INTRODUCTION

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Airway inflammation and hyperresponsiveness are characteristic features of asthma. Stimuli such as allergen, ozone, and viral infections can cause airway hyperresponsiveness in human and animals, and the development of this airway hyperresponsiveness has been associated with an influx of inflammatory cells into the airways (1). In addition to eosinophilic airway inflammation in asthma, infiltration of neutrophils is detected in the airways of acute severe asthma (2, 3). The precise mechanism of inflammatory cell accumulation and hyperresponsiveness remains unknown, but is considered to be dependent on the generation of various inflammatory mediators, including prostaglandins, leukotrienes, cytokines, and nitric oxide (NO).

NO has been shown to play an important role in diverse physiological processes. It is produced by the oxidation of L-arginine by an enzyme, NO synthase (NOS). Neuronal and endothelial NOS are generally constitutively present in the cells where they are expressed (4). Inducible NOS (iNOS), the third form of NOS, is usually only produced after stimulation of cells by agents such as tumor necrosis factor- (TNF-), interleukin-1 (IL-1), interferon- (IFN-), or lipopolysaccharide (5). In the respiratory tract, NO is produced by a variety of cells, including, neurons, epithelial cells, macrophages, and inflammatory cells (6), and NO may play a crucial role in inflammatory airway diseases such as asthma. The concentration of NO in exhaled air is increased in asthmatics (7), and an elevation of exhaled NO in patients with acute asthma exacerbation decreases with glucocorticoid treatments (8). NO generated by constitutive NOS is consider to be a bronchoprotective factor that is a neurotransmitter of the inhibitory nonadrenergic noncholinergic nerves (9), whereas the large amount of NO generated from iNOS possibly amplifies the inflammatory process. Thus, oxidant stress upregulates the expression of IL-8, a proinflammatory cytokine with potent chemotactic activity for neutrophils (10), and the free radical NO may also upregulate gene expression of proinflammatory cytokines in vivo (11, 12). The high IL-8 concentrations observed in sputum from patients with asthma in acute exacerbation, and IL-8 may mediate airway neutrophilia in acute asthma (2).

Inhaled ozone increases oxygen radical release from bronchoalveolar lavage (BAL) cells (13). Increased NO production and iNOS messenger RNA (mRNA) expression in the lung has also been demonstrated after acute exposure of rats to ozone (14). Ozone exposure increases the production of IL-8 in airway epithelial cells and alveolar macrophages in vitro (15) and in airway lavage fluid of human subjects (16). The administration of IL-8 into the airways induces local infiltration of neutrophils and airway hyperresponsiveness (17). Therefore, we hypothesized that NO could contribute to local neutrophil recruitment and airway hyperresponsiveness after ozone exposure and that IL-8 could be involved in mediating these effects of NO.

Because epithelial cells lining the airways are stimulated selectively to express IL-8 (18), and because NO production by airway epithelial cells is enhanced by cytokines (19), we first examined whether NO donors could induce IL-8 production and whether NOS inhibitors could inhibit increased IL-8 production by a mixture of cytokines (TNF-, IL-1, and IFN-) in human transformed bronchial epithelial cells in vitro. Next, in guinea pigs in vivo, we investigated whether NOS inhibitors could prevent the recruitment of neutrophils, airway hyperresponsiveness, and the upregulation of IL-8 mRNA induced by exposure to ozone. Induction of iNOS involves gene transcription, and increased production of NO occurs several hours after stimulation (19). Ozone-induced airway hyperresponsiveness persists for at least 24 h in guinea pigs (20). Therefore, we studied airway responsiveness, BAL for cell counts and for measurements of NO derivatives, and in situ hybridization for IL-8 mRNA in airway tissues immediately after ozone and 5 h after ozone. To inhibit NOS, we used N^G-nitro-L-arginine methyl ester (L-NAME), N^G-monomethyl-L-arginine (L-NMMA), and aminoguanidine (AG). L-NAME and L-NMMA are arginine analogs that inhibit both constitutive NOS and iNOS, and AG inhibits iNOS (21).

	METHODS

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Reagents

Minimal essential medium (MEM), fetal calf serum (FCS), and penicillin-streptomycin were purchased from Gibco-BRL (Grand Island, NY); S-nitroso-N-acetylpenicillamine (SNAP), sodium nitroprusside (SNP), L-NAME, L-NMMA, AG, and acetylcholine were purchased from Sigma (St. Louis, MO); IL-8 enzyme-linked immunosorbent assay (ELISA) kit was purchased from Amersham (Buckinghamshire, UK). Nitrate reductase from Aspergillus species, nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), and flavin adenine dinucleotide (FAD) were purchased from Boehringer Mannheim (Indianapolis, IN); Sulfanilamide and naphthylethylene-diamine were purchased from Wako Chemicals (Osaka, Japan); Human transformed bronchial epithelial cells, 16-HBE cells, were provided by Dr. Dieter Gruenert (University of California San Francisco).

