Introduction

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Inhaled nitric oxide (NO) has received much attention as a potentially important therapeutic agent for the treatment of pulmonary diseases associated with pulmonary arterial hypertension, including persistent pulmonary hypertension of the newborn, cardiac postoperative respiratory deterioration, and acute respiratory distress syndrome (1, 2). In addition to its vasodilating action, endogenous NO also appears to modulate the activity of cellular elements found in the lung. NO synthesis inhibition enhances neutrophil adhesion (3), implying that endogenous NO is responsible for preventing constitutive neutrophil adhesion. Several studies have also suggested that exogenous NO at high concentrations inhibits oxidative respiratory burst (4, 5), although methodologic issues call these results into question. Thus, increasing endogenous or exogenous NO may harbor the potential to alter neutrophil emigration and subsequent oxidative response that typifies acute lung injury (6).

Once localized to lung parenchyma, neutrophils must be cleared from the pulmonary bed to minimize release of cellular products toxic to surrounding tissue. Aging neutrophils undergo apoptosis (7), a form of programmed cell death distinctly different from necrosis (8, 9), which leads to recognition and phagocytosis by tissue macrophages (10, 11). Apoptosis is characterized by typical nuclear and cytoplasmic condensation in the presence of cell wall integrity, and specific endonuclease-mediated DNA fragmentation into 180 to 200 base pair length segments (8). Necrosis, in contrast, produces organelle swelling, early cell membrane disruption, and subsequent disintegration with random DNA cleavage and diffuse smears on electrophoretic gels. Apoptosis also inhibits functional activity of the neutrophil respiratory burst and phagocytosis (12). Thus, neutrophil apoptosis could play a central role in the physiologic clearance of neutrophils in the lung and in the termination of lung inflammation to limit further tissue injury (7).

The mechanisms inciting neutrophils to undergo apoptosis are still poorly defined. Several studies suggest that NO may play a role in induction of apoptosis in other cell types. Exposure of murine peritoneal macrophages to NO gas produced DNA fragmentation patterns typical of apoptosis (13); macrophage apoptosis was also prevented by addition of NO synthase inhibitors that prevented endogenous cellular NO production. Effects of NO on induction or enhancement of apoptosis in neutrophils have not been reported. We previously found that NO gas decreased neutrophil oxidative function in association with increased cell death (14). We therefore hypothesized that exogenous NO, in gaseous form at therapeutically relevant concentrations, increases neutrophil cell death by enhancing DNA fragmentation consistent with apoptosis.

	Materials and Methods

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Neutrophil Isolation

Blood was obtained after informed consent from normal adult human volunteers by standard venipuncture. Neutrophils were isolated from whole blood as previously described (15). Briefly, heparinized blood was layered over sodium ditriazoate (Histopaque-1077; Sigma Chemical Co., St. Louis, MO) and centrifuged, followed by sedimentation with 6% dextran (Spectrum, Gardenia, CA) and hypotonic lysis of red blood cells. Isolated samples contained > 98% neutrophils by Wright stain and differential count and demonstrated > 98% trypan blue exclusion. Neutrophils were maintained in Dulbecco's phosphate-buffered saline (DPBS) with 0.1% glucose (Gibco, Grand Island, NY). Neutrophils were used for exposure studies immediately following isolation.

Neutrophil Exposure

For time-dependent studies, isolated neutrophils were maintained for zero to 24 h in modified Iscove's modified Dulbecco's medium (Gibco BRL, Gaithersburg, MD) with 10% platelet-rich plasma-derived autologous human serum, penicillin and streptomycin at 100 U/liter, at 37°C in 21% O₂ and 5% CO₂ as described by Savill and associates (10).

In separate experiments, isolated neutrophils were exposed for 2 h to an atmosphere of 21% O₂ (room air [RA]), 80% O₂ (O₂), NO at a concentration of 20 ppm (NO/RA) balanced with RA, or NO at 20 ppm (NO/O₂) balanced with 80% O₂ prior to evaluation. A NO concentration of 20 ppm was chosen to represent clinically relevant concentrations currently in use in human inhaled NO trials. A separate group of neutrophil samples were exposed to the above conditions for 2 h and then washed and maintained for 24 h in Iscove's modified Dulbecco's medium, 10% autologous serum, and antibiotics as above. Similar experiments were also performed using NO concentrations of 5 and 50 ppm.

