disease-related metabolic processes and play an important role in modulating cell activation and function. Increased production of ROS is believed to be deleterious, contributing to accelerated aging and atherosclerosis and to the pathogenesis of cancer, diabetes mellitus, and neurodegenerative diseases (1–5). Although there are multiple different ROS, including superoxide, hydrogen peroxide (H2O2), and hydroxyl radical, because of rapid transition from one reactive oxygen intermediate to another, most studies have assumed that increased generation of any ROS will result in cellular and organ dysfunction. However, recent data suggest that different ROS may have distinct effects on cellular activation. For example, whereas exposure to superoxide appears to increase the release of proinflammatory mediators, such as cytokines, by neutrophils and other cellular populations, elevated intracellular concentrations of H2O2 diminish LPS-induced neutrophil activation (6–9).
Antioxidants are involved in regulating the intracellular concentrations of ROS and are generally considered to have a beneficial role in diminishing oxidant stress and related cellular and tissue injury. Superoxide dismutase and catalase occupy linked functions in ROS elimination, with superoxide dismutase catalyzing the dismutation of superoxide into oxygen and H2O2 and catalase facilitating the decomposition of H2O2 to water and oxygen. A number of studies have shown a protective role for catalase in oxidant-induced cellular damage. For example, catalase overexpression prevents tissue injury in animals subjected to pathophysiologic situations associated with increased ROS generation, such as hypoxia-reoxygenation or doxorubicin-induced cardiotoxicity (10, 11). However, despite the apparent importance of catalase in modulating oxidant-induced cellular stress, mice or humans lacking active catalase are phenotypically normal (12), and some recent studies have shown that reduction in catalase activity is not necessarily associated with worse outcome after oxidant-induced stress. For example, acatalasemic mice are not more susceptible to hyperoxia-induced acute lung injury (ALI), and catalase overexpression failed to prevent allergic airways disease (13, 14).
Neutrophils benefit the host by participating in antimicrobial responses, but an excessive release of proinflammatory mediators by these cells can contribute to harmful inflammatory processes and organ dysfunction in settings, such as ALI, sepsis, or severe hemorrhage, in which activated neutrophils play a major role (15–17). Although activated neutrophils produce large amounts of ROS and are exposed to a pro-oxidant environment during acute inflammatory responses, there is only limited information concerning how ROS modulate neutrophil activity and neutrophil-dependent proinflammatory processes. Recently, we have shown that although increased levels of extracellular or intracellular superoxide have proinflammatory effects in neutrophils, resulting in enhanced nuclear translocation of nuclear factor (NF)-κB and increased generation of NF-κB–dependent cytokines, such as tumor necrosis factor (TNF)-α and macrophage inhibitory protein (MIP)-2, H2O2 appears to have antiinflammatory properties (6, 8, 9). In particular, incubation of neutrophils with H2O2 or H2O2–generating combinations of glucose and glucose oxidase resulted in diminished LPS-mediated NF-κB activation and proinflammatory cytokine production through mechanisms that involve inhibition of proteasomal function and stabilization of cytoplasmic levels of the inhibitory protein IκB-α (9). Such results suggest that increasing intracellular concentrations of H2O2 in neutrophils may be beneficial in pathophysiologic states associated with excessive neutrophil activation and accumulation, such as ALI.
To examine the hypothesis that enhanced intracellular concentrations of H2O2 have antiinflammatory properties in vitro and in vivo, we used acatalasemic mice as well as treatment of neutrophils and mice with the catalase inhibitor aminotriazole. The present studies demonstrate that the absence or blockade of catalase activity decreases LPS-induced neutrophil activation and the severity of ALI.
Male C57BL/6, acatalasemic C3Ga.Cg-Cat b/J and control C3HeB/FeJ mice, 8 to 12 weeks of age, were purchased from Jackson Laboratory (Bar Harbor, ME). The mice were kept on a 12 hour/12 hour light-dark cycle with free access to food and water. All experiments were conducted in accordance with protocols approved by the institutional review board (Institutional Animal Care and Use Committee, UAB).
