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Am. J. Respir. Cell Mol. Biol., Volume 26, Number 4, April 2002 413-419

Deficiency in Inducible Nitric Oxide Synthase Protects Mice from Ozone-Induced Lung Inflammation and Tissue Injury

Ladan Fakhrzadeh, Jeffrey D. Laskin, 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

    Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Inhalation of ozone causes Type I epithelial cell necrosis and Type II cell hyperplasia and proliferation. This is associated with an accumulation of activated macrophages in the lower lung, which we have demonstrated contribute to tissue injury. Nitric oxide (NO) is a highly reactive cytotoxic macrophage-derived mediator that has been implicated in lung damage. In the present studies we used knockout mice with a targeted disruption of the gene for inducible nitric oxide synthase (NOSII) to analyze the role of NO in ozone-induced lung inflammation and tissue injury. Treatment of wild-type control mice with ozone (0.8 ppm) for 3 h resulted in a time-dependent increase in protein and cells in bronchoalveolar lavage fluid, which reached a maximum 24-48 h after exposure. Alveolar macrophages isolated from animals treated with ozone were found to produce increased amounts of NO, as well as peroxynitrite. This was correlated with induction of NOSII protein and nitrotyrosine staining of lung macrophages in tissue sections and in culture. Production of superoxide anion and prostaglandin (PG)E2 by alveolar macrophages was also increased after ozone inhalation. In contrast, alveolar macrophages from NOSII knockout mice did not produce reactive nitrogen intermediates even after ozone inhalation. Moreover, production of PGE2 was at control levels. NOSII knockout mice were also protected from ozone-induced inflammation and tissue injury, as measured by bronchoalveolar lavage protein and cell number. There was also no evidence of peroxynitrite-mediated lung damage in these animals. Taken together, these data demonstrate that NO, produced via NOSII, and potentially, its reactive oxidative product peroxynitrite, play a critical role in ozone-induced release of inflammatory mediators and in tissue injury.

    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Ozone is a highly reactive oxidant present in photochemical smog. Inhalation of toxic levels of ozone causes pulmonary edema, airway hyperresponsiveness, and alveolar epithelial damage (1). This is correlated with an accumulation of macrophages in the lower lung. We have previously demonstrated that these cells are activated following ozone inhalation and release mediators that contribute to toxicity (2). Activated macrophages produce a number of cytotoxic mediators and proinflammatory cytokines that have been implicated in lung injury (5). Of particular interest are reactive nitrogen intermediates, including nitric oxide (NO) and peroxynitrite, which have been reported to be involved in the pathogenesis of pulmonary edema, acute lung injury, emphysema, and damage to pulmonary surfactants (8).

NO is generated in large amounts by alveolar macrophages exposed to inflammatory mediators such as interferon gamma  (IFN-gamma ) and lipopolysaccharide (LPS), via a calcium-independent, inducible form of nitric oxide synthase (NOSII) (2, 13). NO production by macrophages is dependent on L-arginine and is blocked by a variety of inhibitors including aminoguanidine, which is relatively selective for NOSII (14). Once generated, NO can react with superoxide anion to form peroxynitrite, an even more potent and cytotoxic oxidant (15). Acute exposure of rats to ozone results in expression of NOSII mRNA and protein in the lung, and increased production of NO by alveolar macrophages and Type II cells (2, 4, 16). In the present studies we used mice with a targeted disruption of the NOSII gene to analyze the role of reactive nitrogen intermediates in ozone toxicity. The results of our studies provide support for our hypothesis that reactive nitrogen intermediates contribute to inflammation and toxicity in this model.

    Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Animals and Treatments

Female C57/BL6X129 NOSII knockout mice (8-16 wk old) were obtained from Dr. J. Mudget (Merck and Co., Rahway, NJ). Wild-type B6J129SV F2 control mice were from Jackson Laboratories (Bar Harbor, ME). 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 a UV light ozone generator (Orec Corp., Phoenix, AZ). Ozone concentrations within the chamber were maintained by adjusting both the intensity of the UV 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 IFN-gamma was purchased from GIBCO (Grand Island, NY). Salmonella enteritidis LPS and DNase I were from Sigma Chemical Co. (St. Louis, MO), 12-O-tetradecanoyl-phorbol-13-acetate (TPA) from LC Services (Woburn, MA), and dihydrorhodamine 123 from Molecular Probes (Eugene, OR). Rabbit polyclonal antibodies against cyclooxygenase-1 (COX-1) (clone H-62, sc-7950) and NOSII (clone M-19-G, sc-650-G), goat polyclonal antibody against COX-2 (clone M-19, sc-1747), and horse radish 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).

