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Published ahead of print on July 10, 2003, doi:10.1165/rcmb.2003-0044OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 30, pp. 280-287, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0044OC

Superoxide Dismutase–Overexpressing Mice Are Resistant to Ozone-Induced Tissue Injury and Increases in Nitric Oxide and Tumor Necrosis Factor-{alpha}

Ladan Fakhrzadeh, Jeffrey D. Laskin, Carol R. Gardner and Debra L. Laskin

Environmental and Occupational Health Science Institute, Rutgers University, and University of Medicine and Dentistry of New Jersey-Robert Wood Johnson Medical School, Piscataway, New Jersey

Address correspondence to: Dr. Debra Laskin, Rutgers University, Department of Pharmacology and Toxicology, 160 Frelinghuysen Road, Piscataway, NJ 08854. E-mail: laskin{at}eohsi.rutgers.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Reactive oxygen intermediates have been implicated in lung injury induced by inhaled irritants. The present studies used mice overexpressing Cu/Zn-superoxide dismutase (SOD+/+) to analyze their role in ozone-induced lung inflammation and cytotoxicity. Treatment of wild-type mice with ozone (0.8 ppm, 3 h) resulted in increased bronchoalveolar lavage fluid protein, which was maximal after 24–48 h. Significant increases in lung macrophages and 4-hydroxyalkenals were also observed. In contrast, bronchoalveolar lavage fluid protein and macrophage content and 4-hydroxyalkenals were at control levels in ozone-treated SOD+/+ mice. There was also no evidence of peroxynitrite-mediated lung damage, demonstrating that SOD+/+ mice are resistant to ozone toxicity. Whereas alveolar macrophages from wild-type mice produced increased amounts of nitric oxide and expressed more inducible nitric oxide synthase, phospholipase A2, and tumor necrosis factor-{alpha} after ozone inhalation, this was not evident in cells from SOD+/+ mice. Ozone-induced decreases in interleukin-10 were also not observed. In wild-type mice, ozone inhalation resulted in activation of nuclear factor-{kappa}B, which regulates proinflammatory gene activity. This response was significantly reduced in SOD+/+ mice. These data demonstrate that antioxidant enzymes play a critical role in ozone-induced tissue injury and in inflammatory mediator production.

Abbreviations: bovine serum albumin, BSA • cyclooxygenase, COX • Dulbecco's modified Eagle's medium, DMEM • glutathione, GSH • Hanks' balanced salt solution, HBSS • 4-hydroxynonenal, 4-HNE • horseradish peroxidase, HRP • interferon {gamma}, IFN-{gamma} • interleukin-10, IL-10 • lipopolysaccharide, LPS • monochlorobimane, MCB • inducible nitric oxide synthase, NOSII • inhibitory {kappa}B, I{kappa}B • nuclear factor {kappa}B, NF-{kappa}B • prostaglandin E2, PGE2 • phosphate-buffered saline, PBS • superoxide dismutase, SOD • Cu/Zn-superoxide dismutase overexpressor, SOD+/+ • tumor necrosis factor-{alpha}, TNF-{alpha} • 12-O-tetradecanoyl-phorbol-1, TPA


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Ozone is a highly reactive oxidant present in photochemical smog. Exposure of humans and experimental animals to toxic levels of ozone causes alveolar epithelial cell damage. This is associated with an accumulation of macrophages at sites of injury and increased generation of inflammatory mediators, which have been implicated in tissue damage. Of particular interest are reactive oxygen and nitrogen intermediates, which can induce damage to membranes and proteins (1). To prevent or limit oxidative damage, lung cells possess relatively high levels of antioxidants such as superoxide dismutase (SOD), catalase, and glutathione (GSH) (2, 3). However, during states of increased oxidative stress antioxidants become depleted, exacerbating tissue injury (4).