Escherichia coli 0111:B4 endotoxin (LPS), H2O2, or aminotriazole (ATZ) were obtained from Sigma (St. Louis, MO). 2′,7′-dichlorodihydrofluorescein (DCFH-DA) was purchased from Invitrogen (Carlsbad, CA). Cytokine ELISA kits were obtained from R&D Systems (Minneapolis, MN). MG132 and clasto-lactacystein β-lactone were purchased from Calbiochem (San Diego, CA). The antibodies to IκB-α and phospho-Ser32/36 IκB-α were purchased from Cell Signaling (Beverly, MA). Antirabbit horseradish peroxidase and anti-IKKγ were from Santa Cruz Biotechnology (Santa Cruz, CA), and antiactin antibody and pegylated catalase (PEG-CAT) were from Sigma. The Chromogenic LAL kit was obtained from Lonza (Walkersville, MD).
Bone marrow neutrophils were isolated from the femurs and tibias of 8- to 12-week-old mice and purified by negative selection using primary antibodies specific for the cell surface markers F4/80, CD4, CD45R, CD5, and TER119 (StemCell Technologies, Vancouver, BC, Canada) as described previously (9). Neutrophil purity, as determined by Wright-Giemsa–stained cytospin preparations, was consistently greater than 97%. Neutrophils were cultured in RPMI 1640 medium containing FBS (0.5%) and treated as described in the figure legends. The percentage of cells that were viable and not apoptotic or necrotic, as determined by flow cytometry after staining with annexin V FITC and propidium iodide, was consistently greater than 95%.
Catalase-like activity was determined by measuring the time-dependent decomposition of H2O2 by neutrophils treated with or without the catalase inhibitor aminotriazole (ATZ). Neutrophils (3.5 × 106) were incubated with ATZ (0 or 25 mM) in culture media (RPMI 1640, 0.5% FBS) for 90 minutes and treated with H2O2 (150 μM) for 0, 5, 10, 20, and 30 minutes at 37°C, and the H2O2 concentration in the culture medium was determined using the xylenol orange assay (18). The rates of H2O2 decomposition by control or ATZ-treated neutrophils (3.5 × 106 /ml) were ~4.4 μmol/min and 2.6 μmol/min, respectively. Enzymatic activity of catalase was also measured in lung or liver homogenates as described previously (13, 19). Briefly, catalase-like activity was determined by incubation of lung or liver protein extracts in the presence of H2O2 (30 mM) in phosphate buffered saline (PBS) buffer for 5 minutes, and the time-dependent decrease in H2O2 concentration was monitored at 240 nm. The differences between initial rates of H2O2 decomposition were expressed as fold change as compared with control.
Intracellular levels of ROS were determined using the redox sensitive probes DCFH-DA in conjunction with fluorescent microscopy (20, 21). Briefly, neutrophils (1.5 × 106/well) in a four-well chambered cover glass (Nalge, Naperville, IL) were treated as indicated in the figure legends followed by incubation with DCFH-DA (20 μM) for an additional 30 minutes, and images were acquired using a Leica DMIRBE inverted epifluorescence/Nomarski microscope outfitted with Leica TCS NT laser confocal optics (Leica Inc., Exton, PA) fluorescent confocal microscopy. The levels of fluorescence were averaged using SimplePCI software (Compix, Cranberry Township, PA). Images were processed using IPLab Spectrum and Adobe Photoshop (Adobe Systems, San Jose, CA) software.
The extracellular level of H2O2 in neutrophil cultures was determined using the Amplex Red/horseradish peroxidase method (22, 23). Briefly, bone marrow neutrophils (106 cells/well) were cultured with ATZ (0 or 25 mM) for 30 minutes in Ringer's lactate buffer followed by Amplex Red (100 μM) and horseradish peroxidase (0.2 U/ml) inclusion for an additional 30 minutes at 37°C. Supernatants (50 μL from each well) were transferred to 96-well plates, and fluorescence was measured spectrophotometrically (absorption: 544 nm/emission: 590 nm).
