INTRODUCTION

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Inhaled nitric oxide is a potent local vasodilator and a promising therapy for persistent pulmonary hypertension of the newborn (PPHN), as well as for other conditions that result from pulmonary vasoconstriction (1). Infants with severe PPHN exhibit increased oxygenation in response to inhaled nitric oxide, which is associated with decreased duration and intensity of mechanical ventilation, reduced lung barotrauma and oxygen toxicity, and improved clinical outcome. Several randomized multicenter trials have established the efficacy of inhaled nitric oxide in the therapy of PPHN (2, 3). However, the growing knowledge of the ubiquitous nature of nitric oxide as a physiologic and pathophysiologic mediator has heightened concerns regarding the toxicity associated with inhalation of this gas. A number of studies have addressed the potential untoward effects of inhaled nitric oxide. Alterations in airway conductance, humoral immune responses, and host resistance, as well as damage to the alveolar epithelium have been reported in humans and rodents after acute or chronic inhalation of nitric oxide (4- 7). These effects are similar to those observed after inhalation of pulmonary toxicants such as nitrogen dioxide and ozone (7, 8). Both of these gases are also known to modulate various functional responses of alveolar macrophages (8) and a similar response may occur after inhalation of nitric oxide.

Alveolar macrophages represent an early and nonspecific defense system against inhaled pathogens. Many of the functions of these cells are mediated through the release of reactive oxygen and nitrogen intermediates, including superoxide anion, hydrogen peroxide, hydroxyl radicals, and nitric oxide (11). Nitric oxide is synthesized by activated alveolar macrophages through the action of an inducible form of nitric oxide synthase (iNOS) (9, 12). This enzyme is induced in alveolar macrophages by inflammatory mediators such as lipopolysaccharide (LPS), interferon gamma (IFN-), and interleukin-1 (IL-) (9, 12, 13). Increased production of nitric oxide as well as reactive oxygen intermediates by macrophages has been reported in response to endotoxemia (12, 14), and after exposure of animals or humans to pulmonary irritants such as ozone (9), silica (15), and asbestos (16, 17), and it has been hypothesized that this contributes to toxicant-induced lung inflammation and injury (18). The effects of inhaled nitric oxide on lung macrophage function are unknown and represent the focus of the present studies. We found that brief exposure of mice to inhaled nitric oxide resulted in increased expression of iNOS and production of reactive nitrogen and oxygen intermediates by lung macrophages. Alterations in the balance of these mediators in the lung following inhalation of nitric oxide may lead to tissue damage and/or altered host defense.

	METHODS

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Animals and Exposures

Male pathogen-free Balb/c mice (7- to 9-wk old) were purchased from Taconic (Germantown, NY). Animals were housed in microisolator cages and received food and sterile pyrogen-free water ad libitum. Animals were placed in a whole-body plexiglass chamber and exposed to ultrapure compressed air (control), or 10 to 100 ppm nitric oxide gas (Scott Medical Products, Plumsteadville, PA) for 5 h. The nitric oxide tank was equipped with a stainless steel regulator and a low flowmeter for delivering finely gauged flow rates. Nitric oxide gas was blended with ultrapure compressed air and then delivered to the chamber at a rate of 5 L/min. A chemiluminescence nitric oxide/nitrogen dioxide analyzer (model 8840; Monitor Labs, Denver, CO) was used to assess nitric oxide and nitrogen dioxide concentrations in the chamber. Calibration of the system was confirmed with nitric oxide gas concentration at 80% of full-scale range, with measurements at 0 to 100 ppm. Preliminary studies revealed no accumulation of nitrogen dioxide in the chamber.

Reagents

Mouse recombinant interferon gamma (rIFN-) was purchased from GIBCO (Grand Island, NY), LPS (serotype 0128:B12) and DNAse I from Sigma Chemical Co. (St. Louis, MO), and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) from LC Services (Woburn, MA). Rabbit polyclonal antibody against iNOS was from Affinity Bioreagents (Golden, CO). Nitrate reductase and NADPH were from Boehringer Mannheim (Indianapolis, IN).

