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Rationale: Increasing evidence suggests that tumor necrosis factor (TNF)-α plays a key role in pulmonary injury caused by environmental ozone (O3) in animal models and human subjects. We previously determined that mice genetically deficient in TNF response are protected from lung inflammation and epithelial injury after O3 exposure.
Objectives: The present study was designed to determine the molecular mechanisms of TNF receptor (TNF-R)–mediated lung injury induced by O3.
Methods: TNF-R knockout (Tnfr−/−) and wild-type (Tnfr+/+) mice were exposed to 0.3 ppm O3 or air (for 6, 24, or 48 h), and lung RNA and proteins were prepared. Mice deficient in p50 nuclear factor (NF)-κB (Nfkb1−/−) or c-Jun–NH2 terminal kinase 1 (Jnk1−/−) and wild-type controls (Nfkb1+/+, Jnk1+/+) were exposed to O3 (48 h), and the role of NF-κB and mitogen-activated protein kinase (MAPK) as downstream effectors of lung injury was analyzed by bronchoalveolar lavage analyses.
Results: O3-induced early activation of TNF-R adaptor complex formation was attenuated in Tnfr−/− mice compared with Tnfr+/+ mice. O3 significantly activated lung NF-κB in Tnfr+/+ mice before the development of lung injury. Basal and O3-induced NF-κB activity was suppressed in Tnfr−/− mice. Compared with Tnfr+/+ mice, MAPKs and activator protein (AP)-1 were lower in Tnfr−/− mice basally and after O3. Furthermore, inflammatory cytokines, including macrophage inflammatory protein-2, were differentially expressed in Tnfr−/− and Tnfr+/+ mice after O3. O3-induced lung injury was significantly reduced in Nfkb1−/− and Jnk1−/− mice relative to respective control animals.
Conclusions: Results suggest that NF-κB and MAPK/AP-1 signaling pathways are essential in TNF-R–mediated pulmonary toxicity induced by O3.
Nuclear factor-κB and MAPK/AP-1 signaling pathways are essential in tumor necrosis factor receptor–mediated pulmonary toxicity induced by ozone.
NF-κB and MAPK/AP-1 signaling pathways are essential in TNF receptor–mediated lung injury induced by ozone.
Ozone (O3) is a principal oxidant in air pollution. Elevated ambient O3 levels have been associated with increased hospital visits and respiratory symptoms in epidemiologic studies (1, 2). Subjects with preexisting allergic/inflammatory airway disorders, such as asthma and rhinitis, are known to be particularly vulnerable to O3 and at risk of exacerbations (3). Recent evidence also suggested that O3 enhances the effect of inhaled allergen in patients with asthma (4). Acute O3 toxicity in rodent airways includes predominant neutrophilic inflammation accompanied by airway hyperresponsiveness, chemokine/cytokine production, mucus overproduction and hypersecretion, and cell death and proliferation. However, the mechanisms of O3-induced effects on the lung are not completely understood.
A number of investigations have focused on the potential roles of inflammatory mediators, including tumor necrosis factor (TNF)-α, in the pathogenesis of O3-induced lung inflammation and injury. TNF-α is a member of the trimeric cytokine family (5), which has diverse bioregulatory activities engaged in inflammation/immunity responses, cell proliferation/differentiation, and apoptosis. TNF-α has a critical role in many acute and chronic inflammatory diseases, and anti-TNF strategies have proven to be clinically effective (6). TNF-α binds to two distinct cellular membrane receptors (TNF-R1p55, TNF-R2p75). Ligand binding to TNF-R1 induces sequential recruitment of intracellular adaptor proteins, including TNF-R1–associated death domain protein (TRADD) and TNF-R–associated factor 2 (TRAF2) to the membrane. TRAF2 is also a well-defined intracellular adaptor for TNF-R2. The interaction of TRAF2 in the TNF-R complex with the inhibitor of κB (IκB) kinase (IKK) and subsequent phosphorylation of IKK and IκB eventually activates the transcription factor, nuclear factor (NF)-κB (7). Another pathway that becomes activated by the TRADD/TRAF2 complex is the mitogen-activated protein kinase (MAPK) cascade, which induces nuclear transactivation of activator protein (AP)-1 transcription factors (7). TNF-α signaling directs transcriptional regulation of inflammatory mediator genes, including early-response cytokines (e.g., interleukin [IL]-1β), chemokines (e.g., macrophage inflammatory protein [MIP]-2), and adhesion molecules (e.g., intercellular adhesion molecule [ICAM]-1) in various airway cells (8, 9).