SOD is generally considered one of the first lines of antioxidant defense (5). This enzyme converts superoxide anion into hydrogen peroxide, which is then removed by catalase. Several classes of SOD have been identified that differ in their metal binding ability, distribution, and sensitivity to various xenobiotics. These include copper/zinc (Cu/Zn)-SOD, manganese-SOD, and extracellular SOD. The most prominent and widely distributed form is Cu/Zn-SOD. Following exposure of animals to ozone, SOD increases in the lung (6–8). Moreover, transgenic mice overexpressing manganese-SOD or Cu/Zn-SOD (SOD+/+) have been reported to be resistant to oxygen-induced toxicity (9–11). These findings demonstrate the importance of SOD in limiting lung injury induced by oxidants. In the present studies we used SOD+/+ mice to evaluate the effects of altered oxidant/antioxidant balance in ozone toxicity and inflammatory mediator production. We found that this antioxidant enzyme plays a critical role in the regulation of the redox-sensitive transcription factor nuclear factor (NF)-{kappa}B and several downstream gene products involved in production of inflammatory mediators induced after ozone inhalation. This activity may be important in protecting against ozone toxicity in SOD+/+ mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and Treatments
Female C57BL/6xCBA/J SOD+/+ mice (12) and C57BL/6 wild-type control mice (8–16 wk old) were obtained from Dr. Oleg Mirochnitchenko (UMDNJ-Robert Wood Johnson Medical School, Piscataway, NJ). Animals were housed in microisolator cages and received food and sterile pathogen-free water ad libitum. Animals 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 generator (Orec Corp., Phoenix, AZ). Ozone concentrations within the chamber were maintained by adjusting both the intensity of the ultraviolet light and the flow rate of ozone into the chamber. Concentrations of ozone were continuously monitored using an ozone analyzer (Model 1008 AH; Dasibi Environmental Corp., Glendale, CA).

Reagents
Mouse recombinant interferon-{gamma} (IFN-{gamma}) was purchased from GIBCO (Grand Island, NY). Salmonella enteritidis lipopolysaccharide (LPS), DNase I and D,L-buthionine-(S, R)-sulfoximine (BSO) were obtained from Sigma Chemical Co. (St. Louis, MO) and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) from LC Services (Woburn, MA). Dihydrorhodamine-123 and monochlorobimane (MCB) were from Molecular Probes (Eugene, OR). Rabbit polyclonal antibodies against inducible nitric oxide synthase (NOSII) (sc-650), cyclooxygenase-1 (COX-1) (sc-7950), phospholipase A2 (cPLA2) (sc-438) and interleukin-10 (IL-10) (sc-1783), goat polyclonal antibody against COX-2 (sc-1747) and tumor necrosis factor-{alpha} (TNF-{alpha}) (sc-780), and horseradish peroxidase (HRP)-conjugated anti-rabbit and anti-goat IgG were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit polyclonal antibody against nitrotyrosine was purchased from Upstate Biotechnology (Lake Placid, NY).

Isolation of Lung Macrophages and Preparation of Extracts
Alveolar macrophages were isolated from the lung as previously described (13). Briefly, the lung was 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 Hanks' balanced salt solution (HBSS) containing 0.005% DNase I for 30 min. This was followed by mixing using a Vortex Genie 2 (Fisher Scientific, Pittsburgh, PA) at speed 3 for 3 min. The cells released during these steps were filtered through a 220-µm filter, washed, and subjected to 18% metrizamide gradient centrifugation for elimination of red blood cells, dead cells, and debris. The cells recovered were 98% viable as determined by trypan blue dye exclusion and > 98% 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. Nonident-40 (final concentration 0.1%) was added and the cells incubated for an additional 5 min on ice. Cells were then centrifuged at 4°C (16,000 x g) for 5 min and supernatants containing cytoplasmic extracts collected and aliquots frozen at -70°C. The pellet was then 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 (16,000 x g) at 4°C for 5 min, supernatants containing nuclear extracts collected, and aliquots frozen at -70°C. Protein was assayed using a BCA protein assay kit (Pierce, Rockford, IL) with bovine serum albumin (BSA) as the standard.

Quantitation of Bronchoalveolar Lavage Cell Number and Protein
Animals were killed and the trachea cannulated with polyethylene tubing (PE-90; Clay Adams, Parsippany, NJ) attached to the 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 then centrifuged (350 x g, 10 min, 4°C) and protein in supernatants quantified using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA) with BSA as the standard. Cells recovered from the lavage fluid were washed twice (350 x g, 10 min), resuspended in Ca2+/Mg2+-free PBS, and viable cells enumerated by trypan blue dye exclusion using a hemocytometer. Differential analysis of stained cells showed that the population was > 98% macrophages.

Quantitation of Malondialdehyde and 4-Hydroxyalkenals
Lungs were homogenized (10% wt/vol) in ice-cold phosphate buffer (25 mM, pH 6.0) containing EDTA and 0.5% hexadecyltrimethyl ammonium bromide. Samples were then sonicated for 15 s and centrifuged at 3,000 x g for 30 min. Malondialdehyde (MDA) and 4-hydroxyalkenals (4-HAK) were assayed in supernatants using a commercial lipid peroxidation kit (Calbiochem-Novabiochem Corp, San Diego, CA).

