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Alveolar macrophages (AM) and inflammatory mediators including nitric oxide and peroxynitrite contribute to ozone-induced lung injury. The generation of these mediators is regulated, in part, by the transcription factor NF-κB. We previously demonstrated a critical role for NF-κB p50 in the ozone-induced injury. In the present studies mechanisms regulating NF-κB activation in the lung after ozone inhalation were analyzed. Treatment of wild type (WT) mice with ozone (0.8 ppm, 3 h) resulted in a rapid increase in NF-κB binding activity in AM, which persisted for at least 12 h. This was not evident in mice lacking TNFα which are protected from ozone-induced injury; there was also no evidence of nitric oxide or peroxynitrite production in lungs from these animals. These data demonstrate that TNFα plays a role in NF-κB activation and toxicity. TNFα signaling involves PI-3-kinase (PI3K)/protein kinase B (PKB), and p44/42 MAP kinase (MAPK) which are important in NF-κB activation. Ozone Inhalation resulted in rapid and transient increases in p44/42 MAPK and PI3K/PKB in AM from WT mice, which was evident immediately after exposure. Caveolin-1, a transmembrane protein that negatively regulates PI3K/PKB and p44/42 MAPK signaling, was downregulated in AM from WT mice after ozone exposure. In contrast, ozone had no effect on caveolin-1, PI3K/PKB or p44/42 MAPK expression in AM from TNFα knockout mice. These data, together with our findings that TNFα suppressed caveolin-1 in cultured AM, suggest that TNFα and downstream signaling mediate activation of NF-κB and the regulation of inflammatory genes important in ozone toxicity, and that this process is linked to caveolin-1.
Tumor necrosis factor alpha (TNFα) is a macrophage-derived proinflammatory cytokine implicated in the pathogenesis of oxidant-induced tissue injury (Mukhopadhyay et al., 2006). The biological actions of TNFα are mediated by two structurally related, but functionally distinct receptors: TNFR1 (p55) and TNFR2 (p75) (Locksley et al., 2001). TNFα signaling through the p55 receptor is the primary pathway leading to inflammatory responses (Vandevoorde et al., 1997; Peschon et al., 1998). Binding of TNFα to TNFR1 causes recruitment of cytoplasmic adaptor proteins important in initiating signal transduction. This leads to activation of various signaling molecules, including phosphatidylinositol 3’-kinase (PI3K)/protein kinase B (PKB), and p44/42 mitogen activated protein kinase (MAPK), and downstream transcription factors such as nuclear factor-κB (NF-κB), which is known to regulate genes controlling proinflammatory mediator and antioxidant production, as well as apoptosis and cellular proliferation (Sebban and Courtois, 2006; Perkins, 2007). In the lung, TNFα is thought to contribute to a variety of inflammatory conditions including acute respiratory distress syndrome, idiopathic pneumonia, chronic obstructive pulmonary disease and asthma (reviewed in Mukhopadhyay et al., 2006)
Ozone is a highly reactive oxidant present in photochemical smog. Inhalation of toxic levels of ozone leads to airway inflammation and damage to the alveolar epithelium in the lower lung (Laskin et al., 2003; Uysal and Schapira, 2003; Hollingsworth et al., 2007). A number of studies have suggested that at least one mechanism underlying the toxicity of ozone involves excessive production of cytotoxic mediators including reactive oxygen and nitrogen intermediates by activated lung macrophages (reviewed in (Laskin et al., 2003)). In this regard, we have demonstrated that acute exposure of rodents to ozone results in increased production of nitric oxide and peroxynitrite by lung macrophages (Pendino et al., 1993b; Fakhrzadeh et al., 2002; Fakhrzadeh et al., 2004a; Fakhrzadeh et al., 2004b). Moreover, transgenic mice lacking nitric oxide synthase II (NOSII) or over expressing superoxide dismutase are protected from ozone toxicity (Fakhrzadeh et al., 2002; Fakhrzadeh et al., 2004a). TNFα expression has also been reported to be upregulated in mouse lung following ozone inhalation (Cho et al., 2001; Fakhrzadeh et al., 2004b; Lu et al., 2006). The present studies were aimed at analyzing the role of TNFα in ozone-induced nitric oxide production and toxicity. Mechanisms regulating TNFα signaling in ozone-exposed animals were also investigated. We found that macrophages from transgenic mice with a targeted disruption of the gene for TNFα were unable to generate reactive nitrogen intermediates and that these mice were protected from ozone toxicity. This was associated with a marked attenuation of ozone-induced activation of PI3K/PKB and p44/42 MAPK and suppression of caveolin-1 (Cav-1), a negative regulator of TNFα signaling (Williams and Lisanti, 2004). These findings are novel and may lead to the identification of new targets for therapeutic intervention aimed at regulating lung inflammation induced by pulmonary irritants.