Bronchoalveolar Lavage

The mice were killed and the lungs and trachea exposed. The trachea was then cannulated with polyethylene tubing (PE-90; Clay Adams, Parsippany, NJ) attached to a syringe and the lung lavaged by slowly instilling and withdrawing 1 ml of phosphate-buffered saline (PBS) three times (19). The protein content of lung lavage fluids was quantified using the Bradford protein determination assay (Bio-Rad Laboratories, Hercules, CA).

Cell Isolation

Alveolar macrophages were isolated from mouse lung as previously described (19, 20). Briefly, the lungs were excised, the trachea and major bronchi removed, and the lungs cut into uniform 500-µm slices (McIlwain Tissue Chopper; Brinkmann Instruments, Westbury, NY). These were incubated in ice cold Ca²⁺/Mg²⁺-free Hanks' balanced salt solution (HBSS) containing 0.005% DNAse I (HBSS-DNAse) for 30 min with periodic agitation. The tissue slices were then washed with HBSS-DNAse using a Vortex Genie 2 (Fisher Scientific, Pittsburgh, PA) at shaking speed 3 for 3 min. The cells released during these steps were filtered through a 15-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 for leukocyte differentials were prepared using a Cytospin 2 (Shandon, Cheshire, UK) and stained with Giemsa (Fisher Scientific, Springfield, NJ).

Measurement of Nitrite and Nitrate Production

Cells were cultured in 96-well dishes (1.5 × 10⁵ cells/well) in the presence of LPS and/or IFN-, or medium control. The accumulation of nitrite in the culture medium was quantified 24 to 72 h later using the Griess reaction with sodium nitrite as the standard (9). Supernatants were mixed with equal volumes of 1% sulfanilamide and 0.1% N-1-naphthylethylenediamine hydrochloride in 50% H₃PO₄. After 5 min, absorbance was measured at 540 nm. For nitrate determinations, samples were treated with nitrate reductase and NADPH for 30 min prior to analysis. We found that the ratio of nitrate:nitrite produced by alveolar macrophages was 1.8:1 and that this ratio was unaffected by nitric oxide inhalation.

Western Blot Analysis

Macrophages were cultured in 35-mm dishes (1 × 10⁶ cell/dish) in the presence of LPS (100 ng/ml) and IFN- (100 U/ml) or medium control. After 24-h incubation, the cells were lysed in buffer containing 10 mM EDTA, 150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 1 mM phenylmethylsulfonyl fluoride, and 1% Nonidet P-40. Proteins (10 µg/lane) were fractionated on 10% sodium dodecyl sulfate (SDS)-polyacrylamide, transferred to nitrocellulose and incubated with a 1:1,000 dilution of anti-mouse macrophage inducible nitric oxide synthase (iNOS). The blots were then incubated with alkaline-phosphatase-labeled anti-rabbit immunoglobulin and developed using an enhanced chemoluminescence (ECL) detection kit (Amersham Life Science, Arlington Heights, IL).

Immunofluorescence Localization of iNOS

Macrophages were cultured in 8-well slide chambers (1.5 × 10⁵ cells/ well) in the presence of LPS (100 ng/ml) and IFN- (100 U/ml) or medium control. After 24 h, the cells were fixed in 1% formalin and permeabilized with lysophosphatidylcholine (40 ng/ml). Cells were washed in blocking buffer (PBS containing 1% bovine serum albumin [BSA] and 0.05% sodium azide) and then incubated overnight with rabbit antibody against macrophage iNOS or pooled normal rabbit serum. Affinity-purified fluorescein isothiocyanate (FITC)-conjugated goat anti-rabbit IgG Fab₂ was used as the secondary antibody. Cell-associated fluorescence was quantified on a Meridian ACAS 570 Anchored Cell Analysis System (Meridian, Okemos, MI). At least five random 120 × 120 µm fields were scanned for each analysis.

Measurement of Superoxide Anion Production by Alveolar Macrophages

Superoxide anion release by alveolar macrophages was measured as the superoxide dismutase (SOD) inhibitable reduction of ferricytochrome C (12). Cells were washed and resuspended (5 × 10⁵ cells/ml) in balanced salt solution 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 nM¹cm¹ at 550 nm) obtained from samples containing SOD.