Neutrophils exposed to NO gas were maintained in a buffer containing NO in solution as previously described (16). NO buffer solution was prepared in a sterile flask by evacuating air by vacuum, bubbling DPBS with pure nitrogen gas for 20 min, and then bubbling pure (99.5%) NO gas (Scott Medical, Plumsteadville, PA) through DPBS for another 20 min at room temperature. Buffer was then diluted immediately before neutrophil exposure to obtain NO concentrations equivalent to 5 to 50 ppm. NO was not added to the buffer in the RA and O₂ exposure groups.

Neutrophils (10⁶ cells per sample) in buffer solution were placed in sterile polypropylene bullet vials in an incubation chamber designed to allow gas flow across neutrophil specimens through entry and exit ports for 2 h. Neutrophils were exposed in approximately 0.5-ml volumes to maximize surface area to volume gas exposure. Neutrophils used for 2-h studies were immediately removed for viability and apoptosis studies, while neutrophils evaluated for 24-h studies were immediately removed from the reaction chamber and incubated overnight at 37°C in a 5% CO₂ environment. Oxygen concentrations in solution were evaluated by a blood gas monitor (Corning, Cambridge, MA). Mean oxygen partial pressure with RA was 149 mm Hg, and in 80% O₂ was 413 mm Hg after 2 h of incubation of samples. NO gas concentrations were monitored continuously throughout the exposure period by chemiluminescence analyzer (Model 42H; Thermoenvironmental Instruments, Inc., Franklin, MA). Using this analyzer, NO gas was balanced with RA or O₂ to maintain ambient NO concentrations at 5, 20, or 50 ppm for the duration of each experiment. Nitrogen dioxide (NO₂) levels were also continuously monitored with an electrochemical detector (Dräger, Inc., Pittsburgh, PA). NO₂ levels remained  1 ppm throughout most exposures and were never higher than 2 ppm. NO concentrations in aqueous solution were measured by chemiluminescence analyzer (Antek Instruments, Houston, TX) in our previous study (14) and found to decrease from 20 ppm to 8.9 ± 1.4 ppm at 90 min and 8.5 ± 1.7 ppm at 2 h of ambient 20 ppm NO/O₂ exposure.

In some experiments, neutrophils were exposed to gases in the presence or absence of the neutrophil stimulant n-formyl methionyl leucine peptide (f MLP; final concentration of 10 nM; Sigma), added prior to exposure, to determine the effects of the activation state on induced cell death.

To determine if peroxynitrite produced by interactions between superoxide radicals and NO played a role in cell death, neutrophils were also coincubated with superoxide dismutase (SOD) (Sigma; final concentration of 100 µg/ ml) during NO exposures. To evaluate the possible effects of NO₂ formed by NO and oxygen during incubation, neutrophils were exposed in a separate set of experiments to nitrogen dioxide gas (National Welders, Inc., Research Triangle Park, NC) in the reaction chamber at a concentration of 2 ppm for 2 h and then examined for viability and DNA fragmentation.

Viability Assessment

Cell viability was evaluated by fluorescence viability/cytotoxicity assay (Eukolight; Molecular Probes, Eugene, OR) (14, 17). Briefly, after exposures, cells were stained with a mixture of the fluorescent probes calcein AM and ethidium homodimer. After uptake, only viable cells containing functioning esterases can cleave the ester group on calcein AM to generate a characteristic green fluorescence under fluorescent microscopy. Ethidium homodimer penetrates the permeable membranes of nonviable cells and binds with nucleic acids identifiable by red-orange fluorescence. Neutrophils with green and red fluorescence were counted from three high power fields, averaged, and expressed as percentage of calcein (green) positive cells/total calcein and ethidium (red) positive cells counted.

Apoptosis Assessment

Samples were evaluated for evidence of apoptosis by the following techniques.

Enzyme-linked immunoabsorbent assay (ELISA). We first used a recently developed apoptotic cell death detection ELISA (Boehringer Mannheim, Indianapolis, IN) to quantitatively determine cytoplasmic histone-associated DNA oligonucleosome fragments associated with apoptotic cell death. This ELISA has demonstrated correlation with gel electrophoresis and TUNEL assays in measurement of apoptotic cell death in multiple cell types with histologic evidence of apoptosis (18, 19). Briefly, neutrophil samples were sonicated to obtain cytoplasmic lysates. Samples were incubated with microtiter plates adsorbed with mouse anti-histone antibody (clone H11-4) to bind histone-associated DNA oligonucleosomes uncovered by endonuclease-mediated DNA nicking. Plates were washed, and nonspecific binding sites saturated with blocking buffer. Bound samples were then reacted with anti-mouse DNA monoclonal antibody (MCA-33) and then conjugated with peroxidase. To determine the amount of retained peroxidase, 2,2'-azino-di-{3-ethylbenzthiazoline sulfonate} (ABTS) was added as a substrate and the complex measured by spectrophotometer at 405 nm. Results were expressed as the ratio of sample absorbance to absorbance of RA control performed daily.