An essential role for TNF-α has been recently documented in animal models of pulmonary inflammation and oxidative injury responses caused by bleomycin and several environmental toxicants, including hyperoxia, endotoxin, and cigarette smoke (10–14). Inhaled O3 also enhances TNF-α release and TNF-R expression in the airway cells or tissues (15, 16). Our positional cloning studies in inbred mice identified Tnf as a candidate susceptibility gene for lung inflammation induced by subacute exposures to 0.3 ppm O3 (17). In support of this hypothesis, we and others have demonstrated that lack of TNF response provided significant protection from O3-induced inflammation and airway hyperreactivity in rodent lungs (16–20). Moreover, recent studies (21, 22) demonstrated in human subjects an association of O3-induced lung functional changes with a TNF polymorphism haplotype including −308A, which is also known to be involved in increased risk of asthma (23). In the present study, we elucidated molecular mechanisms underlying TNF-R–mediated pulmonary pathogenesis of subacute O3 toxicity. Some of the results of this study have been previously reported in abstracts (24, 25).
Male Tnfr−/− (B6;129S-Tnfrsf1atm1ImxTnfrsf1btm1Imx/J), Nfkb1−/− (B6;129P2-Nfkb1tm1Bal/J), and Jnk1−/− (B6.129-Mapk8tm1Flv/J) mice and their respective wild-type mice (6–8 wk) were purchased from Jackson Laboratories (Bar Harbor, ME). After acclimation, mice were placed in individual stainless-steel wire cages within a Hazelton 1000 chamber (Lab Products, Maywood, NJ) equipped with a charcoal and high-efficiency particulate air–filtered air supply. Mice had free access to water and pelleted open-formula rodent diet NIH-07 (Zeigler Brothers, Gardners, PA.). Mice were exposed continuously (for 6, 24, or 48 h) to 0.3 ppm O3. On the basis of National Ambient Air Quality Standards for ambient O3 (0.12 ppm for 1 h and 0.08 ppm for 8 h) (26) and dosimetry studies in which rodents require four- to fivefold higher doses of O3 than humans to create an equal deposition and pulmonary inflammatory response (27), the O3 concentration used in the current study is a reasonable exposure level from which to make comparisons with humans. O3 was generated from ultra-high-purity air (< 1 ppm total hydrocarbons; National Welders, Inc., Raleigh, NC) using a silent arc discharge O3 generator (model L-11; Pacific Ozone Technology, Benicia, CA). Constant chamber air temperature (72 ± 3° F) and relative humidity (50 ± 15%) were maintained. O3 concentration was continually monitored (Dasibi model 1008-PC; Dasibi Environmental Corp., Glendale, CA). Parallel exposure to filtered air was done in a separate chamber for the same duration. Immediately after each exposure, mice were killed by sodium pentobarbital overdose (104 mg/kg). All animal use was approved by the National Institute of Environmental Health Sciences Animal Care and Use Committee.
Left lung tissues were fixed by 10% neutral buffered formalin under constant pressure (25 cm H2O) and sections were processed for histopathology. Immunohistologic staining was done using an anti-TRAF2 antibody (sc-877; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or a proliferating cell nuclear antigen (PCNA) staining kit (Zymed Laboratories, Inc., South San Francisco, CA).
Whole lungs from mice exposed to O3 or air (48 h) were lavaged, and lung cellular inflammation and hyperpermeability were assessed as described previously (16).