Measurement of Peroxynitrite Production by Alveolar Macrophages

Peroxynitrite production by alveolar macrophages was quantified using dihydrorhodamine 123, as previously described (12). Cells were cultured overnight in 8-well slide chambers (1.5 × 10⁵/well) with LPS (100 ng/ml) and IFN- (100 U/ml) or medium control. TPA (170 nM) was added to the wells containing the cells 30 min prior to analysis. Supernatants were then removed, the cells washed with PBS-HEPES buffer and incubated for 30 min at room temperature with dihydrorhodamine 123 (0.5 µg/ml) (Molecular Probes, Eugene, OR). The cells were then washed with PBS-HEPES buffer and analyzed on a Meridian Insight Plus confocal microscope (Meridian, Okemos, MI).

Measurement of Superoxide Anion and Peroxynitrite In Situ

Superoxide anion production was analyzed in situ in histologic sections as described previously (12). Briefly, lungs were perfused sequentially with 15 ml of Ca⁺²/Mg⁺²-free HBSS, 15 ml of serum-free Dulbecco's modified Eagle medium (DMEM) containing 0.5 µg/ml TPA, 20 ml of 0.05% nitro blue tetrazolium (NBT) in DMEM and TPA, and 15 ml of Ca⁺²/Mg⁺²-free HBSS, with or without SOD (60 U/ml). Lungs were then inflated with 10% buffered formalin, histologic sections prepared and stained with Kernechtrot solution. In the presence of superoxide anion, NBT is converted to insoluble formazan, which can be visualized microscopically. To estimate peroxynitrite production in situ, tissue sections were stained with a specific antibody directed against nitrotyrosine (Upstate Biotechnology, Lake Placid, NY). For these studies, histologic sections, prepared as described previously, were deparaffinized and preincubated for 30 min in 3% hydrogen peroxide to quench endogenous peroxidase activity. Sections were then incubated for 20 min with nonimmune goat serum, followed by overnight incubation with rabbit antibody against nitrotyrosine (2 µg/ml) or with nonimmune rabbit IgG. A Vectastain ABC kit (Vector Laboratories, Burlingame, CA) was utilized to visualize antibody binding.

Statistics

All experiments were repeated 3 to 6 times using 3 to 5 animals per experiment. Data were analyzed using a nonpaired, two-tailed Student's t test. In all statistical comparisons a p value of  0.05 was considered significant.

	RESULTS

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Effects of Inhaled Nitric Oxide on Lung Leukocyte Number, Composition, and Lavage Fluid Protein

In initial studies we determined if acute exposure of mice to inhaled nitric oxide modified the number or composition of cells recovered from the lung. Approximately 6.5 × 10⁵ viable cells were obtained from each control mouse (Table 1). Differential staining revealed that 95 to 97% of these cells were macrophages. Although inhalation of nitric oxide (10 to 100 ppm) had no effect on the number of cells recovered from the lungs or on the cellular composition, a dose-related increase in the amount of protein in lung lavage fluid was observed (Table 1).

Effects of Inhaled Nitric Oxide on Production of Reactive Nitrogen and Reactive Oxygen Intermediates by Alveolar Macrophages

Irritant-induced lung injury is associated with increased production of reactive intermediates, including nitric oxide and superoxide anion, by alveolar macrophages (9, 15). To determine if inhalation of nitric oxide primes alveolar macrophages to respond to inflammatory stimuli, we quantified production of these mediators by the cells. In the absence of stimulation, alveolar macrophages from both control and nitric oxide exposed animals did not produce measurable levels of nitrite or nitrate and did not express iNOS protein (Figure 1 and not shown). Treatment of the cells with IFN- resulted in a time- and dose-dependent stimulation of nitric oxide production (Figure 2). Whereas LPS by itself had no effect on the cells, the combination of IFN- and LPS resulted in a small but reproducible increase in nitric oxide production when compared with IFN- treatment alone (Figure 2). Production of nitric oxide by alveolar macrophages was found to be blocked by the nitric oxide synthase inhibitor N-nitro-L-arginine methyl ester (L-NAME), and dependent on the presence of L-arginine in the culture medium (Figure 2). Western blot analysis of cell lysates confirmed that alveolar macrophages expressed iNOS protein following treatment with LPS and IFN- (Figure 1). Treatment of mice with nitric oxide resulted in a 3-fold increase in expression of iNOS protein by stimulated alveolar macrophages, as determined by densitometry. These results were confirmed by immunofluorescence analysis (Figure 3). In these studies iNOS protein could also be visualized in cytoplasmic regions of the cells. Increased expression of iNOS protein by alveolar macrophages following inhalation of nitric oxide was correlated with increased production of nitric oxide by the cells in response to LPS and IFN-, measured as the accumulation of nitrite and nitrate in the culture media (Figure 4 and not shown). The priming effects of inhaled nitric oxide on alveolar macrophages were dose-related in the range of 20 to 100 ppm.