Cell Isolation

Alveolar macrophages were isolated from the lung as previously described (17, 18). 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 (HBSS-DNase) 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 Metrizamide gradient centrifugation for elimination of red blood cells, dead cells, and debris. The recovered cells were 98% viable as determined by trypan blue dye exclusion. Slides were prepared using a Cytospin 2 (Shandon, Cheshire, UK) and stained with Giemsa (Fisher Scientific, Springfield, NJ) for leukocyte differentials.

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 a syringe. The lung was then instilled with 1 ml 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 × g for 10 min, 4°C) and protein content in supernatants quantified using the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA) with bovine serum albumin (BSA) as the standard. Cells recovered from the lavage fluid were washed twice (350 × g, 10 min), resuspended in Ca2+/Mg2+-free PBS, and viable cells enumerated by trypan blue dye exclusion using a hemacytometer.

Measurement of NO Production

Cells were cultured in 96-well dishes (2 × 105 cells/well) in phenol red-free Dulbecco's modified Eagle's medium (DMEM) in the presence or absence of LPS (100 ng/ml) and IFN-gamma (100 U/ml) or medium control. NO was quantified after 24-72 h by the accumulation of nitrite in the culture medium using the Griess reaction with sodium nitrite as the standard (19). 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.

Measurement of Prostaglandin E2 Production

Cells were cultured in 12-well dishes (1 × 106 cells/well) in phenol red-free DMEM in the presence or absence of LPS (100 ng/ml) and IFN-gamma (100 U/ml) or medium control. Prostaglandin (PG)E2 was quantified in culture supernatants after 48 h using a commercial enzyme-linked immunosorbent assay kit (Amersham Pharmacia Biotech, Piscataway, NJ).

Measurement of Superoxide Anion Release

Superoxide anion release by alveolar macrophages was measured spectrophotometrically by the superoxide dismutase (SOD) inhibitable reduction of ferricytochrome C (18, 20). Cells were washed and resuspended (5 × 105 cells/ml) in HBSS containing 44 µm ferricytochrome C, with or without 1 µM SOD and 170 nM TPA. Absorbance was determined spectrophotometrically at 550 nm. 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 dihydrorhodamine 123 (21, 22). Cells were cultured in 8-well slide chambers (1.5 × 105/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 wells containing the cells 30 min before analysis. Supernatants were then removed, and the cells washed with PBS and incubated for 10 min at room temperature with dihydrorhodamine 123 (0.5 mg/ml). The cells were then washed with PBS and analyzed on a Meridian Insight Plus confocal microscope (Meridian, Okemos, MI).

Western Blot Analysis

Cytoplasmic extracts were prepared as previously described (23). Briefly, 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. NP-40 (10%, 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 × g) for 5 min and supernatants containing cytoplasmic extracts collected. Aliquots were frozen at -70°C until analysis. Protein determinations were performed using a BCA protein assay kit (Pierce, Rockford, IL) with BSA as the standard. Extracts were run on 7.5% sodium dodecyl sulfate-polyacrylamide gels (5 µg protein/lane), transferred to nitrocellulose and blocked overnight at 4°C with 5% powdered milk. The nitrocellulose membrane was then incubated with a 1:200 dilution of anti-NOSII, anti-COX-1, or anti-COX-2 antibody for 3 h at room temperature followed by HRP-conjugated anti-rabbit or anti-goat immunoglobulin (1:5,000) for 1 h. The blots were developed using an Enhanced Chemiluminescence detection kit (Amersham Life Science, Arlington Heights, IL).

Immunostaining

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 (2, 3). Sections were deparaffinized before analysis. Alveolar macrophages were prepared for analysis by incubation in 1% buffered formalin prepared in PBS containing 0.5% Triton-X 100 for 10 min at 37°C. Cells were then washed and resuspended (2 × 105 cells/ml) in HBSS. 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 then overnight incubation with rabbit antibody against nitrotyrosine (1:2,000) or with nonimmune rabbit IgG. A Vectastain ABC kit (Vector Laboratories, Burlingame, CA) was utilized to visualize antibody binding. In some experiments the anti-nitrotyrosine antibody (1:2,000) was incubated overnight with 2 mg/ml nitrotyrosine before use as a control.