Culture and Incubation of Human Bronchial Epithelial Cells

The 16-HBE cells were grown to confluence in MEM with 10% FCS and 100 units per ml each of penicillin and streptomycin on 35-mm plates coated with collagen. The cells were studied when they reached confluence. Before incubation, the cells were washed twice with phosphate-buffered saline (PBS) at 37° C, and then they were incubated with reagents up to 24 h.

SNAP and SNP that spontaneously release NO in aqueous medium were used as the NO donors. To test the effect of NO donors on IL-8 production by airway epithelial cells, we incubated 16-HBE cells with several dilutions of SNAP or SNP for 24 h. To examine the effects of NOS inhibitors on IL-8 production by airway epithelial cells, the cells were pretreated with 1 mM of L-NAME, L-NMMA, AG, or culture medium alone for 1 h before stimulation with cytokine mixture (22), a combination of TNF- (10 ng/ml), IL-1 (10 ng/ml), and IFN- (100 IU/ml). The incubation medium of the cells was examined for IL-8 concentration and for lactate dehydrogenase activity. Epithelial cells were then harvested by incubation in 0.3 ml of 0.1% trypsin. The digestion was stopped by the addition of 10% FCS in PBS, and the sample was centrifuged at 1,000 × g for 5 min. The cells were resuspended in the original volume of serum-free MEM before counting cell number with a hemocytometer. Lactate dehydrogenase release from the cells was measured to assess cell viability (LDHcII; Wako Chemicals, Osaka, Japan). In our experiments, it never exceeded 5% release.

Animal Experiments In Vivo

Experimental protocol. Four groups (each group had 6 to 10 animals) of male Hartley-strain guinea pigs (450 to 550 g) were used in this study: (1) sham-exposed animals pretreated with vehicle, the control group; (2) sham-exposed animals pretreated with NOS inhibitors; (3) ozone-exposed animals pretreated with vehicle; (4) ozone-exposed animals pretreated with NOS inhibitors. Because AG has been reported to inhibit iNOS (21), and because of the inhibitory effect of AG plus L-NAME or AG L-NMMA on IL-8 production on airway epithelial cells in vitro, a combination of AG (123 mg/kg, intraperitoneally) (23) and L-NAME (270 mg/kg, intraperitoneally) was used in vivo to examine the effect of NOS inhibitors on airway hyperresponsiveness and on inflammation after ozone.

Animals were pretreated with vehicle (saline) or with AG plus L-NAME 1 h before ozone exposure. Ozone exposures were performed as previously described (24). The animals inhaled dry air or 3.0 ± 0.1 ppm (mean ± SD) of ozone for 2 h while awake and spontaneously breathing in a 2.4-L exposure chamber. Ozone was generated by passing 100% oxygen through an ozonator (Model 0-1-2; Nihon Ozone, Tokyo, Japan) regulated by a variable-voltage supply. The concentration of ozone in the chamber was continuously monitored by an ultraviolet analyzer (Model 1500; Dasibi, Glendale, CA). Immediately after or 5 h after exposure, the measurement of airway responsiveness to acetylcholine and BAL were performed. For in situ hybridization and immunohistochemical studies, animals were euthanized immediately after or 5 h after exposure. The lungs were removed after thoracotomy and were fixed in 4% paraformaldehyde for 30 min at 4° C, and washed in PBS. The lung tissues were cryoprotected with 30% sucrose, embedded in ornithine carbamyl transferase compound (Miles, Elkhart, IN), and frozen. For in situ hybridization, the number of animals in each group was three.