View larger version (24K): [in this window] [in a new window]   Figure 1.   Effects of inhaled nitric oxide on iNOS protein expression. Alveolar macrophages, isolated from animals exposed to air (lanes 1 and 2) or 100 ppm nitric oxide (lanes 3 and 4), were cultured for 24 h in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of LPS (100 ng/ml) and IFN- (100 U/ml). Cell lysates were prepared and analyzed by Western blotting using anti-iNOS antibody. The arrow shows the 130 kD iNOS protein band. The lower band on the gel is nonspecific binding.

View larger version (15K): [in this window] [in a new window]   Figure 2.   Characterization of nitric oxide production by alveolar macrophages. Alveolar macrophages were isolated 48 h after exposure of mice to nitric oxide (40 ppm). Left panel: Cells were incubated with 100 ng/ml LPS (closed squares), 100 U/ml IFN- (closed diamonds), or LPS + IFN- (closed circles). Nitric oxide production was quantified 24 to 72 h later. Right panel: Cells were incubated with LPS + IFN- in the presence of increasing concentrations of L-NAME. Inset: Cells were plated in DMEM containing 10% fetal bovine serum. After 24 h, the cells were washed and the medium was replaced with serum-free DMEM supplemented with increasing concentrations of L-arginine, in the presence of LPS + IFN-. Nitric oxide production was quantified after 48 h. Each value represents the mean ± SE of three samples from one representative experiment.

View larger version (35K): [in this window] [in a new window]   Figure 3.   Immunofluorescence localization of iNOS. Alveolar macrophages, isolated from animals exposed to air (A and B) or 100 ppm nitric oxide (C and D) were cultured for 24 h in the presence (B and D) or absence (A and C ) of LPS (100 ng/ml) and IFN- (100 U/ml). Cells were then fixed in 1% formalin, permeabilized with lysophosphatidylcholine, and incubated with rabbit antibody against macrophage iNOS. Affinity-purified FITC-conjugated goat anti-rabbit IgG Fab2 was used as the secondary antibody. Cell-associated fluorescence was analyzed on a Meridian ACAS 570 Anchored Cell Analysis System. The color bar represents relative fluorescence on a 4-decade log scale. At least five random 120 × 120 µm fields were scanned for each analysis. No fluorescence was observed in samples incubated with nonimmune serum (not shown).

View larger version (26K): [in this window] [in a new window]   Figure 4.   Effects of inhaled nitric oxide on nitrite production by alveolar macrophages. Cells isolated 48 h after exposure of mice to air or nitric oxide were incubated with LPS (100 ng/ml) and IFN- (100 U/ml). Supernatants were assayed for nitrite accumulation 24 h and 48 h later. Each bar is the average ± SE of four to six samples. *Significantly different (p  0.05) from cells from control (air) animals.

We next examined the effects of inhaled nitric oxide on production of reactive oxygen intermediates by lung macrophages. In our initial studies, we evaluated superoxide anion production in situ in histologic sections. A small subset of lung macrophages from control animals were found to produce superoxide anion, as measured by formazan deposition in the cells (Figure 5). Inhalation of nitric oxide was associated with a marked increase in the number of macrophages undergoing a respiratory burst. This response was inhibitable by SOD demonstrating that it was due to superoxide anion production. Following nitric oxide inhalation, formazan deposition was also observed in some Type II cells. This is consistent with our previous findings that inhalation of pulmonary irritants augments production of reactive oxygen and nitrogen intermediates by these cells (21). In contrast to our in situ studies, no significant differences were observed in superoxide anion production by alveolar macrophages isolated from control and nitric oxide treated animals (Table 2).