Statistics

All experiments were repeated 3-6 times using 3-12 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

In initial experiments we compared the effects of ozone inhalation on the production of inflammatory mediators by alveolar macrophages isolated from wild-type control and NOSII knockout mice. In the absence of stimulation alveolar macrophages from wild-type animals did not produce detectable levels of NO. Treatment of the cells with LPS and IFN-gamma caused a time-related increase in NO production (Figure 1). Ozone inhalation resulted in a significant increase in the response of the cells to LPS and IFN-gamma . These effects were time-dependent, reaching a maximum in cells isolated 24-48 h following ozone exposure. By 72 h after exposure, alveolar macrophage NO production began to decline toward control levels. Ozone inhalation also caused a marked induction of NOSII protein expression, which was evident in freshly isolated alveolar macrophages and in cultured cells treated with LPS and IFN-gamma (Figure 2 and not shown). In contrast, NOSII protein was not detectable in freshly isolated macrophages from air-exposed animals. As expected, alveolar macrophages from NOSII knockout mice did not express NOSII protein or produce NO even after ozone inhalation (Figure 2 and Table 1). Treatment of these cells with LPS and IFN-gamma also had no effect on NOSII expression (not shown).


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  		Figure 1.   Effects of ozone inhalation on NO production by alveolar macrophages. Cells were isolated from the lungs of wild-type mice 24-72 h after exposure to air or ozone. After culturing with IFN-gamma (100 U/ml) and LPS (100 ng/ml) for 24-72 h, supernatants were collected and analyzed for nitrite content. Each point is the mean ± SEM of three to five experiments. *Significantly different (P =< 0.05) from air control. Open circles, air (control); closed triangles, 24 h after exposure; closed squares, 48 h after exposure; closed circles, 72 h after exposure.


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  		Figure 2.   Effects of ozone inhalation on NOSII protein expression. Alveolar macrophages isolated 48 h after exposure of wild-type control mice (WT) or NOSII knockout (NOSII-/-) mice to air or ozone were analyzed for NOSII expression by Western blotting. The 130-kD NOSII band is indicated by the arrow. +, positive control for NOSII antibody binding. One representative blot from four separate experiments is shown.

                              
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TABLE 1
Comparison of nitric oxide production by alveolar macrophages from wild-type and NOSII knockout mice

Peroxynitrite is thought to be an important mediator of NO-induced cellular damage (11, 15, 24, 25). We next determined if ozone inhalation primed alveolar macrophages to produce this reactive nitrogen intermediate. Macrophages from ozone-treated but not control animals were found to produce significant quantities of peroxynitrite after stimulation with LPS and IFN-gamma (Figure 3). As observed with NO production, alveolar macrophages from NOSII knockout mice did not produce peroxynitrite, and this was unaltered by ozone inhalation. Peroxynitrite has been shown to act as a nitrating agent leading to the formation of nitrotyrosine residues in proteins, a marker of peroxynitrite-mediated tissue injury (26, 27). Significant nitrotyrosine staining was observed in freshly isolated alveolar macrophages isolated from wild-type mice treated with ozone (Figure 4). In contrast, nitrotyrosine residues were not evident in macrophages from air exposed animals or from NOSII knockout mice even after ozone inhalation (Figure 4 and not shown). Immunohistochemical staining of lung sections also revealed that nitrotyrosine was formed in vivo following ozone inhalation. Thus, after ozone treatment of wild-type mice, diffuse nitrotyrosine staining was noted throughout the lower lung. Alveolar macrophages stained more intensely for nitrotyrosine, when compared with other cells in the tissue. Preincubation of the nitrotyrosine antibody with nitrotyrosine blocked immunostaining in isolated cells and in tissue sections, demonstrating that the antibody was specific. No nitrotyrosine staining was evident in lung sections from air-exposed animals or in sections stained with nonimmune IgG (Figure 5). Nitrotyrosine staining was also not evident in lung sections from NOSII knockout from either air control or ozone-exposed animals (Figure 5).