Measurement of Nitric Oxide Production
Cells were cultured in 96-well dishes (2 x 105 cells/well) in phenol red-free Dulbecco's modified Eagle's medium (DMEM) with and without LPS (100 ng/ml) and IFN-{gamma} (100 U/ml). 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 (14). For nitrate determinations, samples were treated with nitrate reductase and NADPH for 30 min before 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. In the absence of LPS and IFN-{gamma}, macrophages produced negligible quantities of nitrite and nitrate (not shown).

Measurement of Superoxide Anion Release
Superoxide anion release by alveolar macrophages was measured spectrophotometrically by the SOD-inhibitable reduction of ferricytochrome C (15). Cells were washed and resuspended in HBSS (5 x 105 cells/ml) containing 44 µm ferricytochrome C, with or without 1 µM SOD and 170 nM TPA. Absorbance was determined spectrophotometrically at 550 nm 45 min later. The amount of superoxide anion released was calculated using a baseline value (E = 21.1 nM-1 cm-1 at 550 nm) obtained from samples containing SOD.

Measurement of Peroxynitrite Production
Peroxynitrite production by alveolar macrophages was quantified using the fluorescence indicator, dihydrorhodamine 123. This dye is sensitive to peroxynitrite, and to a lesser extent hydroperoxides (16). Cells were cultured in 8-well slide chambers (1.5 x 105 cells/well) with LPS (100 ng/ml) and IFN-{gamma} (100 U/ml) or medium control for 24 h. TPA (170 nM) was added to the cultures 30 min before analysis. Supernatants were then removed, and the cells washed with PBS and incubated with dihydrorhodamine 123 (0.5 mg/ml). After 10 min at room temperature, the cells were washed with PBS and analyzed on a Meridian Insight Plus confocal microscope (Meridian Instruments, Okemos, MI).

Measurement of Intracellular GSH
The fluoresence indicator dye, MCB was used to quantify intracellular GSH (17). Cells were cultured overnight in Nunc (Naperville, IL) coverglass chambers (2 x 106 cells/chamber) in the presence or absence of BSO. The cells were then washed and refed with HBSS containing 20 µM MCB with or without BSO. After 30 min incubation at 37°C, the cells were washed three times and analyzed for fluorescence on a Meridian ACAS 570 anchored cell analysis system (Meridian Instruments).

Immunostaining
Tissue sections (6 µm) were prepared from paraffin-embedded perfused lungs that were inflation-fixed with 3% paraformaldehyde for 4 h at 4°C. Sections were deparaffinized before staining. Alveolar macrophages were incubated in PBS containing 1% buffered formalin and 0.1% Triton-X 100 for 10 min at 37°C. Cells were then washed and resuspended (2 x 105 cells/ml) in HBSS. Slides were prepared using a Cytospin 2 (Shandon, Cheshire, UK). For immunostaining, slides containing tissue sections or cells 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 rabbit antibody against nitrotyrosine (1:2,000) or IL-10 (1:200), with goat antibody against NOSII (1:400) or TNF-{alpha} (1:1,000), or with nonimmune rabbit or goat IgG. A Vectastain ABC kit (Vector Laboratories, Burlingame, CA) was used to visualize antibody binding. In some experiments, the anti-nitrotyrosine antibody (1:2,000) was incubated overnight with 2 mg/ml nitrotyrosine before use. This treatment was found to block immunostaining, demonstrating that the antibody was specific.

Western Blot Analysis
Cytoplasmic extracts were run on 8% SDS-polyacrylamide gels (5 µg protein/lane), transferred to nitrocellulose, and blocked for 1 h at room temperature with 5% powdered milk. The nitrocellulose membrane was then incubated overnight with a 1:200 dilution of anti–COX-1, anti–COX-2, or anti-cPLA2 antibody followed by HRP-conjugated anti-rabbit or anti-goat IgG (1:5,000) for 1 h. The blots were developed using an Enhanced Chemiluminescence detection kit (Amersham Life Science, Arlington Heights, IL). Blots were then stained with Ponceau S (Sigma Chemical Co., St. Louis, MO) to confirm equal loading of proteins on the gel.