Measurement of airway responsiveness. Airway responsiveness to acetylcholine was assessed as previously described (25). Animals were anesthetized with pentobarbital sodium (50 mg/kg, intraperitoneally), and the trachea was cannulated. The animals were then ventilated mechanically with a respirator (7 ml/kg tidal volume, 60 breaths/min; Model 680, Harvard Apparatus, South Natick, MA). A catheter was inserted into a carotid artery, through which blood pressure was measured by an electric manometer (LPU-01; Nihon Kohden, Tokyo, Japan). The animals were placed supine in a body plethysmograph. Plethysmograph airflow was measured with a Fleisch pneumotachograph (TV-132T; Nihon Kohden) and a differential pressure transducer (TP-602T; Nihon Kohden). To evaluate pleural pressure, a fluid-filled catheter was introduced into the esophagus so that the maximal amplitude of pressure was obtained. Transpulmonary pressure was estimated from the difference between pleural and airway opening pressure, as measured by a differential pressure transducer (TP-603T; Nihon Kohden). Total pulmonary resistance (RL) was calculated from the transpulmonary pressure and plethysmograph airflow. Airway responsiveness was assessed by RL changes after inhalation of increasing concentrations of acetylcholine (0.03 to 4.0 mg/ml). Acetylcholine aerosols were generated by an ultrasonic nebulizer (20 µl/min, output at the port of the tracheal cannula; TUR-3200; Nihon Kohden) placed in line with the ventilator. The concentration of acetylcholine required to produce a 200% increase in RL (PC₂₀₀) was calculated by log-linear interpolation from individual animals.

BAL. Guinea pigs were euthanized and BAL was performed after the determination of airway responsiveness as previously described (24, 25). Both lungs were gently lavaged three times with normal saline via the tracheal cannula at a pressure of 25 cm H₂O. Total cell counts were determined under light microscopy using a standard hemocytometer. The BAL fluid (BALF) was centrifuged at 200 × g for 10 min at 4° C. The cell pellet was resuspended in saline, and Cytospin preparations (Cytospin 3; Shandon, Pittsburgh, PA) were made. Differential counts on 200 cells were performed under light microscopy using a single-blind method following a modified Wright-Giemsa stain (Diff-Quik; Baxter, McGaw Park, IL). In the supernatant from the BALF, nitrate/nitrite concentration was measured.

Nitrite and Nitrate Analysis

NO release was determined spectrophotometrically by measuring the accumulation of its stable degradation products, nitrite and nitrate. Nitrite in the incubation medium of 16-HBE cells and in BALF samples was measured using the Giess reagent (1% sulfanilamide / 0.1% naphthylethylene diamine dihydrochloride / 2% phosphoric acid), with the formation of a chromophore absorbing at 540 nm. Nitrate was reduced to nitrite by 0.1 U/ml nitrate reductase from Aspergillus species in the presence of 50 µM NADPH and 5 µM FAD (26), and the products were then measured as nitrite. The combination of nitrite and reduced nitrate was NO₂/NO₃. Background levels were determined in saline and were subtracted for calculation of total amount. Data were presented as nanomoles per liter of BALF.

Measurement of IL-8 Concentration

The concentration of human IL-8 protein in the incubation medium of 16-HBE cells was quantified using an IL-8 ELISA kit and was normalized for cell number and expressed as pg IL-8/10⁶ cells.

Northern Blot Hybridization

32P-labeled probe preparation. Using a 158-bp fragment of complementary DNA (cDNA) for IL-8, containing the sequence from the PstI site of exon I to the HindIII site of exon III, as a template, a ³²P-labeled probe for Northern blot hybridization was generated by random-prime synthesis (Gibco BRL, Gaithersburg, MD), as reported previously (18). Unincorporated label was separated by chromatography over a Sephadex G-50 column (NucTrap push column; Stratagene, La Jolla, CA).

Northern blot analysis. Total RNA from 16-HBE cells was extracted by the guanidinium thiocyanate-phenol-chloroform method (RNAstat-60; Tel-test "B", Inc., Friendwood, TX). A volume of 10 µg of total RNA was separated in 1.2% agarose gel containing deionized glyoxal, transferred to nylon membranes (Genescreen; Dupont, New England Nuclear, Boston, MA) by capillary action, and cross-linked with ultraviolet light. Membranes were prehybridized in 10% dextran sulfate, 0.2% bovine serum albumin (BSA), 0.2% polyvinyl-pyrrolidone, 0.2% ficoll, 50 mM TRIS, 0.1% sodium pyrophosphate, 1% sodium dodecyl sulfate (SDS), and sheared salmon sperm DNA (100 µg/ml) at 42° C for 2 h. Then, membranes were hybridized overnight at 42° C with denatured ³²P-labeled cDNA probe. Membranes were washed (once in 2× saline sodium citrate [SSC] containing 0.1% SDS at room temperature for 5 min, twice in 2× SSC containing 0.1% SDS at 50° C for 30 min, and once in 0.1× SSC containing 0.1% SDS at room temperature for 30 min), and subjected to autoradiography. Densitometry with image analysis program NIH Image 1.52 Macintosh was used to quantify IL-8 transcripts.