Nuclear proteins were prepared from pulverized pieces of right lung. Briefly, lung tissues were pulverized in liquid nitrogen using a mortar and pestle, and were homogenized in a hypotonic buffer (10 mM N-2-hydroxyethylpiperazine-N′-ethane [HEPES], pH 7.9; 0.5 M sucrose; 1.5 mM MgCl2; 10 mM KCl; 10% glycerol; 1 mM ethylenediaminetetraacetic acid [EDTA], pH 8.0; 1 mM dithiothreitol [DTT]; 1 mM phenylmethylsulfonyl fluoride [PMSF]) using a dounce homogenizer. Homogenates were treated with Nonidet P-40 (0.25%), incubated on ice for 15 minutes, and centrifuged (14,000 g, 20 min, 4°C). After collecting supernatants containing soluble cytoplasmic fraction, pellets were resuspended in a hypertonic lysis buffer (20 mM HEPES, pH 7.9; 420 mM NaCl; 1.5 mM MgCl2; 10% glycerol; 0.2 mM EDTA, pH 8.0; 0.5 mM DTT; 0.5 mM PMSF; protease inhibitor cocktail), incubated in ice on a rocking platform (150 rpm, 30 min), and centrifuged (14,000 g, 15 min, 4°C). Supernatants including nuclear proteins were collected and stored at −80°C. DNA binding activity of NF-κB or AP-1 was determined by electrophoretic mobility (gel) shift analysis of nuclear proteins (5–10 μg) as described previously (28). Specific binding activity was determined by preincubation of nuclear proteins with anti–p65NF-κB (sc-372X), anti–p50NF-κB (sc-1190X), or anti-pan Jun AP-1 (sc-44X) antibody followed by electrophoretic mobility shift analysis. The gel was autoradiographed using an intensifying screen at −70°C, and autoradiograph images were scanned and quantified by a Bio-Rad Gel Doc 2000 System (Hercules, PA).
Total lung proteins from right lung tissues were prepared in radioimmunoprecipitation (RIPA) buffer (phosphate-buffered saline, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 10 μg PMSF per ml, 1 mM sodium orthovanadate, protease inhibitor cocktail). Lung cytosolic soluble fractions were acquired during nuclear protein extraction as described above. Proteins (20–100 μg) were analyzed by Western blotting using specific antibodies against TRAF2 (sc-877), TRADD (sc-7868), IκB-α (sc-371), phosphorylated IκB-α (p-IκB; sc-8404), IKK (sc-7607), phosphorylated IKK (p-IKK; sc-21661-R), c-Jun–NH2 terminal kinase (JNK1; sc-571), p-JNK (sc-6254), extracellular signal-regulated kinase (ERK; sc-154), p-ERK (sc-7383), and actin (sc-1615). PCNA was detected in nuclear protein (20 μg) using an anti-PCNA antibody (sc-56). To determine TRADD-bound TRAF2 (an indicator of TNF-R1 signaling complex formation) or TNF-R2–bound TRAF2 (an indicator of TNF-R2 signaling complex formation), 200 μg of total lung protein were immunoprecipitated with anti-TRADD antibody (sc-7868) or anti-TNF-R2 antibody (sc-1074), respectively, and each immunoprecipitate was processed for Western blot analysis with the anti-TRAF2 antibody. Bands were scanned and quantified using the Bio-Rad Gel Doc 2000 System.
Total RNA was isolated from right lung homogenate, and reverse transcriptase–polymerase chain reaction was performed with mouse-specific primers for TNF-α, lymphotoxin (LT)-β, MIP-2, and IL-1β (29). Forward and reverse primers used for ICAM-1 (GI:194077) amplification were 5′-atggcttcaacccgtgccaa-3′ and 5′-gttacttggctcccttccga-3′, respectively. Forward and reverse primers used for IL-6 (GI:198367) amplification were 5′-aagaacgatagtcaattcca-3′ and 5′-gatctcaaagtgacttttag-3′, respectively. Each mRNA abundance was quantified by the Bio-Rad Gel Doc 2000 System as described previously (28) using 18S ribosomal RNA as an internal control.