Electrophoretic Mobility Shift Assay
Binding reactions were performed at room temperature for 30 min in a total volume of 15 µl and contained 2–5 µg nuclear extracts, 5 ml of 5x gel shift binding buffer (20% glycerol, 5 mM MgCl2 EDTA, 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 x 104 cpm/ml {gamma}[32]P-labeled NF-{kappa}B (AGTTGAGGGTTTCCCAGGC) (Promega Gel Shift Assay Systems, Madison, WI) consensus oligonucleotides. Probes were labeled using {gamma}[32]P-ATP (3,000 Ci/mmol; NEN, Boston, MA). Protein–DNA complexes were separated on 5% nondenaturing polyacrylamide gels run at 250 V in 0.5x Tris-borate/EDTA and visualized after the gels were dried and autoradiographed. For supershift reactions, the mixtures were incubated on ice for 20 min with 5 µg antibody to NF-{kappa}B p50 or p65 before the labeled oligonucleotide. For competitor reactions, the mixtures were preincubated with a 100-fold excess of unlabeled oligonucleotide.

Statistics
All experiments were repeated 3–5 times using 6–12 animals per experiment. Data were analyzed using a nonpaired, two-tailed Student's t test. A P value of <= 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
SOD+/+ Mice Are Protected from Ozone-Induced Lung Injury
Treatment of wild-type mice with ozone resulted in significant lung injury as measured by increases in bronchoalveolar lavage fluid protein. This was most prominent 24–48 h after exposure and was correlated with increased numbers of macrophages in lavage fluid (Figures 1A and 1B). In contrast, ozone inhalation had no effect on bronchoalveolar fluid protein or macrophage content in SOD+/+ mice. Production of the lipid peroxidation products, MDA and 4-HAK, was also assessed. Low levels of MDA and 4-HAK were detectable in lung homogenates from air exposed wild-type mice (Figures 1C and 1D). Whereas ozone inhalation had no effect on MDA, a significant increase in 4-HAK was observed in wild-type mice. This was not evident in lung homogenates from SOD+/+ mice. We also analyzed GSH content in alveolar macrophages, which is known to decrease following ozone-induced oxidative stress (18). Relatively high levels of GSH were detected in alveolar macrophages from wild-type mice (Figure 2). Preincubation of the cells with BSO, an inhibitor of GSH biosynthesis (17), reduced intracellular GSH content (not shown). Interestingly, constitutive GSH levels in alveolar macrophages from SOD+/+ mice were reduced when compared with wild-type mice. Following ozone inhalation, GSH levels in macrophages from wild-type mice decreased. In contrast in cells from SOD+/+ mice, GSH levels were not altered by ozone exposure.



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  		Figure 1. Protection of SOD+/+ mice from ozone-induced lung injury. Wild-type or SOD+/+ mice were exposed to air or ozone. (A) Bronchoalveolar lavage fluid protein levels were assessed 24–72 h after exposure (n = 6–12). Open bars, wild-type mice; filled bars, SOD+/+ mice. (B) Lung lavage cells were quantified 48 h after exposure (n = 3). (C and D) Lung homogenates were assayed for MDA and 4-HAK, respectively, 48 h after exposure (n = 3). Open bars, air; filled bars, ozone. Each bar represents the mean ± SE of the number of animals (n) indicated in parentheses. *Significantly different (P <= 0.05) from air control. **Significantly different (P <= 0.05) from wild-type mice.

 


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  		Figure 2. Effects of ozone inhalation on intracellular GSH. Macrophages were isolated 48 h after exposure of wild-type or SOD+/+ mice to air or ozone. After overnight incubation, the cells were washed, stained with MCB, and analyzed on the Meridian ACAS 570 (A). The color bar represents relative fluorescence on a four-decade log scale. (B) Values represent the mean relative fluorescence intensity of 30–40 cells as determined by Meridian ACAS 570 software. One of three similar experiments is shown. Open bars, air; filled bars, ozone. *Significantly different (P <= 0.05) from air control. **Significantly different (P <= 0.05) from wild-type mice.