In Situ Hybridization

Digoxigenin-labeled probe. A digoxigenin-labeled probe for in situ hybridization was obtained by polymerase chain reaction (PCR). Total RNA from guinea pig liver was converted to cDNA using reverse transcription (RT) of 4 µg RNA sample in a 20-µl reaction volume containing 10 mM TRIS HCl (pH 8.8), 50 mM KCl, 4 mM MgCl₂, 0.1% Triton X-100, 1 mM dithiothreitol (DTT), 0.25 mM deoxyribonucleoside triphosphates (dNTPs), 5 µM random hexamer primers, 0.1 U/µl of ribonuclease inhibitor (Promega Corp., Madison, WI), and 10 U/µl of Moloney murine leukemia virus reverse transcriptase (MMLV-RT) (Gibco BRL). The reaction mixture was incubated at 42° C for 1 h, and at 95° C for 5 min. The cDNA was then diluted to 100 µl and was used as a template in PCRs. The primers, which were specific for guinea pig IL-8 cDNA (27) or pUC19 DNA, used were as follows: guinea pig IL-8: sense 5'-TGGTCGTGACAAAGTTGGTC-3', antisense 5'-CCTGCACCCACTTCTTCTTG-3'; pUC 19: sense 5'-CATTTTGCCTTCCTGTTTTT-3', antisense 5'-GCTTTTCTGTGACTGGTG-3'.

The digoxigenin-labeled PCR probes for guinea pig IL-8 mRNA (double-stranded antisense/sense, single-stranded antisense, or single-stranded sense) and for pUC 19 DNA were synthesized in 50-µl reactions containing 70 µM digoxigenin 11-deoxyuridine triphosphate (dUTP), 10 mM TRIS HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl₂, 0.1% Triton X-100, 0.2 mM each unlabeled deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), 0.13 mM unlabeled deoxythymidine triphosphate (dTTP), primer pairs, antisense primer or sense primer alone, 5 µl of cDNA or of pUC 19 DNA as template, and 1.25 U of Taq polymerase (Nippon Gene, Tokyo, Japan). The PCR products were precipitated with 70% ethanol and 0.3 M sodium acetate for 2 h at 20° C. After ethanol precipitation, the DNA was dried. The pellet was resuspended in 2 parts of hybridization mix, which contained 1.2 M NaCl, 20 mM TRIS HCl (pH 7.5), 4 mM ethylenediaminetetraacetic acid (EDTA), 2× Denhardt's solution, 1 mg/ml yeast transfer RNA (tRNA) and 200 µg/ml poly(A) (Pharmacia Diagnostic AB, Uppsala, Sweden), 2 parts of deionized formamide, and 1 part of 50% dextran sulfate. To verify that the incorporation of digoxigenin of each of the probes was similar, 1 µl of the probe was fixed to a Hybond-N membrane (Amersham) and immunologically detected with an antidigoxigenin antibody using the DIG Nucleiacid Detection Kit. The signal was developed using nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP). The intensities of the color signals of the double-stranded probes for guinea pig IL-8 and pUC19, and those of single-stranded antisense and sense probes for guinea pig IL-8 were equal.

In situ hybridization. Frozen sections (5 µm) of guinea pig lung tissues were cut and mounted on positively charged glass slides (Fisher Scientific, Pittsburgh, PA). The negative control for the detection system was obtained by comparing a section treated with the double-strand antisense/sense probe or the antisense probe for guinea pig IL-8 with an adjacent section on the same slide treated with pUC 19 probe or the sense probe for guinea pig IL-8. The cryosections were covered with 4% paraformaldehyde for 5 min. The section was flooded with 3× PBS for 5 min, washed with PBS twice, dehydrated with ethanol, and dried. The section was immersed in 20 µg/ml of proteinase K in 50 mM TRIS HCl (pH 7.5) and 5 mM EDTA for 10 min at 37° C. The slide was rinsed in PBS containing 2 mg/ml of glycine for 30 s and in PBS for 30 s. The slide was immersed in freshly prepared triethanolamine (TEA) buffer for 5 min, and in TEA buffer containing 0.25% acetic anhydride (Sigma) for 10 min. The slide was washed with 2× SSC, dehydrated in ethanol, and dried. The PCR probe hybridization mixture was incubated for 5 min at 93° C, and then chilled in an ice bath. DTT was added to the mixture to yield a final concentration of 50 mM. Each section was covered with 5 µl of the PCR probe mixture and covered with a coverslip. The slide was incubated at 42° C overnight in a well-sealed chamber containing moist chamber solution, which consisted of 50% formamide, 0.6 M NaCl, 10 mM TRIS HCl (pH 7.5) and 2 mM EDTA. After hybridization, the slide was washed four times with a solution containing 0.6 M NaCl, 10 mM TRIS HCl (pH 7.5), 1 mM EDTA, 50% formamide, and 0.1% b-mercaptoethanol for 10 min each at 37° C. The slide was washed twice with Buffer 1 containing 0.1 M TRIS HCl (pH 7.6), 0.1 M NaCl, 2 mM MgCl₂, and 0.05% Triton X-100 for 5 min, then incubated with 3% BSA in Buffer 1 for 10 min. It was then incubated with antidigoxigenin antibody-alkaline phosphatase conjugate (Boehringer Mannheim) in 3% BSA in Buffer 1 for 30 min. The slide was washed with Buffer 1, immersed in AP 9.6 solution containing 0.1 M TRIS HCl (pH 9.6), 0.1 M NaCl, and 0.05 M MgCl₂, and incubated in NBT/BCIP color reaction solution in AP 9.6 for 10 min in a dark room. The color reaction was stopped by washing with 2 mM EDTA in PBS, then rinsed with dH₂O. After the slides were dried, coverslips were applied and the slides were examined under light microscopy.