TUNEL assay. Specific 3'-hydroxyl ends of DNA fragments generated by endonuclease-mediated apoptosis are preferentially repaired by terminal deoxynucleotidyl transferase (TdT) (20). The TdT-mediated dUTP nick end labeling (TUNEL) assay has been developed to label these strand breaks with fluorescent nucleotides and provide a sensitive and specific measure of DNA fragmentation consistent with apoptosis within individual cells (20, 21). Cells were fixed in 4% paraformaldehyde and permeabilized with 1% Triton X-100 and 0.1% sodium citrate. Samples were then incubated for 60 min at 37°C in the absence or presence of exogenous TdT and incubated with fluorescein-conjugated dUTP for repair of nicked 3'OH DNA ends. Mean cell fluorescence intensity (MFI) of 10,000 neutrophils was assessed by flow cytometry (FACScan; Becton Dickinson, Bedford, MA) for each condition.

DNA fragmentation gel electrophoresis. DNA fragmentation was also examined by a modification of gel electrophoresis techniques developed by Oberhammer and coworkers (22). Briefly, 3 × 10⁷ neutrophils from each exposure condition were washed in DPBS and resuspended in a lysis buffer (10 mM Tris-HCl at pH 7.6, 100 mM EDTA, 20 mM NaCl) at 37°C. After resuspension, cells were combined with 1% low melting point agarose on ice for 10 min. Samples were transferred into 1% N-lauroyl-sarcosine (Sigma) with proteinase K (150 µg/ml; Sigma) and ribonuclease A (100 µg/ml; Sigma) and incubated overnight at 37°C. Extracted DNA samples were then loaded and run on a 1% agarose gel for 16 to 18 hours at 40 V in running buffer (89 mM Tris-acetate, 2 mM Na₂EDTA, 89 mM boric acid), stained with ethidium bromide, and photographed. Samples were run in tandem with a DNA molecular weight ladder (Life Technologies, Gaithersburg, MD) providing molecular size markers of 0.5 to 12 kilobase pairs. Gel photographs were evaluated for typical ladder patterns of low molecular weight DNA fragments in multiples of 180 to 200 base pairs, a hallmark of apoptosis (8). Camptothecin-treated HL60 (ATCC) cells were used initially as positive controls and confirmed typical DNA fragmentation patterns seen with cell apoptosis (not shown).

Statistics

Assays were performed in triplicate for each sample exposure, and an average value was determined. Results were expressed as mean ± SD. Statistical analysis was performed using one-way analysis of variance (ANOVA) and Student-Newman-Keuls' comparison for parametric data sets. Data not meeting parametric characteristics were analyzed using Kruskal-Wallis one-way ANOVA on ranks and Student-Newman-Keuls' or Dunn's tests for comparisons. P values of < 0.05 were considered significant.

	Results

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Time-dependent Neutrophil Cell Death

Neutrophils exhibited a decrease in viability in cell culture conditions over a 24-h period in RA, manifested by increased calcein uptake over time (Table 1). During this 24-h period, viability decreased from 95 ± 5% to 80 ± 10% from baseline to 24 h (P = 0.048). Decreased cell viability was accompanied by increasing neutrophil DNA fragmentation over time. A marked increase in histone-associated oligonucleosome fragments consistent with apoptosis was seen by ELISA at 24 h (median: 1,700% increase from t₀; range: 269 to 21,525%) but not at 2, 4, or 6 h (Figure 1; P < 0.0001 by Kruskal-Wallis and Dunn's, n = 7 to 9 in each group). This marked increase in DNA fragmentation was also seen by TUNEL assay at 24 h (median MFI: 203% of 2-h baseline; range: 167 to 270%; P < 0.01; representative histograms are shown in Figures 4A and 4B). Gel fragmentation assay confirmed DNA banding at 24 h but not at 2, 4, or 6 h (data not shown). DNA fragments were not seen in neutrophil supernatants at any time point. This data suggested that the 2- to 6-h time period could be studied without interference from intrinsic neutrophil apoptosis or necrosis.