View larger version (79K): [in this window] [in a new window]   Figure 5.   In situ localization of superoxide anion. Lungs from mice exposed to air (A) or 100 ppm nitric oxide (B and C ) were perfused with TPA and NBT with (C ) or without (A and B) SOD and fixed in buffered formalin. Lung sections (6 µm) were prepared and stained with Kernechtrot solution. Arrows indicate lung macrophages.

Nitric oxide is known to react rapidly with superoxide anion forming peroxynitrite, a potent oxidizing agent (22). To determine if inhaled nitric oxide primes alveolar macrophages to produce peroxynitrite, we used dihydrorhodamine 123 in conjunction with fluorescence image analysis (12). Alveolar macrophages from control animals did not produce detectable levels of peroxynitrite (Figure 6). In contrast, following inhalation of nitric oxide, there was a dramatic increase in peroxynitrite production by these cells. Peroxynitrite production by alveolar macrophages was blocked when L-NAME was added to the cultures, demonstrating that the response was dependent on nitric oxide synthesis. Peroxynitrite can act as a nitrating agent, resulting in the formation of nitrotyrosine residues in proteins. This has been used as a marker of peroxynitrite-mediated tissue injury (23, 24). To determine if peroxynitrite was formed in vivo after inhalation of nitric oxide, we used a specific antibody directed against nitrotyrosine residues. No staining with antibody to nitrotyrosine was observed in tissue sections from control animals or in sections stained with nonimmune IgG (Figure 7). In contrast, following inhalation of nitric oxide, we observed nitrotyrosine staining of lung macrophages. These data suggest that nitric oxide formed by lung macrophages is converted, in large part, to peroxynitrite. This observation is consistent with previous studies on these cells (22).

View larger version (100K): [in this window] [in a new window]   Figure 6.   Effects of inhaled nitric oxide on peroxynitrite production by alveolar macrophages. Cells, isolated 48 h after exposure of mice to air (A and B) or 100 ppm nitric oxide (C and D), were cultured overnight with LPS (100 ng/ml) and IFN- (100 U/ml), in the absence (A and C ) or presence (B and D) of 1 mM L-NAME. Cells were then stimulated with TPA and assayed for peroxynitrite production using dihydrorhodamine 123 and fluorescence image analysis as described in METHODS.

View larger version (65K): [in this window] [in a new window]   Figure 7.   Immunohistochemical localization of nitrotyrosine in the lung. Lungs from mice exposed to air (B) or 100 ppm nitric oxide (A and C ) were perfused with TPA and NBT and fixed in buffered formalin. Lung sections (6 µm) were then stained with antibody to nitrotyrosine (B and C ) or with nonimmune serum (A). Antibody binding was visualized using a Vectastain ABC kit. Arrows indicate lung macrophages.

	DISCUSSION

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The present studies demonstrate that acute exposure of mice to inhaled nitric oxide primes alveolar macrophages to respond to inflammatory mediators. Thus, following inhalation of nitric oxide, alveolar macrophages produce increased quantities of reactive nitrogen and oxygen intermediates in response to LPS and IFN- or TPA, respectively. Both reactive oxygen and reactive nitrogen intermediates have been implicated in the pathogenesis of tissue injury (18). These findings, together with the observation of increased protein content in lung lavage fluid and nitrotyrosine staining in histologic sections, demonstrate that clinically relevant doses of nitric oxide (up to 40 ppm) have the capacity to induce pulmonary injury. Similar increases in production of reactive nitrogen and oxygen intermediates by lung macrophages, as well as lavage fluid protein, have been observed following treatment of animals with endotoxin, ozone, and silica, pulmonary irritants known to induce lung inflammation and injury (9, 12, 14). These data suggest that nitric oxide is a pulmonary irritant. However, in contrast to inhaled ozone or nitrogen dioxide, which induce marked inflammatory responses (7, 9), we did not observe increased numbers of macrophages or neutrophils in the lung after nitric oxide exposure. Thus, the actions of nitric oxide appear to be distinct from these toxicants.