Statistical Analysis

Data are presented as means ± SEM. The effects of NO donors and NOS inhibitors on IL-8 production and the effects of NOS inhibitors on PC₂₀₀ value, cell counts, and the amounts of nitrite/nitrate in BALF were compared by analysis of variance (ANOVA), and the significance of differences between values was assessed with the Bonferroni correction for multiple comparisons. The time-dependent effects of cytokine mixture with or without NOS inhibitors on IL-8 production by epithelial cells were assessed by multiple linear regression. p Values less than 0.05 were considered to indicate statistical significance.

	RESULTS

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Nitrite/Nitrate Concentrations and IL-8 Production from Airway Epithelial Cells In Vitro

IL-8 protein concentration in the supernatant of 16-HBE cells was increased 12 h after incubation with SNAP (10⁶ to 10⁴ M) and with SNP (10⁶ to 10⁴ M) dose-dependently (Figure 1). We also incubated airway epithelial cells with cytokine mixture, TNF- plus IL-1 plus IFN-, in the presence or absence of NOS inhibitors for 24 h. Either AG, L-NAME, or L-NMMA alone did not significantly decrease IL-8 production from 16-HBE cells. However, the combination of AG plus L-NAME, or AG plus L-NMMA, significantly attenuated the cytokine mixture-induced IL-8 production from airway epithelial cells (Figure 2A). The combination of AG plus L-NAME, or AG plus L-NMMA, attenuated cytokine mixture- induced IL-8 production time-dependently. The slopes of the three curves were compared by multiple linear regression. The time course of IL-8 production after incubation of the epithelial cells with AG plus L-NAME and after the incubation with AG plus L-NMMA were significantly different from the IL-8 production without NOS inhibitors after incubation with cytokine mixture alone (p < 0.05 and p < 0.05, respectively; Figure 2B). The inhibitory effect of NOS inhibitors on IL-8 production from epithelial cells in vitro was significant after 6 h of application. Therefore, the effects of NOS inhibitors on ozone- induced airway hyperresponsiveness and neutrophil recruitment were evaluated 5 h after the end of (7 h after the beginning of) ozone exposure. Cytokine mixture caused an increase in the amount of nitrite/nitrate in the incubation medium of 16-HBE cells 24 h after incubation from 2.0 ± 0.1 µM to 5.7 ± 1.0 µM. In the presence of AG plus L-NMMA the amount of nitrite/nitrate 24 h after incubation with cytokine mixture was 2.2 ± 0.2 µM, and NOS inhibitors inhibited the increase in nitrite/nitrate concentration (p < 0.01).

View larger version (31K): [in this window] [in a new window]   Figure 1.   Effect of NO donors, SNAP (closed bars) or SNP (stippled bars), on IL-8 production by airway epithelial cells. Cultured 16-HBE cells were incubated with medium alone as control or with serial dilutions of SNAP or SNP for 12 h. Data are expressed as picograms of IL-8 per 106 cells; values are mean ± SEM; n = 5. *Statistically significant difference from control (p < 0.05).