TNFα knockout mice (C57BL/6X129) were kindly provided by Dr. Michael Marino (Memorial Sloan Kettering Cancer Center, NY). Wild type B6J129SV F2 mice were purchased from Jackson Laboratories (Bar Harbor, ME). Animals were housed in microisolator cages and received food and sterile pathogen-free water ad libitum. Female mice, 8–12 weeks of age, were placed in whole-body Plexiglas chambers and exposed to ultra-pure air (control) or 0.8 ppm ozone for 3 h. Ozone was generated from oxygen gas via an ultraviolet light ozone generator (Orec Corp., Phoenix, AZ). Ozone concentrations in the chamber were maintained by adjusting both the intensity of the ultraviolet light (mJ/cm2) and the flow rate (ml/min). Concentrations of ozone in ppm were continuously monitored using an ozone analyzer (Model 1008 AH; Dasibi Environmental Corp., Glendale, CA). All animals received humane care in compliance with the institution's guidelines, as outlined in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.
Mouse recombinant interferon-gamma (IFN-γ) was purchased from GIBCO (Grand Island, NY). Salmonella enteritidis lipopolysaccharide (LPS) and DNAse I were obtained from Sigma Chemical Co. (St. Louis, MO). Rabbit polyclonal antibodies against NOSII and Cav-1, goat polyclonal antibodies against TNFα PI3K-p85, and horseradish peroxidase (HRP) conjugated anti-rabbit and anti-goat IgG were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Rabbit polyclonal antibody against nitrotyrosine was from Upstate Biotechnology (Lake Placid, NY) and rabbit polyclonal antibodies against NF-κB p50 and p65 from Stressgen (Victoria BC, Canada). Rabbit antibodies against PKB/AKT, phospho-PKB/AKT, p44/42 MAPK, and phospho-p44/42 MAPK were from Cell Signaling Technology (Danvers, MA). FITC-conjugated anti-rabbit and anti-goat IgG were from Molecular Probes (Eugene, OR).
Alveolar macrophages were isolated from the lung as previously described (Fakhrzadeh et al., 2002). Briefly, mice were anesthetized, the lung excised, and the trachea and major bronchi removed. The lung was then cut into uniform 500 µm slices (MacIlwain Tissue Chopper, Brinkmann Instruments, Westbury, NY) and incubated in ice cold Ca2+/Mg2+-free Hank’s balanced salt solution (HBSS) containing 0.005% DNAse I (HBSS-DNAse) for 30 min. This was followed by mixing, using a Vortex Genie 2 (Fisher Scientific, Pittsburgh, PA) at speed 3. The cells released during these steps were filtered through a 220 µm filter, washed and subjected to Metrizamide gradient centrifugation using a Beckman TJ-6 centrifuge for elimination of red blood cells, dead cells and debris. Cell viability was greater than 98%, as assessed by trypan blue dye exclusion. Purity was greater than 97% macrophages based on differential staining with Giemsa (Fisher Scientific, Springfield, NJ). To prepare extracts, cells were lysed in buffer (10 mM HEPES, pH 7.4, 10 mM KCl, 2 mM MgCl2, 2 mM EDTA) on ice for 10 min with intermittent mixing for 2 sec using a Vortex Genie at a setting of 3. NP-40 was added (final concentration 0.1%) and the cells incubated for an additional 5 min on ice. Cells were then centrifuged at 4 °C (16,000 × g) using an Eppendorf microcentrifuge for 5 min and supernatants containing cytoplasmic extracts collected and aliquots stored at −70 °C. The pellet was resuspended in buffer (50 mM HEPES pH 7.4, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA and 10% glycerol). After 20 min on ice, the sample was centrifuged at 4 °C (16,000 × g) for 5 min, supernatants containing nuclear extracts collected, and aliquots stored at −70 °C. Protein determinations were performed using a BCA protein assay kit (Pierce, Rockford, IL) with bovine serum albumin (BSA) as the standard.