Data were expressed as the group mean ± SEM. Two-way analysis of variance (ANOVA) was used to evaluate the effects of exposure and genotype in all experiments except p50 NF-κB binding activity in Nfkb1+/+ mice in which one-way ANOVA was used. The Student-Newman-Keuls test was used for a posteriori comparisons of means (p < 0.05). All of the statistical analyses were performed using SigmaStat 3.0 software program (SPSS, Inc., Chicago, IL).
Intracellular TNF-R signal protein complex was measured as an indicator of TNF-R activation after exposure to O3. TRAF2 is a common intracellular signal transducer that mediates TNF-R1 and TNF-R2 responses, and it has recently been found to be essential for early recruitment of downstream kinases for NF-κB and AP-1 activation (30–32). Immunoprecipitation/Western blotting of total lung protein indicated that TRADD-bound TRAF2 (an indicator of intracellular TNF-R1 signal transducer complex formation) was elevated after 6 hours of exposure, before the onset of inflammation (Figure 1A). Complex formation was significantly attenuated in Tnfr−/− mice compared with Tnfr+/+ mice after O3 exposure (Figure 1A, Table 1). TRAF2 bound to TNF-R2 (an indicator of TNF-R2 signaling complex formation) was significantly increased by O3 in Tnfr+/+ mice, but not in Tnfr−/− mice (Figure 1A, Table 1). Soluble TRAF2 was also relatively higher in Tnfr+/+ mice than in Tnfr−/− mice basally and after O3 exposure, and O3 reduced soluble TRAF2 levels in both genotypes in a time-dependent manner (Figure 1A, Table 1). O3-induced early increases in TRAF2–TRADD and TRAF2–TNF-R2 complexes were concurrent with depletion of soluble TRAF2 before lung pathology developed in the wild-type mice, and suggested the recruitment of “free” cytoplasmic TRAF2 to form membrane complex in response to O3.
Lung TRAF2 was detected constitutively by immunohistochemical staining in cytoplasm and membranes of ciliated and basal bronchial epithelial cells, endothelium, and smooth muscle, and in alveolar macrophages of Tnfr+/+ mice and Tnfr−/− mice (Figure 1B). TRAF2 was also detected in infiltrating inflammatory cells and in terminal bronchiolar cells of the centriacinar region, which was undergoing significant proliferation and reconstitution in O3-exposed mice (Figure 1B) as demonstrated previously (16, 17). Consistent with immunoprecipitation/Western blot data (Figure 1A), relatively fewer TRAF2-positive cells were found in these pathologic regions of Tnfr−/− mice, compared with Tnfr+/+ mice (Figure 1B).
As a dimeric transcription factor, the activity of NF-κB is regulated by its interaction with IκB, a family of cytoplasmic NF-κB inhibitors. Activation of the NF-κB pathway requires sequential phosphorylation of the upstream kinase complex IKK and its substrate IκB, which leads to phosphorylational degradation of IκB and nuclear translocation of NF-κB after having been liberated from NF-κB–IκB complexes. After O3 exposure, lung IKK(α/β) and IκB-α were enhanced similarly in both genotypes (Figure 2A). However, p-IKK(α/β) level normalized by IKK(α/β) was significantly lower in Tnfr−/− mice than in Tnfr+/+ mice basally and after O3 (Table 1). A time-dependent increase of phosphorylated IκB-α (p-IkB-α/IκB-α) by O3 was evident in Tnfr+/+ mice but marginal and significantly lower in Tnfr−/− mice (Figure 2A, Table 1). Baseline DNA binding activities of total NF-κB and p50 κB subunits were significantly suppressed in Tnfr−/− mice compared with Tnfr+/+ mice (Figure 2B, Table 1). O3 significantly enhanced the binding activity of total NF-κB and specific p50 κB over the constitutive level in both genotypes (Figure 2B, Table 1). However, O3-induced total (6 and 24 h) and specific p65 (24 h) and p50 (24 h) NF-κB activity was significantly lower in Tnfr−/− mice compared with Tnfr+/+ mice (Figure 2B, Table 1).