View larger version (24K): [in this window] [in a new window]   Figure 2.   (A) Effect of the inhibitors of NOS, L-NAME (1 mM), L-NMMA (1 mM), and AG (1 mM), on cytokine mixture-induced IL-8 production by airway epithelial cells. Cultured 16-HBE cells were incubated with medium alone as control or with cytokine mixture, TNF- (10 ng/ml) plus IL-1 (10 ng/ml) plus IFN- (100 IU/ml), in the presence or absence of NOS inhibitors for 24 h (closed bars). 16-HBE cells were also incubated with low-dose cytokine mixture in the presence or absence of NOS inhibitors (hatched bars). Data are expressed as percent inhibition of cytokine mixture-induced IL-8 production. (B) Time-dependent IL-8 protein production by 16-HBE cells during incubation with medium alone as control (open cycles) or with cytokine mixture in the presence (closed squares) or absence (open squares) of L-NMMA (1 mM) plus AG (1 mM). Data are expressed as picograms of IL-8 per 106 cells; values are mean ± SEM; n = 5. *Statistically significant difference from cytokine mixture alone (p < 0.05).

Northern blot analysis of 16-HBE cells revealed low levels of IL-8 mRNA expression in the control state, but IL-8 mRNA expression was increased 6 h after incubation with the cytokine mixture. The levels of IL-8 gene expression after the cytokine mixture with NOS inhibitor pretreatment, AG plus L-NAME, or AG plus L-NMMA, was lower than those after the cytokine mixture alone (Figure 3).

View larger version (30K): [in this window] [in a new window]   Figure 3.   IL-8 mRNA expression in 16-HBE cells 6 h after incubation. 16-HBE cell were incubated with medium alone as control (lane 1) or with cytokine mixture, TNF- (10 ng/ml) plus IL-1 (10 ng/ml) plus IFN- (100 IU/ml), in the absence (lane 2) or in the presence of NOS inhibitors, L-NAME (1 mM) plus AG (1 mM) (lane 3), L-NMMA (1 mM) plus AG (lane 4). Total RNA was extracted and subjected to Northern blot hybridization. The blot was hybridized with and human IL-8 cDNA probe (top panel ). The middle panel shows corresponding 18S and 28S ribosomal RNA of the autoradiographs shown in each upper panel, indicating equivalent loading of total RNA. Data shown are derived from typical experiments. The bottom panel shows the densitometric study of IL-8 mRNA transcript levels. Data from three experiments are reported and expressed as percent of maximal response; values are means ± SEM.

Airway Inflammation and Hyperresponsiveness after Ozone in Guinea Pigs In Vivo

There were no significant differences in baseline values of RL among groups.

Immediately after exposure, PC₂₀₀ value in ozone-exposed guinea pigs was significantly lower than that in sham-exposed animals or that in vehicle-treated sham-exposed animals (p < 0.01 or p < 0.01). Five hours after ozone exposure, this airway hyperresponsiveness to acetylcholine remained but was less. PC₂₀₀ value in ozone-exposed animals 5 h after exposure was still significantly lower than that in vehicle-treated sham- exposed animals (p < 0.05).

Pretreatment of guinea pigs with AG plus L-NAME did not influence the PC₂₀₀ value in sham-exposed animals. Pretreatment with AG plus L-NAME had no effect on PC₂₀₀ value immediately after ozone exposure. However, 5 h after ozone exposure, PC₂₀₀ value in the guinea pigs pretreated with AG plus L-NAME was significantly higher than the value in animals pretreated with vehicle (p < 0.05; Figure 4).

View larger version (30K): [in this window] [in a new window]   Figure 4.   Effect of inhibitors of NOS, L-NAME (270 mg/kg) plus AG (123 mg/kg), pretreatment on airway responsiveness to inhaled acetylcholine in guinea pigs measured immediately after and 5 h after ozone exposure. The animals were pretreated with vehicle (open bar and closed bars) or with NOS inhibitors (hatched bars). Data are means ± SEM of 5 animals. *Statistically significant difference from vehicle-pretreated group (p < 0.05).

Ozone exposure caused a significant increase in neutrophil numbers in BALF as compared with those from dry air- exposed animals, and this neutrophilia persisted at least 5 h after ozone exposure. Pretreatment with AG plus L-NAME had no effect on the neutrophil numbers in BALF immediately after ozone, but it significantly attenuated the ozone-induced neutrophil accumulation into BALF 5 h after exposure (p < 0.05; Figure 5).

View larger version (21K): [in this window] [in a new window]   Figure 5.   Effect of L-NAME plus AG pretreatment on cell counts in BALF in guinea pigs, performed immediately after (left panel ) and 5 h after (right panel ) ozone exposure. A significant increase in neutrophils was observed in ozone-exposed vehicle-treated group (closed bars) compared with sham-exposed group (open bar) both immediately after and 5 h after exposure. Pretreatment with L-NAME plus AG (hatched bars) significantly inhibited ozone-induced neutrophilia immediately after exposure, but not 5 h after exposure. Data are means ± SEM of 5 animals. *Statistically significant difference (p < 0.05).