Animals were sacrificed and the trachea cannulated with polyethylene tubing (PE-90; Clay Adams, Parsippany NJ) attached to a 3 cc Bectin-Dickinson (Franklin Lakes, NJ) single use syringe. The lung was then instilled with 1 ml of Ca2+/Mg2+-free phosphate-buffered saline (PBS) at 37°C and the fluids slowly withdrawn and instilled three times. The lavage fluid was centrifuged (350 × g for 10 min, 4°C) and protein content in supernatants quantified using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA) with BSA as the standard.
Cells were cultured in 96-well dishes (2 × 105 cells/well) in phenol red-free Dulbecco’s modified Eagle’s medium in the presence or absence of LPS (100 ng/ml) and IFN-γ (100 U/ml) or medium control. Nitric oxide was quantified after 48 h by the accumulation of nitrite in the culture medium using the Griess reaction with sodium nitrite as the standard as previously described (Fakhrzadeh et al., 2002). For nitrate determinations, samples were treated with nitrate reductase and NADPH for 30 min prior to analysis. We found that the ratio of nitrate to nitrite produced by alveolar macrophages was 1:1 and that this ratio did not change in cells from ozone treated mice.
Alveolar macrophages, lysed in buffer containing 10 mM Tris-HCl (pH 7.4) and 1% sodium dodecylsulfate (SDS), were run on 10% or 15% SDS-polyacrylamide gels (5 µg protein/lane) as described previously (Fakhrzadeh et al., 2002; Fakhrzadeh et al., 2004b). Proteins were then transferred to nitrocellulose and blocked overnight at 4°C with 5% non-fat powdered milk. The nitrocellulose membrane was then incubated overnight with anti-Cav-1 (1:1000), anti-PKB (1:500), or anti-PI3K (1:200) antibodies, followed by HRP-conjugated anti-rabbit IgG (1:5000) for 1 h. The blots were developed using an Enhanced Chemiluminescence detection kit (Amersham Life Science, Arlington Heights, IL). Blots were stained with Ponceau S (Sigma) to confirm equal loading of proteins on the gels.
Binding reactions were carried out at room temperature for 30 min in a total volume of 15 µl and contained 2–5 µg of nuclear extract protein, 5 µl of the 5X gel shift binding buffer (20% glycerol, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 50 mM Tris-HCl pH 7.5, 2 µg poly (dI-dC) and 3 × 104 cpm/ml of a γP-labeled NF-κB (AGTTGAGGGTTTCCCAGGC) (Promega Gel Shift Assay Systems) consensus oligonucleotide. Probes were labeled using γP-ATP (3000 Ci/mmol, NEN, Boston, MA). Protein-DNA complexes were separated on 5% non-denaturing polyacrylamide gels run at 250 V at 400 mAmps for 1.5 h in 0.5X TBE running buffer and visualized after the gels were dried and autoradiographed. For supershift reactions, the samples were incubated with antibodies (1 µg) to NF-κB p50 or p65 on ice for 20 min prior to the labeled oligonucleotide. For competitor reactions, samples were preincubated with a 100-fold excess of the respective unlabeled oligonucleotides.
Tissue sections (6 µm) were prepared for immunohistochemistry from paraffin-embedded perfused lungs that were inflation-fixed with 3% paraformaldehyde for 4 h at 4°C (Pendino et al., 1993b; Pendino et al., 1994; Pendino et al., 1995). Sections were deparaffinized prior to analysis. For immunostaining, slides containing tissue sections or cells cytocentrifuged onto slides, were preincubated for 30 min in 3% hydrogen peroxide to quench endogenous peroxidase. This was followed by incubation for 20 min in PBS containing 1% BSA and 0.05% sodium azide and overnight incubation with antibody to TNFα (1:1000), Cav-1, PKB/AKT, phospho-pKB/AKT, nitrotyrosine, or with the appropriate nonimmune IgG. A Vectastain ABC kit (Vector Laboratories, Buringame, CA) was utilized to visualize antibody binding.
Macrophages were washed in PBS, permeabilized and fixed with a Leucoperm kit (Serotec, NC) according the manufacturers protocol. Cells (1 × 106 cells/ml) were labeled with 1:50 dilution of antibody to TNFα, Cav-1, total-p44/p42, phospho-p44/p42 or IgG control for 1 h at room temperature. Cells were then washed with PBS and incubated with FITC-conjugated secondary antibody. After 45 min, the cells were washed with PBS and analyzed by flow cytometry on a Coulter Cytomics FC500 flow cytometer (Beckman Coulter). For each analysis, at least 10,000 events were collected and analyzed using CXP software. The percentage positive cells were calculated using Overton’s cumulative subtraction routine (Coulter software).