Nuclear proteins were purified from 7 × 106 neutrophils, whereas 3.5 × 106 cells were used to obtain whole cell lysates as previously described (9). The protein concentration of the supernatants was determined using Bradford reagent (BioRad, Hercules, CA) with bovine serum albumin as a standard. For Western blots, samples were mixed with Laemmli sample buffer and boiled for 5 minutes. Equal amounts of proteins were resolved by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride membrane (Immobilon P; Millipore, Billerica, MA). The membranes were probed with specific antibodies to IκB-α, phospho-Ser 32/36 IκB-α (Cell Signaling), or actin (Sigma), followed by detection with horseradish peroxidase–conjugated goat antirabbit IgG. Bands were visualized by enhanced chemiluminescence (SuperSignal; Pierce Biotechnology, Rockford, IL) and quantified by AlphaEaseFC Software (Alpha Innotech, San Leandro, CA). Each experiment was performed two or more times using cell populations obtained from separate groups of mice.
Cell lysates (100 μl/sample) were obtained from neutrophil cultures (7.5 × 106/well) using buffer containing 10 mM Tris (pH 7.5), 1 mM EDTA, 20% glycerol, 0.5% Triton X-100, and protease inhibitors (50 μM PMSF, 50 μM TPCK, 2 μg/ml aprotinin, and 2 μg/leupeptin). Cell extracts (15 μg/100 μl) were then incubated with ATP (1 mM) or SDS (0.6%) and the fluorogenic peptide substrates Suc-Leu-Leu-Val-Tyr-AMC (100 μM) or Boc-Leu-Arg-Arg-AMC (100 μM) to determine 26S or 20S proteasomal chymotrypsin-like or trypsin-like activity, respectively (24–27). Fluorescence was measured in a microtiter plate fluorometer (Biorad) at 2-minutes intervals over a 60-minute 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 U/min) and standard AMC concentration curves, with calculated values expressed as fold rates of control. The rates of chymotrypsin-like and trypsin-like activity (fluorescence U/min) were derived by subtracting the rates obtained in the presence of the proteasome inhibitors (MG132 or lactacystein) from the values obtained in their 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).
Nuclear extracts were obtained from bone marrow neutrophils, and electrophoretic mobility shift assays were performed as reported previously (28–30). In brief, the κB DNA sequence of the Ig gene was used. Synthetic double-stranded sequences (with enhancer motifs underlined) were filled in and labeled with [32P]dATP (GE Healthcare, Piscataway, NJ) using Sequenase DNA polymerase: B sequence 5′-GCCATGGGGGGATCCCCGAAGTCC-3′ (Geneka Biotechnology).
ELISA were used to measure cytokines in bronchoalveolar lavage fluid (BALF) or in culture media from LPS-stimulated neutrophils. Levels of TNF-α, MIP-2, IL-6, or KC were determined using commercially available ELISA kits (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions and as previously described (8, 31).
IKKβ activity in neutrophils was determined using a direct kinase assay as previously described (32). Briefly, IKKβ was immunoprecipitated from cellular extracts (300 mg protein per sample) using anti-IKKγ antibodies followed by determination of in vitro phosphorylation of recombinant GST-IκB-α. The reaction was performed in standard kinase buffer (Cell Signaling) containing ATP (10 μM), GST-IκB-α (200 ng/sample), and [32P]ATP (5 μCi) (Amersham, Piscataway, NJ) for 30 minutes at 30°C. The samples were then subjected to SDS-PAGE, and gels were exposed to radiographic film for 24 hours.
To induce ALI, E. coli 0111:B4 LPS (1 mg/kg) in PBS was administered into the oropharynx (33). Briefly, mice were anesthetized with isoflurane, the tongue was gently extended, and LPS in PBS (50 μl) was instilled into the distal part of the oropharynx. ATZ (500 mg/kg) in saline or saline alone was given intraperitoneally 2 hours before the administration of LPS (1 mg/ml) to C57BL/6 mice. This dose of ATZ was previously used in murine models examining the efficacy or toxicity of ATZ (34, 35). In additional experiments, control C3HeB/FeJ or acatalasemic C3Ga.Cg-Cat b/J mice were given LPS (1 mg/kg intratracheally). There were no deaths associated with ATZ or LPS administration.
Lungs were harvested 24 hours after LPS administration. BAL was obtained by cannulating the trachea with a blunt 20-gauge needle and then lavaging the lungs three times with 1 ml of iced PBS.