Ozone also caused a significant increase in the amount of nitrite/nitrate in BALF 5 h after exposure but not immediately after exposure. AG plus L-NAME significantly inhibited the increase in nitrite/nitrate level in BALF 5 h after ozone (Figure 6).

View larger version (30K): [in this window] [in a new window]   Figure 6.   Effect of L-NAME plus AG pretreatment on nitrite and nitrate levels in BALF, performed immediately after and 5 h after ozone exposure. The animals were pretreated with vehicle (open bar and closed bars) or with NOS inhibitors (hatched bars). Data are means ± SEM of 5 animals. *Statistically significant difference from vehicle-pretreated group (p < 0.05).

IL-8 Expression after Ozone in Guinea Pig Airways In Vivo

In the airways of guinea pigs 5 h after ozone exposure, in situ hybridization revealed that IL-8 mRNA was detected in the airway epithelial cells and the accumulated cells, mainly neutrophils, in the airway lumen using a single-stranded antisense probe for IL-8 mRNA (Figure 7B) and double-stranded antisense/sense probe for IL-8 mRNA (data not shown), but not using single-stranded sense probe for IL-8 (Figure 7C) and pUC19 probe (data not shown). In contrast, in the airways of the animals pretreated with AG plus L-NAME 5 h after ozone exposure, only low levels of IL-8 expression were detected in the airway epithelial cells and some accumulated cells in the airway lumen (Figure 7D). In the airways of vehicle-treated sham-exposed animals, no signals were detected with antisense probe for IL-8 mRNA (Figure 7A).

View larger version (98K): [in this window] [in a new window]   Figure 7.   Comparison of IL-8 mRNA in bronchial tissues from guinea pig 5 h after sham exposure as control (A), 5 h after ozone exposure (B), 5 h after ozone exposure with L-NAME plus AG pretreatment (D). Bronchial tissue is hybridized with guinea pig IL-8 antisense probe (A, B, and D). For the specificity of the hybridization signals, bronchial tissue from vehicle-pretreated animals 5 h after ozone is hybridized with guinea pig IL-8 sense probe (C ). Bronchial tissue 5 h after ozone exposure is stained with modified Wright-Giemsa (E and F ) and panel F shows intraluminal cells. Arrows indicate examples of IL-8 mRNA signals. A to E, bar = 50 µm; F, bar = 10 µm.

	DISCUSSION

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The present study shows that NO-generating compounds induce IL-8 production in airway epithelial cells in vitro, and that NOS inhibitors inhibit cytokine mixture-induced IL-8 production. In guinea pigs in vivo, NOS inhibitor pretreatment attenuated ozone-induced airway hyperresponsiveness and neutrophil recruitment 5 h after exposure, but not immediately after exposure. In addition, the pretreatment with NOS inhibitors attenuated the increase in nitrite/nitrate concentrations in BALF and the increase in IL-8 mRNA expression in airway epithelial cells and neutrophils 5 h after ozone exposure. These findings suggest that endogenous NO, or its derivatives, facilitate persistent airway inflammation and hyperresponsiveness after ozone, presumably partly through the production of IL-8 in the airways.

Superoxide and NO generated by iNOS combine to form peroxynitrite, a potent cytotoxic oxidant. Peroxynitrite was shown to induce airway hyperresponsiveness in guinea pigs (28). It has been also demonstrated that an iNOS inhibitor attenuates the inflammatory demyelination in the central nervous system of mice and the plasma extravasation in rat skin (23). The late asthmatic response to allergen is associated with elevated exhaled NO concentrations, and this increase in NO is considered to be caused by the induction of iNOS in the airways (29). In the present study, NOS inhibitors attenuated ozone-induced airway inflammation 5 h after exposure, but not immediately after exposure. These results and the present findings suggest that endogenous NO, probably derived from iNOS, may amplify the inflammatory process. Acute exposure of rats to ozone has been reported to induce an influx of neutrophils and macrophages into the lung and also to increase NO production from lavage cell in vitro; neutrophils contribute to this NO production in the lung (14). Therefore, epithelial cell and neutrophils may contribute to NO production after ozone exposure.