All experiments were repeated 3–5 times using 3–6 animals per experiment. Data were analyzed using a non-paired, two-tailed student’s t test. A P value of ≤ 0.05 was considered statistically significant.
We have previously demonstrated that macrophages play an important role in ozone toxicity (Pendino et al., 1995). Macrophages are known to release a number of mediators that have been implicated in tissue injury (Duffield, 2003; Laskin et al., 2003). Of particular interest is TNFα which promotes inflammation and cytotoxicity (Mukhopadhyay et al., 2006). In the present studies we analyzed the role of TNFα in ozone-induced lung injury. Immunohistochemical evaluation of lung sections revealed low levels of TNFα in alveolar macrophages from air-exposed mice (Fig. 1, upper panels). Ozone inhalation resulted in a rapid induction of TNFα in alveolar macrophages, which was most pronounced immediately after exposure. Subsequently, expression of TNFα began to decline. These findings were confirmed by flow cytometric analysis of isolated alveolar macrophages stained with anti-TNFα antibody. Whereas low basal levels of TNFα were detectable in cells from air-exposed mice, expression increased 3-fold after ozone inhalation (Fig. 1, lower panels). These experiments also revealed a single homogeneous population of alveolar macrophages that expressed TNFα from both control and ozone-treated mice. To analyze the role of TNFα in ozone-induced tissue injury, we used transgenic mice with a targeted disruption of the gene for this cytokine. TNFα was not detectable in lung sections, or in isolated alveolar macrophages from TNFα knockout mice, even after ozone inhalation (Fig. 1). Whereas lung injury, as measured by significant increases in bronchoalveolar lavage fluid protein, was observed in wild type mice treated with ozone, this was not evident in TNFα knockout mice (Fig. 2). These findings demonstrate that TNFα knockout mice are protected from ozone-induced toxicity.
Following ozone inhalation, alveolar macrophages generate increased quantities of nitric oxide and peroxynitrite which contribute to lung toxicity (Pendino et al., 1993b; Fakhrzadeh et al., 2002; Fakhrzadeh et al., 2004a; Fakhrzadeh et al., 2004b). We next assessed the effects of loss of TNFα on this response. Consistent with our previous studies (Fakhrzadeh et al., 2002; Fakhrzadeh et al., 2004a; Fakhrzadeh et al., 2004b), we found that macrophages isolated from wild type mice readily generated nitric oxide in response to the inflammatory stimuli, LPS and IFNγ (Fig. 3, upper panel). This activity increased two-fold in macrophages from wild type mice following ozone inhalation. In contrast, macrophages from TNFα knockout mice were unable to generate significant quantities of nitric oxide, and ozone had no major effect on this activity. Nitric oxide can rapidly react with superoxide anion to form peroxynitrite, a potent nitrating species (Koppenol et al., 1992). We found that ozone inhalation resulted in the appearance of nitrotyrosine residues in proteins in the lung, an in vivo marker of peroxynitrite formation (Fig. 3, lower panel). This was prominent in alveolar macrophages. Nitrotyrosine staining was not detectable in TNFα knockout mice.
TNFα receptor binding activates several downstream signaling molecules including the transcription factor NF-κB, which regulates the activity of inflammatory genes implicated in ozone toxicity including nitric oxide synthase-2 and cyclooxygenase-2 (Hazucha et al., 1996; Fakhrzadeh et al., 2002). We have previously shown that alveolar macrophages from mice with a targeted disruption of NF-κB p50 do not generate reactive nitrogen intermediates and that these mice are protected from ozone toxicity (Fakhrzadeh et al., 2004b). In further studies we analyzed the role of TNFα in ozone-induced NF-κB activation. NF-κB nuclear binding activity was not detectable in alveolar macrophages from air exposed wild type mice (Fig. 4). Following ozone inhalation, a time-related increase in NF-κB binding activity was observed in alveolar macrophages. This was evident immediately after ozone exposure and persisted for at least 12 h. In contrast, no major changes in NF-κB nuclear binding activity were observed in cells from TNFα knockout mice following ozone exposure. In activated macrophages from wild type mice, NF-κB binding activity was blocked by incubating the samples with a 100-fold excess of unlabeled probe, demonstrating the specificity of the probe. Moreover, supershift assays using antibodies to p50 or p65 slowed the migration of the NF-κB complex in the gels indicating that both of these proteins were involved in the responses to ozone.