 
Effects of Overexpression of Cu/Zn-SOD on Ozone-Induced Production of Reactive Oxygen and Nitrogen Intermediates
We previously demonstrated that alveolar macrophages produced increased quantities of inflammatory mediators following exposure of mice to ozone (18–20). In further studies we assessed the effects of overexpression of Cu/Zn-SOD on this response. Initially we analyzed nitric oxide production. Alveolar macrophages generate nitric oxide from L-arginine via the enzyme NOSII (21). In lung sections from air-exposed mice, only low levels of NOSII were detectable (Figure 3, upper panel). As reported previously (19), treatment of wild-type animals with ozone resulted in a marked induction of NOSII protein throughout the lower lungs, which was most prominent in alveolar macrophages (Figure 3, upper panel). This was not observed in the lungs of SOD+/+ mice. These finding suggested that ozone-induced priming of alveolar macrophages for excessive production of reactive nitrogen intermediates may be abrogated in the transgenic mice. To evaluate this possibility, we compared the ability of alveolar macrophages isolated from wild-type and SOD+/+ mice to release nitric oxide into the culture medium following ozone inhalation. Macrophages from both mouse strains were found to readily generate nitric oxide in response to inflammatory mediators (Figure 3, middle panel). This activity was significantly increased in macrophages from wild-type mice following ozone inhalation. In contrast, ozone had no effect on nitric oxide production by cells from SOD+/+ mice. Similarly, whereas alveolar macrophages from wild-type mice generated significant quantities of peroxynitrite after ozone exposure, as measured by intracellular fluorescence of oxidized dihydrorhodamine 123, this was not observed in cells from SOD+/+ mice or in cells from mice exposed to air control (Figure 4, upper panel). Peroxynitrite has been shown to act as a nitrating agent leading to the appearance of nitrotyrosine residues in proteins, a marker of peroxynitrite-mediated tissue injury (22). Immunohistochemical staining of lung sections revealed that peroxynitrite was formed in vivo following ozone inhalation. Thus, after ozone treatment of wild-type mice, diffuse nitrotyrosine staining was noted throughout the lower lung (Figure 4, lower panel). Alveolar macrophages stained more intensely for nitrotyrosine when compared with other cells in the tissue. In contrast, nitrotyrosine was not detectable in lung sections from SOD+/+ mice even after ozone inhalation. These findings are consistent with our results on peroxynitrite generation by macrophages isolated from wild-type and SOD+/+ mice.



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  		Figure 3. Ozone-induced effects on NOSII expression and alveolar macrophage production of nitric oxide and superoxide anion. Upper panel: Histologic sections were prepared 48 h following exposure of wild-type or SOD+/+ mice to air or ozone. Sections were stained with anti-NOSII antibody (A, B, D, E) or IgG control (C, F). Arrows indicate alveolar macrophages. Original magnification: x1,000. Middle and lower panels: Alveolar macrophages were isolated 48 h after exposure of wild-type or SOD+/+ mice to air (open bars) or ozone (filled bars). Nitrite was quantified in culture supernatants after 48 h incubation of the cells with IFN-{gamma} (100 U/ml) and LPS (100 ng/ml) (n = 4–6). Superoxide anion release was assessed after stimulating the cells for 45 min with 170 nM TPA (n = 3). Each bar represents the mean ± SEM of the number of experiments indicated in parenthesis. *Significantly different (P <= 0.05) from air control. **Significantly different (P <= 0.05) from wild-type mice.

 


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  		Figure 4. Effects of ozone inhalation on peroxynitrite production and nitrotyrosine staining of the lung. Upper panel: Alveolar macrophages, isolated 48 h after exposure of wild-type or SOD+/+ mice to air or ozone, were incubated for 24 h with LPS (100 ng/ml) + IFN-{gamma} (100 U/ml). The cells were then stimulated with TPA for 30 min and peroxynitrite production analyzed using dihydrorhodamine 123. Fluorescence is proportional to peroxynitrite production. In the absence of stimulation alveolar macrophages did not generate peroxynitrite (15). Lower panel: Histologic sections were prepared 48 h following exposure of wild-type or SOD+/+ mice to air or ozone. Sections were stained with anti-nitrotyrosine antibody (A, B, D, E), IgG control (C), or anti-nitrotyrosine antibody preincubated with nitrotyrosine (F). Arrows indicate alveolar macrophages. Original magnification: x400.

 
We also analyzed production of superoxide anion by alveolar macrophages from wild-type and SOD+/+ mice following ozone inhalation. Superoxide anion release into the culture medium by alveolar macrophages from air-exposed SOD+/+ mice was significantly reduced when compared with cells from wild-type animals (Figure 3, lower panel). Interestingly, whereas ozone inhalation had no effect on superoxide anion release by macrophages from wild-type mice, this activity increased to control levels in cells from SOD+/+ mice.