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  		Figure 3.   Effects of ozone inhalation on peroxynitrite production by alveolar macrophages. Cells isolated 48 h after exposure of wild-type control mice (A, B, E, F) or NOSII knockout (NOSII-/-) mice (C, D, G, H) to air (A, B, C, D) or ozone (E, F, G, H) were incubated for 24 h with medium (A, C, E, G) or LPS + IFN-gamma (B, D, F, H). The cells were then stimulated with TPA and assayed for peroxynitrite production using dihydrorhodamine 123. Fluorescence is proportional to peroxynitrite production.


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  		Figure 4.   Effects of ozone inhalation on nitrotyrosine staining of alveolar macrophages. Cells were isolated 48 h after exposure of wild-type control mice to air (A) or ozone (B, C, D). Freshly isolated macrophages were stained with anti-nitrotyrosine antibody (A, C), with anti-nitrotyrosine antibody preincubated with nitrotyrosine (B), or with control IgG antibody (D). Original magnification: ×200. Inset, magnification: ×1,000.


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  		Figure 5.   Effects of ozone inhalation on nitrotyrosine staining of the lung. Histologic sections were prepared 48 h following exposure of wild-type control mice (A, B, C, F ) or NOSII knockout mice (D, E) to air (A, D) or ozone (B, C, E, F ). Sections were stained with anti-nitrotyrosine antibody (A, B, D, E), IgG control (C), or anti-nitrotyrosine antibody preincubated with nitrotyrosine (F ). Original magnification: ×1,000.

We next analyzed the effects of ozone inhalation on superoxide anion production by alveolar macrophages from wild-type and NOSII knockout mice. In wild-type mice, inhalation of ozone resulted in decreased superoxide anion production by stimulated alveolar macrophages isolated immediately after exposure (Figure 6). However, these effects were transient, and by 24 h, superoxide anion release was increased when compared with control. Interestingly, superoxide anion release by alveolar macrophages from air-exposed NOSII knockout mice was significantly greater than by cells from wild-type control animals. Ozone inhalation caused a 50% reduction in this activity, a response which was maintained for at least 48 h after exposure.


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  		Figure 6.   Effects of ozone inhalation on superoxide anion release by alveolar macrophages. Cells were isolated 0-48 h after exposure of wild-type control mice (open bars) or NOSII knockout (NOSII-/-; solid bars) mice to air or ozone. After culturing with TPA (170 nM) for 45 min, supernatants were analyzed for superoxide anion release. Each bar represents the mean ± SEM of three to four experiments. *Significantly different (P =< 0.05) from wild-type control animals. tSignificantly different (P =< 0.05) from air-exposed animals.

As observed with superoxide anion, constitutive production of PGE2 was upregulated in macrophages from air-exposed NOSII knockout mice when compared with wild-type mice (Figure 7). In addition, unlike macrophages from wild-type mice, macrophages from NOSII knockout mice were unresponsive to LPS + IFN-gamma . Ozone inhalation caused a significant increase in basal, as well as LPS + IFN-gamma -induced PGE2 production by alveolar macrophages from wild type-mice (Figure 7). In contrast, in alveolar macrophages from NOSII knockout mice, a significant decrease in constitutive PGE2 production was observed following ozone inhalation which returned to control levels in the presence of LPS + IFN-gamma . Freshly isolated alveolar macrophages from air-exposed NOSII knockout mice also expressed greater quantities of both COX-1 and COX-2 proteins than cells from wild-type mice (Figure 8). In cells from both mouse genotypes, expression of these proteins increased after ozone inhalation.


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  		Figure 7.   Effects of ozone inhalation on PGE2 production by alveolar macrophages. Cells, isolated 48 h after exposure of wild-type control mice or NOSII knockout mice (NOSII-/-) exposed to air or ozone, were cultured in the presence of LPS (100 ng/ml) + IFN-gamma (100 U/ml) (solid bars) or medium (open bars). PGE2 production was quantified by ELISA. Each bar represents the mean ± SEM of three to four experiments. *Significantly different (P =< 0.05) from medium. tSignificantly different (P =< 0.05) from air-exposed mice.


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  		Figure 8.   Effects of ozone inhalation on COX-1 and COX-2 protein expression. Alveolar macrophages isolated 48 h after exposure of wild-type control (WT) or NOSII knockout (NOSII-/-) mice to air or ozone were analyzed for COX-1 and COX-2 expression by Western blotting (upper panel). The 70-kD COX-1 and 72-74-kD COX-2 bands are indicated. +, positive control for antibody binding. Data were quantified by densitometry (OneD-Scan) and presented in arbitrary units (lower panel). One representative blot from four separate experiments is shown.