A number of biochemical signaling molecules have been implicated in TNFα-induced activation of NF-κB including PI3K and its downstream target, PKB (Hazucha et al., 1996; Wallach et al., 1999; Grivennikov et al., 2006), and expression of these proteins was analyzed next. Alveolar macrophages from air-exposed wild type mice were found to express low levels of PI3K and PKB as determined by western blotting and immunocytochemistry (Fig. 5). Ozone inhalation caused a marked increase in expression of both total and phospho-PI3K and PKB. Whereas expression of PI3K reached a maximum after 3 h, peak PKB expression was observed after 6 h. Subsequently, expression of both of these proteins declined toward control levels. To determine if PI3K is involved in ozone-induced increases in nitric oxide production by alveolar macrophages, we used wortmanin and LY294002, two structurally distinct inhibitors of this enzyme (Walker et al., 2000). As described above, following ozone inhalation, alveolar macrophages produced two-fold more nitric oxide in response to LPS and IFNγ than cells from air-exposed mice (Fig. 6). Pretreatment of cells from ozone exposed mice with wortmanin and LY294002 prevented this increase. These inhibitors also blocked ozone-induced increases in NF-κB activation (Fig. 4). In contrast, wortmanin and LY294002 had no effect on nitric oxide production or NF-κB binding activity in macrophages from air-exposed animals (Fig. 6 and not shown).
NF-κB activation is also induced via mitogen activated protein kinases such as p44/42 (Wallach et al., 1999; Chen et al., 2001; Grivennikov et al., 2006). Flow cytometric analysis revealed the presence of one homogeneous population of alveolar macrophages from air-exposed wild type mice that expressed low levels of phospho- and total p44/42 MAPK. Ozone inhalation resulted in a rapid increase in phospho-p42/44 MAPK in alveolar macrophages, which was evident immediately after exposure and peaking after 6 h (Fig. 7 upper panel). In contrast, in TNFα−/− mice, constitutive expression of MAPK was not detected, and ozone had no effect on expression of this protein.
We next analyzed the effects of ozone inhalation on expression of Cav-1, a membrane scaffolding protein known to negatively regulate PI3K and p44/42 MAPK (Williams and Lisanti, 2004; Schwencke et al., 2006). Western blot analysis revealed that alveolar macrophages from air exposed wild type mice constitutively expressed Cav-1 (Fig. 8, upper panel). Ozone inhalation caused a dramatic reduction in Cav-1 protein expression, which was observed immediately following exposure and persisted for at least 6 h. Subsequently levels of Cav-1 began to return towards control. These findings were confirmed in intact cells by flow cytometry (Fig. 8, middle panel). This analysis showed that the macrophages from air and ozone-exposed mice consisted of one homogeneous population of cells that expressed Cav-1. Following ozone inhalation, expression of Cav-1 was not detectable. The rapid and transient inhibitory effects of ozone exposure on constitutive Cav-1 expression in alveolar macrophages were also evident in histologic sections stained with anti-Cav-1 antibody (Fig. 8, lower panel). In contrast to the effects observed in wild type mice, ozone had minimal or no effects on Cav-1 expression in TNFα knockout mice. These data suggest that TNFα is required for ozone-induced suppression of Cav-1 (Fig. 8). To analyze this further, the effects of TNFα on Cav-1 expression was assessed in alveolar macrophages isolated from wild type mice. Figure 9 shows that alveolar macrophages from control mice expressed significant quantities of Cav-1. Moreover, treatment of the cells with TNFα for 20 min suppressed Cav-1 expression.