Phosphorylational activation of the MAPK regulates nuclear AP-1 transactivation. Total JNK and ERK MAPK levels were not significantly changed by O3 in either genotype (Figure 3A). Total activated levels of ERK and JNK (determined by ratio of phosphorylated level to nonphosphorylated level) were significantly attenuated basally and after O3 in Tnfr−/− mice compared with Tnfr+/+ mice, although O3 also significantly enhanced activation of these MAPKs in Tnfr−/− mice (Figure 3A, Table 1). Basal and O3-induced (6 h) DNA binding activity of nuclear total AP-1 was significantly suppressed in Tnfr−/− mice, compared with wild-type mice (Figure 3B, Table 1). Specific DNA–binding activity of AP-1 Jun proteins was constitutively lower in Tnfr−/− than in Tnfr+/+ mice, and was not significantly enhanced by 24 hours after O3 in Tnfr−/− mice (Figure 3B, Table 1).
Transcriptional induction of several O3-inducible genes containing cis-acting elements for NF-κB and/or AP-1 binding in their promoters (33–35) was compared in Tnfr+/+ and Tnfr−/− mice. Constitutive expression of TNF-α and IL-1β mRNA was significantly lower in Tnfr−/− compared with Tnfr+/+ mice (Figure 4). O3 caused a significant increase of IL-1β mRNA expression at 24 and 48 hours over the control level, whereas TNF-α, LT-β, MIP-2, and ICAM-1 mRNA was significantly elevated above respective baseline expression only at 48 hours when inflammation and injury by O3 had reached a peak (Figure 4). Induced levels of these genes were significantly lower in Tnfr−/− mice compared with Tnfr+/+ mice (Figure 4). In contrast, IL-6 transcript level was significantly greater in Tnfr−/− mice than in Tnfr+/+ mice after 24- and 48-hour exposure (Figure 4).
Because NF-κB binding activity was significantly lower in Tnfr−/− mice compared with Tnfr+/+ mice after O3 exposure, we hypothesized that mice deficient in a functional subunit of NF-κB (Nfkb1−/−) would be less responsive to inflammatory effects of O3 compared with wild-type (Nfkb1+/+) control animals. No significant differences in mean total protein concentration or cell differentials were found in bronchoalveolar lavage (BAL) fluid between Nfkb1+/+ and Nfkb1−/− mice after air exposure (Figure 5A). O3-induced increases in the mean total protein concentration and numbers of neutrophils and epithelial cells were significantly attenuated (70–50%) in Nfkb1−/− mice relative to the Nfkb1+/+ control animals (Figure 5A). However, no significant O3 or genotype effects on the numbers of BAL macrophages were found (Figure 5A).
Immunohistologic localization of PCNA indicated a few proliferating cells throughout the lung sections in both genotypes of mice exposed to air (Figure 5B). Cellular proliferation in the injured regions (mainly in terminal bronchioles) caused by O3 was markedly reduced in Nfkb1−/− mice compared with Nfkb1+/+ mice (peak at 48 h, Figure 5B). Western blot analysis (Figure 5B) determined significant differences (twofold) in nuclear PCNA (36 kD) between the two genotypes basally and after O3 (48 h).
O3 significantly stimulated total NF-κB and specific p50 and p65 binding in the lungs of Nfkb1+/+ mice at 6 and/or 24 hours (Figure 6). In Nfkb1−/− mice, total κB activity (SB) was slightly enhanced by O3 at 24 hours (Figures 6A–6D), whereas specific binding activity of p50 κB (SSB) was not detected (Figure 6B). Compared with Nfkb1+/+ mice, total (SB in Figure 6A) and p65 (SSB in Figure 6C) κB binding activity was significantly depressed in Nfkb1−/− mice, basally and after O3 exposure (Figure 6D). This suggested that absence of p50 subunit inhibited heterodimerization of p65–p50 κB for DNA binding.