We also quantified protein and cell accumulation in bronchoalveolar lavage fluid as markers of ozone-induced injury. Treatment of wild-type mice with ozone resulted in a time-dependent increase in bronchoalveolar lavage fluid protein which reached a maximum 24-48 h after exposure and returned to control levels by 72 h (Figure 9). In contrast, ozone inhalation had no effect on bronchoalveolar lavage protein in NOSII knockout mice. Similarly, whereas ozone inhalation caused a significant time-related increase in the number of cells recovered by bronchoalveolar lavage in control animals, no cellular accumulation was observed in NOSII knockout mice. Differential staining revealed that the majority of cells (> 98%) recovered from the lung by lavage in both wild-type and NOSII knockout were macrophages. Ozone inhalation had no effect on the type of cells recovered in the lung lavage in either mouse strain.


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  		Figure 9.   Protection of NOSII knockout mice from ozone- induced lung injury. Wild-type control (open bars) or NOSII knockout (NOSII-/-; solid bars) mice were exposed to air or ozone. Lung injury was assessed 24-72 h later by measuring bronchoalveolar lavage fluid protein and cell number. Each bar represents mean ± SEM of three to six animals. *Significantly different (P =< 0.05) from air control.

    Discussion
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Macrophage-derived NO is known to play an important role in nonspecific host defense (13, 28). However, under pathophysiologic states associated with inflammation, excessive production of reactive nitrogen intermediates can lead to tissue damage (29, 30). In previous studies using a rat model we showed that NOSII expression and NO production by alveolar macrophages and type II cells were upregulated after ozone inhalation (2, 4, 16). We speculated that reactive nitrogen intermediates contribute to the pathogenesis of ozone-induced lung injury. To test this possibility we examined ozone toxicity in NOSII knockout mice, which lack the capacity to generate NO via this enzyme. As observed in the rat (2, 31), acute exposure of wild-type control mice to inhaled ozone at concentrations close to toxic environmental levels (32) resulted in increased numbers of activated macrophages in the lungs. These cells were also primed by ozone to produce increased quantities of NO, as well as peroxynitrite, in response to inflammatory mediators. Our findings that alveolar macrophages from NOSII knockout mice did not produce NO or peroxynitrite, even after ozone inhalation, confirm previous studies demonstrating that ozone-induced NO production does not involve constitutive isoforms of NOS (2, 33). We also found that bronchoalveolar lavage protein levels, as well as cell number, were at control levels in NOSII knockout mice treated with ozone. Similar results on lavage protein have recently been reported by Kleeberger and coworkers (34). These findings, together with the observation that there was no evidence of nitrotyrosine residues in histologic sections or isolated alveolar macrophages, demonstrate that NOSII knockout mice are protected from ozone-induced damage.

NO has been implicated as an important mediator of lung injury in a number of different experimental models, including carrageenan-induced increases in vascular permeability and edema, as well as damage induced by leukotoxin, immune complexes, paraquat, and endotoxin (8, 10, 33). The present studies demonstrate that NO also plays a role in ozone-induced inflammatory cell accumulation, mediator production, and injury. NO is known to complex with electron-rich substrates, including iron sulfur or heme-containing proteins, resulting in either inhibition or activation of enzymes involved in respiration, DNA synthesis, and cytotoxicity (40). These actions may contribute to tissue damage in this model. NO toxicity may also be due to the generation of peroxynitrite, a highly reactive and mutagenic radical (15, 41). Peroxynitrite has been reported to induce lipid peroxidation, oxidation of proteins and nonprotein sulfhydryls, deoxyribonucleic acid and membrane phospholipids, and to alter DNA bases (24, 29, 40, 43). Peroxynitrite also disrupts epithelial cell ion channel function, as well as the activity of pulmonary surfactants (12, 44, 45). Following ozone inhalation, alveolar macrophages were found to produce significant quantities of peroxynitrite. This was associated with nitrotyrosine staining of the cells, which was detected in vitro and in situ in histologic sections. A similar pattern of nitrotyrosine staining of the lung has been described previously following ozone inhalation by rats (46). Production of peroxynitrite by alveolar macrophages was correlated with increased superoxide anion release, which may account for the more prominent immunostaining of these cells in the lung when compared with other cell types. Nitrotyrosine staining of the lung has also been observed in ischemia-reperfusion injury, LPS-induced injury, immune complex-induced pulmonary edema, obliterative bronchitis, emphysema, and following inhalation of cigarette smoke (10, 11, 22, 26, 47).