TNFα is a multifunctional cytokine implicated in the pathogenesis of a number of pulmonary disorders including acute respiratory distress syndrome and idiopathic pneumonia, as well as tissue injury induced by endotoxin, bleomycin, silica and asbestos (Liu et al., 2001; Pryhuber et al., 2003; Shimabukuro et al., 2003; Bhatia and Moochhala, 2004; Zhao et al., 2004; Shukla et al., 2005; Ke et al., 2006; Mukhopadhyay et al., 2006). We found that ozone inhalation was associated with a rapid and transient induction of TNFα in the lung. These findings are in accord with previous reports showing increased TNFα, as well as TNF receptor-1 (TNFR1, p55) and TNFR2 (p75), in the lung and/or in bronchoalveolar lavage fluid of humans and experimental animals exposed to ozone (Pendino et al., 1994; Kleeberger et al., 1997; Peschon et al., 1998; Cho et al., 2001; Shore et al., 2001; Cho et al., 2007). The fact that TNFα and its receptors are detectable in the lung prior to ozone-induced increases in epithelial cell permeability and lavageable inflammatory cells (Ishii et al., 1997), suggests a role of this cytokine in the progression of the pathogenic response. Our findings that mice lacking TNFα are protected from ozone-induced injury are consistent with this idea. A similar protective effect against ozone-induced injury has been reported in TNFR1 and/or TNFR2 knockout mice (Cho et al., 2001; Shore et al., 2001; Cho et al., 2007), and in mice pretreated with anti-TNFα antibodies (Piguet and Vesin, 1994; Young and Bhalla, 1995; Kleeberger et al., 1997; Cho et al., 2001; Bhalla et al., 2002). TNFα plays an important role in inflammatory cell trafficking into sites of injury and activation, and this may contribute to the toxicity of ozone. This is supported by findings that administration of antibodies to TNFα attenuates ozone-induced increases in macrophage adherence, neutrophil accumulation and IL-1 and IL-6 expression in the lung, and that ozone-induced inflammation and macrophage activation are reduced in TNFR1 knockout mice (Young and Bhalla, 1995; Kleeberger et al., 1997; Pearson and Bhalla, 1997; Cho et al., 2001; Bhalla et al., 2002).
Reactive nitrogen intermediates including nitric oxide and peroxynitrite are cytotoxic mediators produced in excessive quantities after ozone inhalation (Pendino et al., 1993b; Laskin et al., 1996; Fakhrzadeh et al., 2002; Fakhrzadeh et al., 2004b). Reactive nitrogen intermediates generated via NOSII have been shown to be important in ozone-induced injury and inflammation (Inoue et al., 2000; Kleeberger et al., 2001; Fakhrzadeh et al., 2002). TNFα stimulates nitric oxide production by macrophages (Lavnikova et al., 1993; Frankova and Zidek, 1998), and this may also be important in the pathogenesis of injury induced by ozone. In this regard, we found that macrophages from TNFα−/− mice were unable to generate nitric oxide, even after ozone inhalation. These findings demonstrate a direct role for TNFα in activating NOSII in the lung in vivo after ozone exposure. Our results are in accord with reports that TNFα is involved in regulating nitric oxide production in idiopathic pneumonia syndrome (Shukla et al., 2005). Consistent with our previous studies (Pendino et al., 1993a; Fakhrzadeh et al., 2002; Fakhrzadeh et al., 2004a; Fakhrzadeh et al., 2004b), we also found that alveolar macrophages from ozone treated mice produced increased quantities of nitric oxide suggesting that these cells are primed to respond to inflammatory mediators. This was correlated with peroxynitrite-induced tissue injury as assessed by nitrotyrosine staining of the lung. The fact that nitrotyrosine staining was not apparent in TNFα-− /− mice provides further support for the concept that these mice are protected from ozone-induced injury and that reactive nitrogen intermediates are critical mediators of toxicity (Inoue et al., 2000; Kleeberger et al., 2001; Fakhrzadeh et al., 2002; Zeidler and Castranova, 2004).
TNFα receptor binding leads to the recruitment of adaptor proteins and activation of a cascade of signaling molecules including PI 3-kinase/PKB, p44/42 MAPK, and NF-κB, a transcription factor important in the regulation of many inflammatory genes and proteins involved in ozone toxicity including NOSII (Aggarwal et al., 2002; Fakhrzadeh et al., 2002; Shishodia and Aggarwal, 2002). The present studies show that increased TNFα expression in the lung following ozone inhalation is correlated with activation of NF-κB which we have previously demonstrated is important in the toxicity of this oxidant (Fakhrzadeh et al., 2004b). Interestingly, this activity was absent in mice lacking TNFα These findings are consistent with recent reports on the effects of continuous exposure of TNFR1−/− mice to ozone for 48 hr (Cho et al., 2007) and provide additional support for the idea that activation of NF-κB in this model of lung injury is dependent on TNFα signaling.