View larger version (13K): [in this window] [in a new window]   Figure 1.   Box plot graph of cell death ELISA results from untreated neutrophils from 0 to 24 h after isolation, as a percentage of daily time zero (t0) controls. Neutrophils demonstrated marked increase in anti-histone-associated oligonucleosome binding consistent with apoptosis at 24 h, but not at 2, 4, or 6 h (P < 0.0001 by Krus-kal-Wallis and Student-Newman-Keuls, n = 9). Tukey box plot graph illustrates median (middle box line), twenty-fifth to seventy-fifth percentile (horizontal box ends), range (bars), and outliers (circles). *P < 0.0001 compared with baseline.

View larger version (16K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   Figure 4.   Representative flow cytometry histograms (FACScan; Becton Dickinson) of TUNEL assay results from neutrophil samples exposed to RA, O2, NO/RA, and NO/O2 at 2 h (A) or incubated for 24 h after these exposures (B). Histograms show relative number of cells (y axis) as a function of mean fluorescence intensity (x axis), representative of fluorescence-labeled dUTP binding to fragmented DNA.

Exogenous NO Exposure

Neutrophil exposure to NO reduced viability in a dose-dependent fashion (Table ). Neutrophils exposed to NO/O₂, NO/RA, and O₂ alone had lower viability than RA control at all NO concentrations (P < 0.05 by Student-Newman-Keuls) at both 2 and 24 h. NO/O₂ exposure at all concentrations decreased viability more than NO/RA or O₂ alone at 2 h (P < 0.05). NO/O₂ exposure at both 20 and 50 ppm reduced viability (P < 0.05) more than 5 ppm NO/O₂. Viability at 24 h in control neutrophils was significantly lower than at 2 h (P < 0.05).

Based on viability studies, 20 ppm NO concentrations were chosen for DNA fragmentation experiments. ELISA showed that 20 ppm NO/O₂ exposure for 2 h induced significant increases in neutrophil histone-associated oligonucleosomes (median: 292% of control; range: 106 to 2,488%) compared with RA control (100%), 20 ppm NO/RA (148%; 112 to 400%) or O₂ alone (220%; 82 to 1,050%) (Figure 2; n = 9; P < 0.01). O₂ alone also produced significant DNA fragmentation compared with RA (P < 0.01), while NO/RA did not induce a significant increase.

View larger version (21K): [in this window] [in a new window]   Figure 2.   Box plot graph of cell death ELISA results in neutrophils exposed for 2 h at 37°C to RA, O2, 20 ppm NO/RA, and 20 ppm NO/O2, expressed as a percentage of daily RA controls. Neutrophils exposed to NO/RA and O2 showed significant increases in apoptosis compared with RA controls (*P < 0.01), while NO/O2 showed significant apoptosis at 2 h compared with RA, NO/RA, or O2 alone (**P < 0.01 by Kruskal-Wallis and Student-Newman-Keuls). Box plot graph illustrates median, twenty-fifth to seventy-fifth percentile, range, and outliers.

By TUNEL assay, 20 ppm NO/O₂ exposure for 2 h increased neutrophil MFI to 118 ± 5% of RA control MFI (P < 0.05, n = 5 per group). Increases with O₂ alone (106 ± 13%) or 20 ppm NO/RA (113 ± 7%) were not significant. Twenty parts per million NO/O₂ increased DNA fragmentation significantly more than O₂ alone (P < 0.05).

Gel electrophoresis of DNA from neutrophils exposed to 20 ppm NO/RA, O₂, and 20 ppm NO/O₂ showed DNA fragmentation associated with apoptotic cell death (data not shown; n = 2), as well as prominent necrotic cell death (demonstrated by smearing in electrophoresis gel lanes).

The effects of 20 ppm NO/O₂ on DNA fragmentation were more pronounced at 24 h of exposure (Figures 3, 4A, and 4B). By TUNEL assay, NO/O₂ exposure significantly increased MFI by 164% (range: 110 to 242%) compared with RA (P < 0.05). The median increase in MFI was also significantly greater for NO/O₂ than for O₂ (101%; 84 to 130%) P < 0.05; or 20 ppm NO/RA (121%; 89 to 153%) exposure alone (n = 6 for each group).