The wet-to-dry ratio was determined as reported previously (28, 36). All mice used for lung wet-to-dry weight ratios were of identical ages. Lungs were excised, rinsed briefly in PBS, blotted, and weighed to obtain the “wet” weight. Lungs were then dried in an oven at 80°C for 7 days to obtain the “dry” weight.
Myeloperoxidase (MPO) activity was assayed as reported previously with minor modifications (28, 37). In brief, lung tissue was homogenized in 1 ml of potassium phosphate buffer (50 mM; pH 6.0) containing a reducing agent (N-ethylmaleimide; 10 mM) for 30 seconds on ice. The homogenate was centrifuged (12,000 × g) for 30 minutes at 4°C and washed twice in ice-cold buffer. The pellet was resuspended and sonicated on ice for 90 seconds in 10× volume of hexadecyltrimethylammonium bromide (HTAB) buffer (HTAB 0.5%) and potassium phosphate (50 mM; pH 6.0). Samples were incubated in a water bath for 2 hours at 56°C and centrifuged (12,000 × g) for 10 minutes. The supernatant was collected for assay of MPO activity as determined by measuring the H2O2-dependent oxidation of 3,3′-dimethoxybenzidine dihydrochloride (λ = 460 nm).
For each experiment, neutrophils were isolated and pooled from groups of mice (n = 3–6), and all conditions were studied at the same time. One way ANOVA, the Tukey-Kramer Multiple Comparisons test (for multiple groups), or Student's t test (for comparisons between two groups) were used. A P value of < 0.05 was considered to be statistically significant.
Previous studies have shown that LPS-mediated activation of neutrophils is diminished after exposure to exogenous H2O2 or the H2O2-generating combination of glucose and glucose oxidase (8, 9). However, the role of intracellular enzymes involved in H2O2 decomposition and, in particular, of catalase in the regulation of neutrophil responses to proinflammatory stimuli has not been described. To address this question, we determined whether catalase inhibition with aminotriazole (3-amino-1,2,4-triazole, ATZ) alters LPS-induced neutrophil activation.
Culture of bone marrow neutrophils with ATZ decreased the rate of H2O2 decomposition by approximately 50% (Figure 1A). These results are consistent with catalase being a major—but not the sole—enzyme involved in the removal of H2O2 (38).
Because catalase facilitates the decomposition of H2O2 to H2O and O2, catalase inactivation should result in increased intracellular H2O2 concentrations. DCF fluorescence increased by approximately twofold in neutrophils treated with ATZ (Figures 1B and 1C). The DCFH-DA probe is not a specific indicator of H2O2 because it is oxidized by reactive nitrogen species and hydroxyl radical (39, 40). Because ATZ inhibits catalase but not other enzymes involved in H2O2 removal, the observed increase in DCF fluorescence in ATZ-treated neutrophils is likely to reflect elevated intracellular H2O2 concentrations. However, to confirm that ATZ does indeed increase H2O2 levels in neutrophils, we used the more specific Amplex Red/horseradish peroxide assay (22, 23). ATZ resulted in significantly increased activity with this assay (Figure 1D). ATZ did not induce neutrophil death, either apoptotic or necrotic, as cell viability was consistently greater than 95% and similar in control and ATZ-treated cultures as determined by annexin V FITC and propidium iodide staining (see Figure E1 in the online supplement). Culturing neutrophils with LPS did not alter DCF or Amplex Red fluorescence (data not shown).
We recently found that exposure of neutrophils to H2O2 decreased LPS-induced nuclear translocation of NF-κB, degradation of IκB-α, and 26S proteasomal activity (9). To determine if the alterations in intracellular H2O2 concentrations achieved by inhibition of catalase could produce similar effects, we examined such NF-κB–related signaling events in ATZ-treated neutrophils.
Inclusion of ATZ in neutrophil cultures inhibited LPS-induced nuclear translocation of NF-κB as well as mRNA and protein expression of the NF-κB–dependent cytokines TNF-α and MIP-2 (Figure 2). The time-dependent decrease in IκB-α levels normally found after neutrophil stimulation with LPS was also inhibited in cells treated with ATZ (Figure 3A). There was a significant decrease in chymotrypsin-like, but not in trypsin-like, 26S proteasomal activity in ATZ-treated as compared with control neutrophils (Figure 3B).