Peroxynitrite formation is dependent on the availability of NO and superoxide anion. Interestingly, we observed significantly increased production of superoxide anion by alveolar macrophages from untreated mice lacking NOSII. The fact that these mice are protected from ozone-induced injury suggests that superoxide anion does not play an important role in the toxicity of this oxidant. Unstimulated alveolar macrophages from air-exposed NOSII knockout mice also produced significantly more PGE2 and expressed greater quantities of COX-1 and COX-2 when compared with unstimulated cells from wild-type mice. This may be due to compensatory increases in inflammatory mediator production in transgenic mice (50). Following ozone inhalation, alveolar macrophages from wild-type mice produced significantly more PGE2 than cells from air exposed animals. These cells also expressed greater quantities of COX-1, as well as COX-2. Thus, both enzymes appear to be involved in the inflammatory response to ozone. In alveolar macrophages from NOSII knockout mice, COX-1 and COX-2 protein levels were increased after ozone inhalation. In contrast, constitutive production of PGE2 by alveolar macrophages was significantly reduced. This may be due to superoxide anion-induced auto-inactivation of the COX enzymes (51). This is consistent with the observation that LPS + IFN-gamma -induced PGE2 production was increased in these cells at a time when superoxide anion generation was reduced.

Our studies also showed that macrophage release of superoxide anion was decreased immediately following exposure of both wild-type and NOSII knockout mice to ozone. These results are similar to those reported previously in alveolar macrophages from rats and mice isolated immediately after ozone inhalation (52). Interestingly, in wild-type mice, macrophage superoxide anion release increased 24 h after ozone exposure. These findings provide further support for the concept that alveolar macrophages are activated in response to ozone (2). The fact that superoxide anion production remained depressed in cells from NOSII knockouts demonstrates that nitric oxide is important in ozone-induced inflammatory mediator production.

The presence of excess protein and increases in the number of inflammatory cells in bronchoalveolar lavage fluid following ozone inhalation is a hallmark of alveolar epithelial injury (1). Increases in protein may be caused by transudation of proteins from the blood into the alveolar space, lysis of injured cells, and/or enhanced protein secretion by cells of the respiratory tract (53). Following ozone exposure, we observed a time-dependent increase in lung lavage fluid protein content and cell number, which was maximal after 24-48 h. Protein content and cell number returned to basal levels by 72 h, indicating recovery. Mice lacking NOSII were protected from ozone-induced damage as shown by basal control levels of protein and cell number in the bronchoalveolar lavage. These data demonstrate that NO generated from NOSII contributes to ozone toxicity.

Injury induced by pulmonary irritants such as ozone causes the release of a cascade of inflammatory mediators, which appear to be important in the pathogenic process. The use of transgenic mice with a targeted disruption of genes for one or more of these inflammatory mediators provides an important approach for assessing the potential role of a particular mediator in toxicity. Our findings that NOSII knockout mice are protected from injury provide direct support for the concept that reactive nitrogen intermediates are involved in tissue damage and inflammation in this model.

    Footnotes

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

(Received in original form January 29, 2001 and in revised form June 19, 2001).

Abbreviations: bovine serum albumin, BSA; cyclooxygenase, COX; Dulbecco's modified Eagle's medium, DMEM; Hanks' balanced salt solution, HBSS; horseradish peroxidase, HRP; interferon-gamma , IFN-gamma ; lipopolysaccharide, LPS; nitric oxide, NO; inducible nitric oxide synthase, NOSII; prostaglandin E2 (PGE2); phosphate-buffered saline, PBS; superoxide dismutase, SOD; 12-O-tetradecanoyl-phorbol-13-acetate, TPA.

Acknowledgments: This work was supported by a Career Development Award from the Burroughs Wellcome Fund awarded to D.L.L., and by National Institutes of Health Grants ES04738, ES06897, GM34310, and ES05022.
    References
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

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