Effects of Ozone Inhalation on cPLA2, COX-1, and COX-2 Expression in Wild-Type and SOD+/+ Mice
Another group of mediators involved in the inflammatory response to irritants are eicosanoids generated via COX-1 and COX-2 (23). The reaction is initiated by cPLA2, which acts on membrane phospholipids to release arachidonic acid, the substrate for these enzymes. In further studies we compared effects of ozone on expression of cPLA2, COX-1, and COX-2 in cells from wild-type and SOD+/+ mice. Alveolar macrophages from air-exposed mice were found to express low levels of cPLA2 (Figure 5, upper panel). Ozone inhalation caused an increase in cPLA2 expression in macrophages from both wild-type and SOD+/+ mice. However, the response of alveolar macrophages from SOD+/+ mice was significantly reduced when compared with cells from wild-type mice. COX-1 and COX-2 proteins were also detectable in alveolar macrophages from air-exposed wild-type and SOD+/+ mice (Figure 5, middle and lower panels). Greater quantities were evident in cells from SOD+/+ mice when compared with wild-type mice. In macrophages from wild-type mice, expression of COX-1 and COX-2 increased after ozone inhalation. An increase in COX-1 expression was also observed in macrophages from SOD+/+ mice.



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  		Figure 5. Effects of ozone inhalation on cPLA2, COX-1, and COX-2 expression. Alveolar macrophages, isolated 48 h after exposure of wild-type or SOD+/+ mice to air or ozone, were analyzed by Western blotting. The 85-kD cPLA2, 70-kD COX-1, and 72- to 74-kD COX-2 bands are indicated by the arrows. +, positive control for antibody binding. One representative blot from three experiments is shown. Data from the blots were scanned and presented in arbitrary units (mean ± SEM). *Significantly different (P <= 0.05) from air control. **Significantly different (P <= 0.05) from wild-type mice.

 
Effects of Overexpression of Cu/Zn-SOD on Ozone-Induced Alterations in TNF-{alpha} and IL-10 Expression in the Lung
TNF-{alpha} is a proinflammatory cytokine produced by activated macrophages (24). It is known to induce oxidative metabolism in phagocytes and has been reported to be upregulated in the lung following ozone inhalation (20). In wild-type mice we found that ozone inhalation induced TNF-{alpha} expression throughout the lower lung in particularly high levels in macrophages (Figure 6, upper panel). In contrast, only low levels of TNF-{alpha} were detected in the lungs of SOD+/+ mice after ozone exposure. TNF-{alpha} was not evident in lung sections from air-exposed mice from either genotype.



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  		Figure 6. Effects of ozone inhalation on TNF-{alpha} and IL-10 expression. Lung sections were prepared 48 h following exposure of wild-type or SOD+/+ mice to air (A, C, D) or ozone (B, E). Sections were stained with TNF-{alpha} or IL-10 antibody (A, B, D, E) or IgG control (C, F). Arrows indicate alveolar macrophages. Original magnification: x400.

 
IL-10 is a potent anti-inflammatory cytokine known to inhibit macrophage functional activity (24). Relatively high levels of IL-10 were detected in lung sections of air-exposed wild-type mice (Figure 6, lower panel). Treatment of the mice with ozone resulted in a significant decrease in IL-10 expression. In contrast, in lung sections from SOD+/+ mice, IL-10 staining was diffuse, and this did not change after ozone inhalation.

Effects of Ozone Inhalation on NF-{kappa}B Nuclear Binding Activity
NF-{kappa}B is a ubiquitous transcription factor known to regulate a number of inflammatory genes important in ozone toxicity, including NOSII, COX-2, and TNF-{alpha} (25). NF-{kappa}B nuclear binding activity was not detectable in alveolar macrophages from air-exposed wild-type or SOD+/+ mice (Figure 7). Following ozone inhalation, a time-dependent induction of NF-{kappa}B binding activity was observed in cells from wild-type mice. This was evident immediately after ozone exposure and reached a maximum within 6–12 h. Although inhalation of ozone also caused an increase in NF-{kappa}B binding activity in cells from SOD+/+ mice, this response was significantly reduced when compared with wild-type animals and was transient.