Although ATZ blocks H2O2 decomposition due to the inactivation of catalase, ATZ can also affect heme synthesis and heme-containing proteins (41, 42), events that may influence intracellular oxidant balance. Therefore, we used neutrophils from acatalasemic mice to examine more specifically the role of catalase in modulating cellular activation. The loss of catalase activity due to a single-point mutation in its gene has previously been shown to decrease H2O2 decomposition in several tissues (43), but such effects on intracellular H2O2 concentrations have not been previously reported in acatalasemic neutrophils.
As expected, catalase-like activity was significantly decreased in neutrophils obtained from acatalasemic mice as compared with control mice (Figure 4A). Similar to results in ATZ-treated neutrophils, total ROS levels were increased in acatalasemic as compared with control neutrophils (Figures 4B and 4C). LPS-induced expression of TNF-α and MIP-2 was significantly decreased in acatalasemic neutrophils (Figures 5A, 5C, and 5D).
PEG-CAT, unlike native catalase, has been shown to accumulate in many types of cells and effectively diminishes the effects mediated by increased levels of intracellular H2O2 (44, 45). Pretreatment of acatalasemic neutrophils with PEG-CAT restored the levels of LPS-induced TNF-α mRNA expression (Figure 5A) and the production of TNF-α and MIP-2 (Figures 5C and 5D). Because the concentrations of endotoxin in the PEG-CAT were less than 0.1 EU/100 U, the ability of PEG-CAT to enhance cytokine expression is not due to further addition of LPS to the neutrophil cultures. Such results indicate that the absence of intracellular catalase is responsible for the alterations in LPS-induced responses found in acatalasemic neutrophils. Pretreatment with ATZ did not alter TNF-α expression in LPS-stimulated acatalasemic neutrophils (Figure 5B), showing that the ATZ-dependent inhibition of neutrophil activation (see Figures 2 and and3)3) was specifically due to catalase inactivation and concomitant increases in intracellular H2O2 levels rather than to other effects of ATZ.
Consistent with the diminished expression of TNF-α and MIP-2 in LPS-stimulated acatalasemic neutrophils, nuclear translocation of NF-κB after LPS stimulation was also decreased as compared with control neutrophils (Figure 6A). LPS-induced degradation of IκB-α was less pronounced in acatalasemic neutrophils (Figure 6B), similar to results found after ATZ treatment (see Figure 3A), and was partially restored by the inclusion of PEG-CAT in the cultures (Figures 6C and 6D). Decreased 26S and 20S proteasomal chymotrypsin-like, but not trypsin-like activity, was present in LPS-stimulated neutrophils from acatalasemic mice as compared with that present in control neutrophils (Figures 6E and 6F). Acatalasemic and control neutrophils had similar IKKβ activity after LPS exposure (Figure E2A). LPS-induced phosphorylation of IκB-α was similar or even slightly increased at early time points in acatalasemic as compared with control neutrophils (Figure E2B).
Activation of NF-κB and enhanced production of NF-κB–dependent proinflammatory mediators contribute to the development of ALI, a pathophysiologic process in which neutrophils play a major role (17, 46–48). Because catalase inhibition or the absence of active catalase diminished LPS-induced nuclear translocation of NF-κB and expression of TNF-α and MIP-2 in neutrophils, we hypothesized that such therapy could reduce the severity of lung injury.
A significant decrease (~ 50%) in catalase-like activity was detected in lung and liver homogenates obtained from mice treated with ATZ (Figure 7A). ATZ treatment resulted in reduced severity of LPS-induced lung injury, as determined by wet-to-dry ratios, MPO activity in lung homogenates, and total cell and neutrophil numbers in BALF (Figures 7B and 7C). The levels of TNF-α, MIP-2, IL-6, and KC in BALF from LPS-treated mice given ATZ were significantly decreased compared with those present in control animals that received LPS alone (Figure 7D). ATZ alone had no significant effects on pulmonary neutrophil accumulation or lung cytokine levels as compared with the values present in saline treated mice (Figures 7C and 7D).
A significant decrease (~60%) in catalase-like activity was detected in lung homogenates obtained from acatalasemic mice (Figure 8A). Similarly, decreased catalase-like activity was present in liver homogenates from acatalasemic as compared with control mice (Figure 8A).