View larger version (21K): [in this window] [in a new window]   Figure 3.   Box plot graph of TUNEL assay results from neutrophils exposed to gas conditions for 2 h and then incubated for 24 h at 37°C in Iscove's media. Unshaded boxes represent mean fluorescence intensity for all neutrophils in sample as a percentage of daily RA controls. Shaded boxes represent the percentage of neutrophils expressing increased fluorescence intensity (second peak) compared with RA control neutrophils. Box plot graph illustrates median, twenty-fifth to seventy-fifth percentile, range, and outliers. NO/O2-exposed neutrophils had significantly increased DNA fragmentation by both methods of analysis of mean fluorescence intensity (*P = 0.05 by Kruskal-Wallis and Student-Newman-Keuls; n = 6).

In addition to measuring the median percent increase in TUNEL fluorescence per cell, we compared the median number of neutrophils with increased log fluorescence. Again, 20 ppm NO/O₂ produced the greatest increase (151%; 127 to 182%) compared with RA control versus O₂ (111%; 87 to 207%) or 20 ppm NO/RA (124%; 87 to 159%) (P < 0.05; n = 6).

Dose dependence for NO effects on DNA fragmentation was evaluated in subsequent experiments. By apoptosis ELISA, 50 ppm NO/O₂ (median: 377% of baseline; range: 196 to 1,413%; n = 8) significantly increased DNA fragmentation compared with RA (P < 0.05 by ANOVA on ranks). The effects of 50 ppm NO were increased but not significantly different from 20 ppm NO/O₂ (median: 292% of RA baseline) (P = 0.07). Five parts per million NO/O₂ (median: 173% of baseline; range: 138 to 181%) did not significantly increase DNA fragmentation compared with RA control.

By ELISA, marked DNA fragmentation was seen in all exposure groups at 24 h, but no significant differences were seen (data not shown). Gel fragmentation electrophoresis demonstrated oligonucleosomal banding in all groups, consistent with previous reports of the natural course of time-dependent neutrophil apoptosis (10, 12). Although NO/RA exposure appeared to slightly increase the intensity of DNA fragment banding on gel electrophoresis, this could not be consistently repeated.

Reduction of superoxide radical generation with SOD mitigated the effects of NO on cell death and DNA fragmentation. Coincubation of neutrophils with SOD significantly decreased cell death in both 20 ppm NO/RA exposure (92.2 ± 1.5% viability compared with 80.2 ± 2.9% in NO/RA alone; n = 6; P < 0.05) and 20 ppm NO/O₂ exposure (84.7 ± 1.7% viability compared with 69.2 ± 2.4% in NO/O₂ alone; n = 6; P < 0.05). The addition of SOD also significantly reduced, but did not abolish, TUNEL DNA fragmentation in neutrophils exposed to 20 ppm NO/RA (median: 88% of NO/RA alone; range: 66 to 100%; P < 0.05) and to 20 ppm NO/O₂ (median: 85% of NO/O₂ alone; range: 68 to 89%; P < 0.05).

Two parts per million NO₂/O₂ exposure did not significantly decrease neutrophil viability (96 ± 1.1%; n = 4) compared with RA (95 ± 1.2%) or O₂ alone (91.8 ± 1.8%) at either 2 h (P = 0.13). No significant differences were seen 24 h after 2 ppm NO/O₂ exposure (86.0 ± 3.0%) compared with RA (91.8 ± 2.6%) or O₂ alone (81.5 ± 3.6%; n = 4; P = 0.10). Twenty-four hours after NO₂/O₂ exposure, DNA fragmentation by apoptosis ELISA (median: 330% of baseline; range: 209 to 746%; n = 4; P < 0.05) was significantly increased from RA baseline (100%) but not compared with O₂ alone (median: 178.5% of baseline; range: 144 to 285%). No differences in DNA fragmentation were seen at 2 h of NO₂/O₂ exposure.

We also examined the effects of neutrophil activation state on the development of apoptosis. Neutrophil stimulation with f MLP prior to 2 h of NO exposure did not alter the degree of neutrophil DNA fragmentation in the absence (89% of RA control; range: 87 to 92%) or presence of NO/O₂ (189% of RA control with fMLP pretreatment versus 179% in unstimulated cells), as measured by ELISA (P = 0.11).

	Discussion

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These results show that exogenous NO exposure by NO gas induces neutrophil cell death, in part by enhancing the induction of apoptosis. Exogenous NO thus may accelerate the natural in vitro course of programmed neutrophil death first demonstrated by Savill and associates (10) and confirmed by our results. NO gas exposure at a clinically relevant concentration induced a decrease in cell viability and increased DNA fragmentation. These findings suggest that the decreased neutrophil function associated with exogenous NO exposure seen in previous studies (4, 14) could arise from enhancement of neutrophil apoptosis. NO could accelerate the development of impaired neutrophil function which occurs in aging neutrophils during apoptosis in vitro (12).