We found that IFN- stimulated nitric oxide production in mouse alveolar macrophages and that this response was augmented in the presence of LPS. The response of mouse alveolar macrophages to inflammatory mediators appears to be distinct from mouse peritoneal macrophages, which require multiple signals for activation (25). Exposure of mice to nitric oxide resulted in increased production of reactive nitrogen intermediates by alveolar macrophages in response to IFN- and LPS, which was correlated with increased expression of iNOS protein. This increase may reflect a general response of alveolar macrophages to tissue injury, in which cytokine-mediated signal transduction pathways are upregulated (26). In contrast to our findings, previous studies have described decreases in nitric oxide production and/or iNOS expression in macrophages (27, 28), as well as in endothelial cells (29), after in vitro exposure of these cells to organic nitric oxide donors. The fact that we did not observe negative feedback regulation of iNOS suggests that in vitro treatment protocols may not accurately reflect the response of cells during in vivo exposure. This latter response is most likely influenced by the multitude of inflammatory mediators produced by cells in the lung after inhalation of nitric oxide.

Nitric oxide is a highly reactive molecule that has been reported to be directly cytotoxic to lung cells and tissue (6, 7, 30). Similarly, following exposure of mice to nitric oxide, we observed increased protein content of bronchoalveolar lavage fluid, which is a hallmark of alveolar epithelial injury (8). Nitric oxide can also induce endothelial cell damage, alter membrane permeability, and disrupt normal lung function (31). After brief inhalation of nitric oxide by healthy humans, pulmonary function changes indicative of small airway edema have been reported (5). Similarly, interstitial edema, alterations in arteriolar endothelial cells, and paraseptal emphysema have been described after chronic exposure of animals to low doses of nitric oxide (4, 6). Taken together, these data suggest that inhalation of nitric oxide induces pathophysiologic changes in the lung that need to be considered in the design of protocols for its administration in clinical settings. In this regard, it has recently been suggested that inhaled nitric oxide may increase mortality in patients with acute respiratory distress syndrome (32), and our results suggest that this may be due, at least in part, to priming of lung macrophages for mediator production.

Macrophages activated by inflammatory mediators release reactive oxygen species including superoxide anion, hydroxyl radical, and hydrogen peroxide, which have been implicated in tissue injury induced by a number of xenobiotics (11, 18). After inhalation of nitric oxide, we observed increased production of superoxide anion by stimulated lung macrophages in situ. Reactive oxygen intermediates produced by activated macrophages can cause cell damage by inducing lipid peroxidation leading to increased membrane permeability and a loss of function (18). Thus, enhanced production of superoxide anion after inhalation of nitric oxide may have deleterious effects on lung tissue. In contrast to our in vivo findings, macrophages isolated from control and nitric oxide exposed animals released similar amounts of superoxide anion in response to TPA. This may be due to the fact that these cells are activated in vivo by inhaled nitric oxide and are refractory to further stimulation in vitro. A similar lack of responsiveness has been observed in activated macrophages isolated from endotoxin-treated rats (33). It is also possible that conditions favoring iNOS-catalyzed superoxide anion generation in vivo are reversed by incubation of isolated cells in arginine-containing medium (34).

Peroxynitrite is a toxic oxidizing agent formed from nitric oxide and superoxide anion. We observed increased peroxynitrite production by alveolar macrophages after inhalation of nitric oxide, which was correlated with peroxynitrite-mediated damage as evidenced by the presence of nitrotyrosine residues in lung macrophages. Peroxynitrite is known to induce lipid membrane peroxidation and to alter DNA bases. In addition, the reaction of peroxynitrite with metals results in the formation of a potent nitrating agent. Recent studies have shown that peroxynitrite inhibits epithelial cell ion channels as well as the activity of pulmonary surfactants (23, 35). Unlike nitric oxide, peroxynitrite is relatively stable at physiologic pH and can diffuse considerable distances. Increased peroxynitrite production may augment oxidative tissue damage mediated by locally produced nitric oxide and superoxide anion.

Alveolar macrophages remove inhaled particulate matter and infectious agents from the lung. They exhibit extensive phagocytic capacity and release soluble mediators and molecular oxidants that regulate immune defense and nonspecific host resistance (11). We have demonstrated that exposure of mice to inhaled nitric oxide results in tissue damage and increased production of nitric oxide, superoxide anion, and peroxynitrite by alveolar macrophages in response to inflammatory mediators. Further studies are required to determine if nitric oxide-induced priming of alveolar macrophages alters nonspecific host defense and/or leads to tissue injury in patients.