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  		Figure 7. Effects of ozone inhalation on NF-{kappa}B nuclear binding activity. Alveolar macrophages were isolated 0–48 h after exposure of wild-type or SOD +/+ mice to air or ozone. NF-{kappa}B binding activity was analyzed by EMSA. Nuclear extracts, prepared 6 h after ozone treatment of wild-type mice, were incubated with anti-p50 or p65 antibody or with 100x excess unlabeled probe (CC) before the labeled probe.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Reactive oxygen intermediates are thought to be primary mediators of macrophage-induced tissue injury, and they have been implicated in the pathogenesis of xenobiotic-induced lung toxicity (4). Thus, following inhalation of irritants, pulmonary levels of reactive oxygen intermediates increase in the lung (4, 26). Moreover, antioxidants have been shown to abrogate tissue injury in several of these models. The present studies focused on analyzing the role of antioxidant enzymes in ozone-induced lung toxicity. For these studies we used a transgenic mouse strain that over-expresses Cu/Zn-SOD (12). Macrophages isolated from these mice have been reported to exhibit reduced ability to generate superoxide anion and nitric oxide (12). Our findings that SOD+/+ mice are protected from lung damage induced by ozone provide support for the idea that macrophage-derived reactive oxygen and nitrogen intermediates are critical cellular mediators of tissue injury and inflammation in this model.

In accord with previous studies (19), we found that acute inhalation of ozone by wild-type mice resulted in significant lung injury, as evidenced by increased numbers of macrophages and levels of protein in bronchoalveolar lavage fluid, as well as lung lipid peroxidation. In contrast, we detected little evidence of tissue damage in SOD+/+ mice. These results demonstrate that SOD+/+ mice are protected from ozone-induced toxicity and suggest that reactive oxidants play an important role in initiating lung injury. This is supported by our observation that there was no evidence of peroxynitrite-mediated tissue injury in lungs from these mice. A similar protective effect of overexpression of Cu/Zn-SOD has been described in a model of oxygen-induced lung injury (10, 11). In contrast to 4-HAK, MDA levels did not change in the lungs of wild-type mice following acute ozone exposure. Increases in lung MDA have been reported after prolonged exposure of mice to ozone (27). Differences between these studies and our results may be due to the different treatment protocols. Alternately, it may be that 4-HAK is a more sensitive marker of acute lung injury than MDA.

GSH is an important regulator of the cellular redox status and can protect against proinflammatory processes in the lung caused by oxidative stress (2, 4). As observed in the rat (18), ozone inhalation resulted in decreased GSH levels in alveolar macrophages from mice. Similar decreases in lung GSH have been described in response to other oxidants (4). In alveolar macrophages from SOD+/+ mice, GSH was significantly reduced when compared with cells from wild-type animals. This may reflect the lack of need for additional antioxidants in SOD+/+ mice. It is also possible that GSH is autoxidized by Cu/Zn-SOD, directly reducing levels of this antioxidant (28). In contrast to wild-type mice, ozone had no effect on GSH in macrophages from SOD+/+ mice. This is consistent with reduced oxidative injury observed in these animals.

As expected, alveolar macrophages from air-exposed SOD+/+ mice released significantly less superoxide anion than cells from wild-type mice. Whereas ozone inhalation had no effect on cells from wild-type mice, superoxide anion production by macrophages from SOD+/+ mice increased to levels similar to those released by the wild-type mice. This may be due to ozone-induced increases in the activity of enzymes such as NADPH oxidase or cytochrome 450, which are known to generate superoxide anion (29). Increased production of superoxide anion in SOD+/+ mice following ozone inhalation may also reflect reduced peroxynitrite production as a consequence of decreased nitric oxide release. It should be noted that superoxide anion data are presented on a per cell basis. Because ozone inhalation increases the number of macrophages in the lower lungs of wild-type mice, this measurement may underestimate the total amount of superoxide anion produced in the tissue.

Our studies also showed that alveolar macrophages from wild-type mice were primed by ozone inhalation to produce increased quantities of both nitric oxide and peroxynitrite. This was correlated with increased expression of NOSII protein and nitrotyrosine staining of the lung. These findings are consistent with previous studies in rodents (18, 19). In contrast, cells from SOD+/+ mice expressed low levels of NOSII and did not produce significant amounts of nitric oxide or peroxynitrite, or stain for nitrotyrosine even after ozone inhalation. However, as indicated above, these macrophages did generate greater quantities of superoxide anion. This suggests that in wild-type animals, the majority of the superoxide anion generated following ozone inhalation reacts with nitric oxide to form peroxynitrite, and indicate that this reactive nitrogen intermediate is a major mediator of toxicity in this model. Peroxynitrite has been implicated in lung injury induced by ischemia-reperfusion, LPS, immune complexes, and cigarette smoke (26). SOD has been reported to inhibit peroxynitrite production by epithelial cells treated with cigarette smoke (30). Similarly, macrophages from rats pretreated with an SOD mimetic and challenged with carrageenan were unable to generate peroxynitrite (31). These findings demonstrate a critical role of superoxide anion in oxidant/antioxidant balance and in the generation of reactive nitrogen species.