TNFα expression in the lung following ozone inhalation was also associated with upregulation of PI3K and PKB in alveolar macrophages. These findings, together with the observation that ozone-induced increases in nitric oxide production by alveolar macrophages were markedly reduced by PI3K inhibitors suggest that this signaling pathway is likely to be involved in controlling TNFα-induced NOSII activity in the lung following ozone inhalation. Similar decrements in nitric oxide production by PI3K inhibitors have previously been described in macrophages, as well as astrocytes and microglia (Jung et al., 1999; Kim et al., 2005; Bani-Hani et al., 2006; Kesherwani and Sodhi, 2007). We also found that culturing macrophages from both air and ozone-exposed animals with PI3K inhibitors blocked NF-κB nuclear binding activity. This indicates that PI3K/PKB signaling plays a role in ozone-induced NF-κB activation. Our findings are in accord with previous reports demonstrating that PI3K inhibitors decrease fMLP-induced nitric oxide production and NF-κB nuclear binding activity in murine peritoneal macrophages (Sodhi and Biswas, 2002). Ozone inhalation also caused upregulation of p44/42 MAPK in alveolar macrophages which is in accord with recent findings in whole lung (Cho et al., 2007), This signaling molecule has been reported to be a key transducer of NF-κB activation and nitric oxide production (Barton and Medzhitov, 2003; Takeda et al., 2003), and it may also be important in ozone-induced toxicity. The fact that p44/42 activation was not evident in TNFα−/− mice which are resistant to ozone-induced injury is consistent with this idea. A similar dependency of ozone-induced MAPK activation on TNFα has recently been described (Cho et al., 2007). Taken together, these data support the idea that the NF-κB and MAPK pathways are important in TNFα mediated pulmonary injury induced by ozone.
Both PI3K and p44/42 MAPK are negatively regulated by the membrane scaffolding protein, Cav-1 (Galbiati et al., 1998; Williams and Lisanti, 2004). By maintaining these molecules in an inactive form, downstream biological responses mediated by PI3K and p44/42 MAPK are inhibited (Engelman et al., 1998; Okamoto et al., 1998). Ozone inhalation caused a rapid and transient decrease in Cav-1 expression in alveolar macrophages. This was correlated with increased expression of PI3K, PKB and p44/42 MAPK activity suggesting a regulatory role for Cav-1 in ozone-induced activation of these signaling pathways. Our findings are in accord with the observation that Cav-1 knockout mice exhibit hyperactivation of PI3K/PKB and p44/42 MAPK (Park et al., 2002; Cohen et al., 2003). Results from the present studies also indicate that TNFα plays a role in down-regulating Cav-1 following ozone inhalation. This is supported by the fact that decreases in Cav-1 are in general not observed in ozone-treated TNFα−/− mice, and that TNFα reduces Cav-1 expression in cultured alveolar macrophages. The mechanism mediating TNFα-induced down-regulation of Cav-1 is unknown. Studies have suggested that phosphorylation of Cav-1 by TNFα via p44/42 MAPK results in a conformational change in Cav-1 leading to dissociation of the scaffolding domain from the inhibitory binding motifs (Engelman et al., 1998; Galbiati et al., 1998; Volonte et al., 2005). Alternatively, TNFα may cause activation of putative kinases resulting in phosphorylation of residues within the scaffolding domain. This would lead to dissociation and activation of p44/42 MAPK. The observation that p44/42 MAPK is rapidly upregulated in the lung following ozone inhalation supports the idea that p44/42 MAPK is a key enzyme involved in this process. Interestingly, a small and transient decrease in Cav-1 expression was observed in alveolar macrophages isolated immediately after exposure of TNFα−/− mice to ozone. These finding suggest that there are TNFα-independent mechanisms mediating Cav-1 down regulation in the lung at early times following ozone inhalation.
The present studies demonstrate that TNFα is a critical mediator of ozone-induced lung injury, NF-κB activation, and macrophage production of nitric oxide. Moreover, TNFα signaling in lung macrophages appears to be negatively regulated by the membrane scaffolding protein, Cav-1. These findings are novel and suggest new potential therapeutic targets that may be useful for abrogating oxidant-induced lung injury.
This work was supported by National Institutes of Health Grants ES004738, GM034310, CA100994, AR055073, and ES005022.
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