The severity of LPS-induced lung injury, including the development of interstitial edema (as measured by wet-to-dry ratio) and neutrophil accumulation into the pulmonary interstitium and airspaces, was significantly reduced in acatalasemic as compared with control mice (Figure 8B and 8C). Levels of proinflammatory cytokines and chemokines, specifically TNF-α, KC, MIP-2, and IL-6, were also significantly decreased in the lungs of LPS-treated acatalasemic mice (Figure 8D).
To examine the role that enhanced intracellular catalase activity might have on LPS-mediated ALI, mice were pretreated with PEG-CAT in saline or saline before being exposed to LPS. PEG-CAT alone had no effects on measures of lung injury or pulmonary cytokine levels. In contrast, administration of PEG-CAT before LPS resulted in increased wet to dry ratios, accumulation of neutrophils into the pulmonary parenchyma (MPO activity) and BALF, as well as increased pulmonary TNF-α and MIP-2 levels as compared with values found in control, LPS-exposed mice (Figure 9).
ROS have generally been considered to be proinflammatory and to contribute to organ dysfunction. A putative mechanism for such effects is ROS-induced activation of transcriptional factors, particularly NF-κB, with resultant enhanced production of cytokines and other proinflammatory mediators. However, despite studies showing that oxidants can enhance NF-κB activation in cell lines and primary cell populations, this has not been a universally observed effect (50–52). For example, although H2O2 can enhance nuclear translocation of NF-κB in some cell populations, particularly those that are macrophage derived (53, 54), in other cell types, H2O2 has been found to inhibit inflammatory responses (55–57). In particular, exposure of neutrophils to extracellular H2O2 diminished LPS-induced activation of NF-κB and expression of NF-κB–dependent proinflammatory cytokines (8, 9). Such findings suggested that interventions that increased intracellular concentrations of H2O2 might be beneficial in neutrophil-driven proinflammatory conditions, such as ALI.
The present experiments are consistent with an antiinflammatory role for increased intracellular concentrations of H2O2. In these studies, inactivation or absence of catalase was associated with increased intracellular H2O2 and resulted in inhibition of activation of the 20S and 26S proteasome, degradation of IκB-α, nuclear translocation of NF-κB, and expression of NF-κB–dependent cytokines in LPS-stimulated neutrophils. In addition, the severity of LPS-induced ALI was decreased in mice that were acatalasemic or in which catalase had been inhibited with aminotriazole and was increased in mice treated with PEG-CAT.
There are a number of intracellular antioxidant enzymes involved in facilitating the decomposition of H2O2 to H2O and O2, with catalase appearing to occupy a major role in this process among most cell populations that have been examined (12, 13, 58). However, there has been only limited information concerning the role of catalase in modulating intracellular levels of H2O2 in neutrophils. In the present studies, increased H2O2 levels were found in LPS stimulated acatalasemic neutrophils and in neutrophils in which catalase was inhibited with aminotriazole. These results indicate that catalase plays a significant role in the removal of endogenously produced H2O2 in neutrophils. However, other antioxidant enzymes, in addition to catalase, can modulate intracellular levels of H2O2 and potentially alter neutrophil activation through H2O2–dependent mechanisms. For example, the inhibitory effects of H2O2 on LPS-stimulated NF-κB activation was recently demonstrated in primary tracheal epithelial cells isolated from glutaredoxin 1 (Grx1)-deficient mice (55).
In the present experiments and in previous studies (9), increases in intracellular H2O2 were associated with decreased 26S and 20S proteasomal activity. Because degradation of IκB-α after TLR4 induced cellular activation occurs through mechanisms involving the 26S proteasome, such inhibitory effects of H2O2 on proteasomal function would be expected to result in increased cytoplasmic levels of IκB-α that would inhibit nuclear translocation of NF-κB through continued association between the two molecules. Previous studies have shown that treatment of resting or LPS-stimulated neutrophils with H2O2 or the H2O2 generating combination of glucose and glucose oxidase is associated with elevations in cytoplasmic concentrations of IκB-α (9). The present experiments also found that the inactivation or absence of catalase resulted in decreased LPS-induced degradation of IκB-α, despite no change in IKKβ activity, consistent with the antiinflammatory actions of increased intracellular H2O2 being due to effects on 26S proteasomal activity. Nevertheless, it is possible that elevated concentrations of H2O2 may inhibit NF-κB activation through other mechanisms, such as by reducing ubiquitination of IκB-α, an event that precedes proteasomal degradation of IκB-α (59), or through modulating NF-κB phosphorylation or association with transcriptional coactivators, such as CBP or p300.