Interestingly, hyperoxia alone increased apoptotic fragmentation to a similar degree as NO alone. The combination of NO and hyperoxia produced a synergistic effect on both necrotic cell death and apoptotic DNA fragmentation. This synergy may result from the formation of peroxynitrite, a potent oxidant formed from the reaction of NO and superoxide and implicated in oxidant lung injury (23). Peroxynitrite has been demonstrated to induce apoptotic cell death in the HL-60 monocytic tumor cell line (24). We did not measure peroxynitrite concentrations in our samples, and it is uncertain whether peroxynitrite is formed under these experimental conditions. However, we have observed significant neutrophil DNA fragmentation and cell death in neutrophils incubated with synthesized peroxynitrite (Fortenberry, preliminary results).

Experiments evaluating DNA fragmentation by ELISA demonstrated wide standard deviations. Such large standard deviation has been seen in other studies using this assay in measuring apoptosis (18, 19), perhaps because of the variability in quantity of DNA fragmentation under way in individual cells at any given time point. The deviation in our experiments, however, was the result of outliers in NO-exposed cells with very high levels of fragmentation, and these experiments did find statistical significance by nonparametric methods.

The percentage of nonviable cells in each exposure group was greater than the percentage of cells undergoing apoptotic DNA fragmentation by TUNEL assay. This discrepancy could suggest that neutrophil cell death from NO gas is primarily necrotic and that apoptotic fragmentation plays a lesser role. Alternatively, the in vitro nature of our experiments may account for this discrepancy. In vivo, apoptosis may occur within minutes (25). In the presence of tissue macrophages, neutrophils undergo rapid phagocytosis and clearance in vitro (10) and in vivo (11). If, however, tissue phagocytes are absent, as with in vitro studies such as ours, or overwhelmed by massive numbers of apoptotic cells, as with in vivo studies of hepatocyte apoptosis induced by Fas antigen (26), secondary necrosis of noningested apoptotic cells may occur. Assays of cell viability and of DNA fragmentation might then underestimate the primary role of apoptotic cell death induced by NO.

Recent reports suggest that exogenous NO may provide a protective effect in inflammation-mediated lung injury. Studies in isolated rabbit lungs have demonstrated substantial reductions in oxidant-induced increase in pulmonary vascular permeability and pulmonary edema by pretreatment with inhaled NO in concentrations of 90 to 120 ppm (27) and 24 ppm (28). Inhalation of 5 to 10 ppm NO also enhanced survival in adult rats exposed to 95% oxygen (29). Guidot and coworkers found that 50 ppm NO decreased oxygen radical-dependent pulmonary capillary leak in isolated rat lungs by an effect that appeared dependent on inhibition of neutrophil function (30).

Our results suggest that effects of NO on neutrophil function may result, in part, from induction of apoptotic cell death and resultant diminution of neutrophil oxidative machinery (12). Some previous reports of depression of oxidative function by NO (4, 5) are hindered by several potential artifacts. Superoxide radicals could have been diverted in these studies to react with NO and form peroxynitrite, thus artifactually diminishing cytochrome c reduction and underestimating superoxide generation by exposed neutrophils. We avoided this problem in our previous study on the effects of NO gas on neutrophil oxidative function (14) by performing superoxide generation assays after removal of NO exposure and washing cells. The concentrations of NO used in previous studies (4, 5) could also have generated NO metabolites not normally observed in significant quantities during biologic reactions. High NO concentrations could also have produced frank displacement of dissolved oxygen in reaction systems following equilibration with these high NO concentrations. We observed NO effects, in both this study and our previous study (14), at clinically relevant concentrations used in current practice that are significantly below those evaluated in the above studies, making such artifacts much less likely in our experiments.

Exposure to exogenous NO and induction of neutrophil apoptosis may also have undesired effects. Excessive clearance of neutrophils could be detrimental in the presence of active bacterial pulmonary infection. This would be particularly concerning in neonates, who possess inherently impaired neutrophil response (31) and are at risk for group B streptococcal pneumonia. Of additional concern, exogenous NO may have deleterious effects on resident lung cells that could potentially outweigh beneficial anti-inflammatory effects (32). Further in vitro and in vivo studies will therefore be critical to characterize the overall risks or benefits of NO exposure with respect to immunomodulatory effects.