NOS inhibitors pretreatment attenuated ozone-induced neutrophil recruitment in the airways in vivo, and NOS inhibitors may affect neutrophil chemotaxis directly. NO has been reported to inhibit neutrophil chemotaxis, and this inhibition is associated with a decrease in intracellular cyclic guanosine monophosphate (cGMP) levels (30). Endogenous NO mediates the chemotaxis of neutrophils in vitro (31). However, in our study pretreatment of NOS inhibitors did not affect neutrophil accumulation in the airways immediately after ozone exposure. Therefore, the inhibitory effects of NOS inhibitors are believed to be dependent on mediators expressed in the course of inflammation.

NOS message and activity have been demonstrated in the airways, especially epithelial cells, and iNOS expression is dependent on stimulation. In contrast, constitutive iNOS expression was reported recently in the airways (32). NOS inhibitors might affect the basal degree of airway responsiveness or responsiveness immediately after ozone exposure. However, in the present study NOS inhibitors attenuated ozone-induced airway hyperresponsiveness and neutrophil recruitment 5 h after exposure, but not immediately after exposure. These observations indicate that iNOS, but not constitutive NOS, may play an important role in airway responsiveness in guinea pigs. Our findings are supported by previous reports on the time course of NO production by iNOS following stimulation, in which approximately 6 h pass before significant amounts of NO are produced (33).

In the present study, AG, L-NAME, or L-NMMA alone did not inhibit IL-8 production significantly. Therefore, the effect of combination of these NOS inhibitors was examined; they attenuated IL-8 production in airway epithelial cells in vitro, and they inhibited ozone-induced neutrophil accumulation and airway hyperresponsiveness in vivo. The possible explanation why NOS inhibitors inhibited IL-8 production only in combination may be that the available inhibitors of NOS are poorly selective and not potent enough (34). In the present study, the combination of AG with L-NMMA was more effective than with L-NAME. The evidence that iNOS is more sensitive to L-NMMA and that constitutive NOS is more sensitive to L-NAME supports our findings (35). Future studies using new selective and potent inhibitors of iNOS will provide further information. In the present study, IL-8 expression induced by cytokine mixture was inhibited by NOS inhibitors partially. Control of IL-8 gene expression has been investigated and the transcription factor nuclear factor kappa B (NF-B) shown to be essential for the activation by proinflammatory cytokines (36). NO induces NF-B activation through G proteins (37). We speculate that cytokines could activate NF-B and subsequently stimulate IL-8 expression directly or indirectly through NO.

Oxidant stress upregulates gene expression of proinflammatory cytokines such as IL-8 (10). In in vitro studies, NO, a nitrogen-free radical, has also been reported to upregulate IL-8 expression in endothelial cells (11), and stimulation of Fc receptors includes the expression of rat analogues of the human IL-8/Gro chemokine (cytokine-induced neutrophil chemoattractant) in rat peritoneal macrophages (12). In the present study, IL-8 production was not only stimulated by NO donors but also inhibited by NOS inhibitors in vitro, and nitrite/nitrate concentrations in BALF of guinea pigs were increased 5 h after ozone exposure in vivo. Therefore, ozone is considered to upregulate IL-8 expression in the airways in vivo, presumably through increased NO production.

The development of airway hyperresponsiveness after exposure to ozone in a variety of animal preparations has been associated with an acute inflammatory response in the airways (24). Eicosanoids, especially leukotriene B₄, has been suggested to be important in the pathogenesis of ozone-induced airway inflammation and airway hyperresponsiveness in dogs. However, a recent study reported that a 5-lipoxygenase-activating protein antagonist (MK-0591) does not inhibit ozone-induced airway hyperresponsiveness and neutrophil accumulation in the airways (38). The production of IL-8 in human airway epithelial cells in vitro is increased after ozone exposure (15). In in vivo studies, exposure to ozone induces mRNA expression of a cytokine-induced neutrophil chemoattractant in rat lung (39) and increases IL-8 protein concentrations in airway lavage fluid of human subjects (16). The administration of IL-8 into the airways induces local infiltration of neutrophils and airway hyperresponsiveness (17). In the present study, IL-8 mRNA signals were detected in guinea pig airways 5 h after ozone exposure, but not sham-exposed animals. These results suggest that IL-8 may play a key role in neutrophil recruitment and in hyperresponsiveness in the airways after ozone.

In conclusion, we showed that pretreatment with NOS inhibitors attenuates ozone-induced airway inflammation and hyperresponsiveness at persistent phase, presumably partly through the inhibition of IL-8 production. Along with such evidence of the anti-inflammatory properties of NOS inhibitors, our findings suggest that NOS inhibitors could exert a beneficial effect in the treatment of inflammatory airway diseases.