These studies used acatalasemic mice and treatment with aminotriazole as two different approaches for exploring the role of catalase in modulating intracellular H2O2 levels and neutrophil-associated inflammatory responses. Both approaches showed similar benefits of catalase inhibition or absence in diminishing LPS-induced neutrophil activation and the severity of ALI. In addition, enhanced intracellular catalase activity, achieved by administration of PEG-CAT, resulted in greater severity of ALI, consistent with decreased intracellular H2O2 being a proinflammatory event.
Although acatalasemic mice lack active catalase, the chronic nature of this cellular alteration likely results in the development of compensatory mechanisms that may be protective from oxidant-induced stress. To address this issue by transiently blocking catalase activity, we used treatment with aminotriazole, a potent and irreversible inhibitor of catalase that has been shown to increase intracellular H2O2 levels in vitro and in vivo (34). Although the effects of aminotriazole on H2O2 decomposition appear to be selective for catalase and not other antioxidant enzymes, such as peroxyreductases, aminotriazole can affect heme synthesis and heme-containing proteins and may indirectly affect intracellular H2O2 levels through catalase-independent mechanisms (41, 42). However, because the effects of aminotriazole on intracellular H2O2 levels, proteasome activity, degradation of IκB-α, activation of NF-κB, and production of proinflammatory cytokines in LPS-treated neutrophils were similar to those found in LPS-stimulated acatalasemic neutrophils, it appears likely that such actions of aminotriazole in neutrophils are due to its inhibitory effects on catalase rather than interactions with other intracellular enzymes. In addition, the protective effects of aminotriazole in reducing the severity of LPS-induced pulmonary inflammation suggest that inhibition of catalase may be useful as a therapeutic approach for ALI and other inflammatory processes in which neutrophils play a major role.
Although preclinical studies have shown that antioxidants can diminish the severity of ALI, such findings were primarily obtained using transgenic mice overexpressing antioxidants or as a pretreatment (49, 60–62). Clinical trials with antioxidants for acute illnesses, such as ALI or sepsis, have been disappointing, and no agent has been shown to be effective in critically ill patients (63–68).
The present results provide a potential mechanism for the lack of efficacy of nonspecific antioxidant therapy in clinical trials enrolling patients with ALI, sepsis-induced organ dysfunction, and other acute inflammatory conditions in which neutrophils play a major role. Because H2O2 appears to be antiinflammatory in neutrophils, the administration of nonspecific antioxidants, even though they may reduce concentrations of some proinflammatory ROS, such as superoxide, may also induce enhanced neutrophil activation and neutrophil-induced tissue damage by decreasing intracellular concentrations of H2O2. More targeted antioxidants that increase intracellular concentrations of H2O2, either by facilitating the dismutation of superoxide to H2O2 or diminishing the decomposition of H2O2 to H2O and O2, would be expected to be more beneficial than nonspecific agents. The present results, showing that inactivation or absence of catalase is antiinflammatory in neutrophils and reduces the severity of ALI, support this hypothesis. Similarly, the efficacy of EC-SOD and EC-SOD mimetics, which facilitate the transition of superoxide to H2O2, in reducing the severity of ALI in experimental models (69, 70), also is consistent with benefit being associated with antioxidant approaches that specifically result in increases in intracellular H2O2 concentrations.
Supported by National Institutes of Health Grants HL62221, HL76206, and HL068743 (E.A.) and by the Société Française d'Anesthésie et de Réanimation and the University Hospital of Amiens (France) (E.L.).
This article has an online data supplement, which is accessible from this issue's table of contents at www.atsjournals.org
Originally Published in Press as DOI: 10.1164/rccm.200806-851OC on January 16, 2009
Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.