Because activated JNK (p-JNK) was also suppressed in Tnfr−/− mice compared with Tnfr+/+ mice after O3 exposure, we compared inflammatory responses to O3 in Jnk−/− and Jnk+/+ mice to address functional relevance of this signaling pathway. No significant differences in mean BAL total protein concentration or numbers of neutrophils and epithelial cells were found between air- and O3-exposed Jnk1−/− mice (Figure 7). These lung injury indices were significantly lower (60–75%) in Jnk1−/− mice compared with those in Jnk1+/+ mice after O3 (Figure 7). O3 did not significantly change the mean numbers of BAL macrophages in either genotype (Figure 7).
The functional importance of TNF-α as a key modulator of O3-induced airway toxicity has been documented by multiple animal studies. Anti–TNF-α antibody pretreatment decreased airway inflammation, hyperpermeability, and cell proliferation after acute or subacute O3 exposure in rodents (17, 19, 20, 36). Lung inflammation and epithelial injury were also reduced after subacute O3 exposure in mice genetically deficient in TNF-R (Tnfr1−/−, Tnfr2−/−, or Tnfr1−/−Tnfr2−/−) (16). Furthermore, acute O3-induced airway hyperreactivity was decreased in these TNF-R knockout mice (16, 18). In the present study, we determined that relative to wild-type mice, activation of NF-κB and MAPK/AP-1 pathways was significantly reduced in Tnfr−/− mice, and significantly lower lung injury and inflammation were found in Nfkb1−/− and Jnk1−/− mice after O3 exposure. Our observations are the first to demonstrate that NF-κB and MAPK/AP-1 pathways are key signaling components of TNF-mediated pulmonary pathogenesis by inhaled O3 (Figure 8).
NF-κB and MAPK/AP-1 pathways are critical in developmental processes and immune responses by orchestrating expression of multiple genes involved in inflammation and immunity, development, lymphoid differentiation, oncogenesis, and apoptosis. Use of NF-κB subunit– or Jnk-deficient mice has supplied direct evidence for the role of NF-κB and MAPK in pulmonary inflammation and allergy models. For example, Nfkb1−/− mice were resistant to allergic airway eosinophilic inflammation and mycobacterial infection (37, 38). c-Rel κB deficiency also reduced airway hyperresponsiveness and chemokine induction after allergen challenge (39). Lack of JNK (Jnk1 or Jnk2) inhibited neutrophilic influx and chemokine expression after mechanical ventilation (40). These two redox-sensitive transcription factor signaling pathways have also been shown to be induced by O3 in airway cells and tissues (41–44). More recently, Fakhrzadeh and colleagues (45) determined a functional role of pulmonary NF-κB in the increase of inducible nitric oxide synthase and TNF-α levels by inhaled O3. Our current observations support NF-κB and MAPK as key mediators of TNF-R responses.