The present studies also show that cPLA2 expression was markedly increased in alveolar macrophages following ozone exposure. This is consistent with reports of enhanced PGE2 production by alveolar macrophages after ozone inhalation (19). Similar increases in cPLA2 have been reported in cultured airway epithelial cells treated with ozone (32). In contrast, ozone caused a much smaller effect on cPLA2 expression in SOD+/+ mice. Expression of cPLA2 has been reported to be regulated by NF-{kappa}B and activator protein-1, which are activated by oxidants (25). High levels of SOD in the transgenic mice may limit oxidant-induced activation of these transcription factors and abrogate cPLA2 expression. cPLA2 activation is associated with increased generation of 4-HAK lipid peroxidation products in neuronal tissue (33). Our findings that ozone-induced increases in cPLA2 were correlated with 4-HAK levels are in accord with this report. Activation of cPLA2 results in the generation eicosanoids via COX-1 and COX-2 (23). We found that ozone-induced increases in cPLA2 expression were correlated with increases in COX-1 in alveolar macrophages from both wild-type and SOD+/+ mice. Although COX-2 also increased in wild-type mice after ozone, there was little effect in SOD+/+ mice. This is likely due to high constitutive expression of COX-2 in these mice. These findings suggest that signaling pathways in addition to NF-{kappa}B and activator protein-1 may be involved in regulating COX-1 and COX-2 expression in SOD+/+ mice.

Oxidants have been shown to induce expression of TNF-{alpha}, a cytokine known to stimulate cPLA2 (24). Ozone inhalation resulted in a significant increase in TNF-{alpha} expression in the lung. The fact that ozone had no effect on TNF-{alpha} in SOD+/+ mice suggests that this antioxidant enzyme plays a role in proinflammatory cytokine generation in this model. Reduced TNF-{alpha} expression may explain the lack of induction of cPLA2 in SOD+/+ mice following ozone inhalation.

Following ozone inhalation, constitutive IL-10 expression decreased in the lungs of wild-type mice. These results are in agreement with previous studies on the effects of ozone on IL-10 expression (34). In contrast, only diffuse low level IL-10 staining was noted in lung sections from SOD+/+ mice, and this did not change following ozone inhalation. These findings indicate that oxidant/antioxidant balance is involved in controlling anti-inflammatory cytokine expression.

Reactive oxygen and nitrogen intermediates are thought to be important in activation of the redox-sensitive transcription factor, NF-{kappa}B (25). NF-{kappa}B controls the activity of numerous genes crucial for immunity, inflammation, and stress responses, including TNF-{alpha}, NOSII, COX-2, and cPLA2. Ozone inhalation resulted in increased NF-{kappa}B nuclear binding activity in alveolar macrophages from wild-type mice. Although increased NF-{kappa}B activity was also observed in macrophages from SOD+/+ mice after ozone exposure, the response was attenuated and transient, decreasing by 6 h. This may be due to direct inhibition of NF-{kappa}B by IL-10, which has been reported to block its DNA-binding activity (35). Decreased NF-{kappa}B activity may underlie reduced NOSII, TNF-{alpha}, and cPLA2 expression in SOD+/+ mice and contribute to the resistance of these mice to ozone. This is supported by our findings that transgenic mice with a targeted disruption of the NF-{kappa}Bp50 subunit are protected from ozone-induced toxicity (unpublished observation).

In summary, the present studies demonstrate that overexpression of Cu/Zn-SOD significantly abrogates the ability of ozone to induce tissue injury and inflammation. Thus, in contrast to wild-type mice, in SOD+/+ mice there were no increases in bronchoalveolar lavage fluid cell or protein content, and alveolar macrophages did not generate excessive quantities of nitric oxide or peroxynitrite, or express significantly greater levels of NOSII, TNF-{alpha}, or PLA2. Evidence of peroxynitrite-mediated tissue injury was also absent. These findings provide support for our hypothesis that macrophages and inflammatory mediators contribute to ozone-induced lung inflammation and toxicity (26).


   Acknowledgments
 
This work was supported by the National Institutes of Health Grants ES04738, GM34310, ES06897, CA100994, and ES05022. The technical support of Dr. Oleg Mirochnitchenko's laboratory for breeding the mice is gratefully acknowledged.

Received in original form February 5, 2003

Received in final form June 10, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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