The present study, however, indicated that TNF signaling does not account for all O3-induced NF-κB and MAPK/AP-1 activities. As depicted in Figures 2 and and33 (also see Table 1), O3 exposure significantly activated signal transducers of NF-κB and MAPK/AP-1 pathways even in the absence of TNF-R. This suggests that receptor-mediated signals other than TNF-R activate these pathways in response to O3. It is possible that greater fold increases of certain NF-κB and MAPK signal proteins in Tnfr−/− mice than in Tnfr+/+ mice compared with genotype-matched air-exposed control animals may be associated with compensatory activation of these non–TNF-R signals in the absence of TNF-R. In support of this concept, Alcamo and associates (46) determined that TNF-R1/NF-κB p65-double deficient mice were significantly more resistant to lung neutrophilic inflammation and chemokine/cytokine expression (e.g., ICAM-1, MIP-2) than TNF-R1 single knockout mice during acute lung injury induced by endotoxin. These studies thus indicated that activation of pulmonary NF-κB may also occur independently of TNF-R signaling after stimulation with exogenous stimuli. It has become clear that proinflammatory responses by the pulmonary innate immune system are partially mediated through pattern recognition receptors including the Toll-like receptor (TLR) family of proteins (47–49). We previously determined that TLR4 contributes significantly to the pulmonary hyperpermeability response to subacute O3 exposure (50), and that mechanisms underlying hyperpermeability are dissociated from those for TNF-R–mediated cellular inflammation (16). Accumulating evidence shows that MAPK and NF-κB signaling pathways are essential in TLR4/MyD88-dependent cell signaling (51, 52). In addition, lung injury induced by a particle (residual oil fly ash) was significantly attenuated in mice with dominant mutant Tlr4 (C3H/HeJ) compared with Tlr4 normal mice (C3H/HeOuJ), and this resistance was shown to be mediated through suppressed activation of downstream MAPK/AP-1 and NF-κB pathways (29). Collectively, these investigations suggest that interaction exists between TNF and TLR4 signaling mechanisms through NF-κB and MAPK pathways during the pathogenesis of pulmonary oxidative injury. In the current study, abolishment of O3-induced hyperpermeability in Jnk1−/− mice (see Figure 7) and Nfkb1−/− mice (see Figure 5A) supports this possibility.
The current study also identified multiple proinflammatory genes that were differentially regulated in Tnfr−/− and Tnfr+/+ mice during O3-induced lung inflammation. Included among these is the potent neutrophil chemoattractant MIP-2, which is also TNF dependent in murine pulmonary models of silica and cigarette smoke toxicity (10, 13). We also observed TNF-R–mediated induction of TNF-α (autoregulation) in O3-exposed lungs. A similar observation was reported in the lungs after cigarette smoke exposure, and mice deficient in TNF-R had decreased expression of TNF-α, whereas TNF-α was induced in wild-type mice after exposure (13). Presence of functional AP-1 and NF-κB binding sites in mouse MIP-2 (34, 53) and TNF-α (54, 55) gene promoters further supports TNF-mediated MIP-2 and TNF-α regulation via these transcription factors. The injurious effects of TNF-dependent IL-1β have also recently been determined after acute O3 exposure (56). Interestingly, in the present study, IL-6 mRNA was overexpressed in O3-resistant Tnfr−/− mice, which may suggest a protective role for this cytokine. IL-6 has been shown to have antiinflammatory properties. For example, IL-6 deficiency augmented hydrogen peroxide–induced murine alveolar epithelial cell death (57), and anti–IL-6 antibody treatment significantly increased neutrophilic inflammation caused by O3 exposure in rats (58). However, converse effects have also been reported, and IL-6 was determined to be proinflammatory during the early phase of O3 exposure in mice (59, 60).
Figure 8 depicts a schematic representation of the molecular mechanisms that we have investigated and identified as putative signal transduction pathways leading to pulmonary toxicity caused by inhaled O3. In summary, we uncovered that NF-κB and MAPK/AP-1 play key roles in subacute O3-induced lung inflammation and injury mediated through TNF-R. Although further investigation is required to clarify the complex link between these two pathways and downstream inflammatory mediator networks, the current study provided details of molecular events underlying pulmonary O3 toxicity. Our observations may have important implications for understanding the pathogenesis of inflammatory sequelae after environmental O3 exposure in normal subjects and individuals with preexisting lung disease.
Ozone exposures were conducted at the National Institute of Environmental Health Sciences (NIEHS) Inhalation Facility under contract to Alion Science and Technology, Inc. The authors thank Mr. Herman Price for coordinating the inhalation exposures. Drs. Farhad Imani and Donald Cook at the NIEHS provided excellent critical review of the manuscript.
Supported by the Intramural Research Program of the National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services.
Originally Published in Press as DOI: 10.1164/rccm.200509-1527OC on January 25, 2007
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.