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Allergic Airway Hyperresponsiveness and Eosinophil Infiltration is Reduced by a Selective iNOS Inhibitor, 1400W, in Mice
Pulmonary Pharmacology & Therapeutics (2000) 13, 267–275 doi:10.1006/pupt.2000.0254, available online at http://www.idealibrary.com on
PULMONARY PHARMACOLOGY & THERAPEUTICS
Allergic Airway Hyperresponsiveness and Eosinophil Infiltration is Reduced by a Selective iNOS Inhibitor, 1400W, in Mice Akira Koarai, Masakazu Ichinose∗, Hisatoshi Sugiura, Shunsuke Yamagata, Toshio Hattori, Kunio Shirato First Department of Internal Medicine, Tohoku University School of Medicine, Sendai, Japan
SUMMARY: Nitric oxide (NO) hyperproduction has been reported in asthmatic airways and may contribute to airway inflammatory responses. The purpose of this study was to examine the role of NO via inducible NO synthase (iNOS) in allergic airway inflammation using a selective iNOS inhibitor, N-[3-(aminomethyl)benzyl] acetamidine (1400W), in ovalbumin (OVA)-sensitized Balb/c mice. Sensitized animals were challenged with aerosolized 0.5% OVA for 1 h on two occasions 4 h apart. 1400W or the vehicle was administered by osmotic mini-pump from 2 h before to 24 h after OVA challenge. Twenty-four hours after OVA challenge, the vehicletreated mice showed a significant airway hyperresponsiveness to intravenous methacholine (P<0.05) as well as an influx of eosinophils into the airways (P<0.05). iNOS immunoreactivity was obvious in the epithelial and, to a lesser extent, the infiltrated inflammatory cells. iNOS protein in the airway assessed by Western blotting also increased. Pretreatment with 1400W almost completely abolished the OVA-induced airway hyperresponsiveness and to a lesser extent eosinophil accumulation into the airways. These results suggest that NO synthesized by iNOS may participate in airway hyperresponsiveness and eosinophil infiltration into the airways after allergic reaction. 2000 Academic Press KEY WORDS: Asthma, Airway resposiveness, Nitric oxide, iNOS protein, iNOS immunoreactivity, Eosinophil infiltration.
reported that the exhaled NO levels are increased in bronchial asthma patients compared with those of healthy subjects,6 and that the increase of exhaled NO is correlated with eosinophilic inflammation,7 which is a prominent feature of bronchial asthma. Therefore, although NO produced by iNOS appears to be involved in the inflammatory process of asthma, the precise role of NO in asthmatic airway inflammation has not yet been fully elucidated. Until now, the effect of iNOS-derived NO has been examined using selective iNOS inhibitors such as aminoguanidine8,9 and -N6-(1-iminoethyl) lysine (NIL).10 However, the selectivity of aminoguanidine and -NIL for iNOS is not very high. Therefore, some results obtained using these compounds may be misleading. Recently, a new, highly selective inhibitor for iNOS, N-[3-(aminomethyl)benzyl] acetamidine (1400W), has been developed by Garvey et al.11 Its inhibitory potency to iNOS/eNOS is at least 1000, indicating the selectivity for iNOS by 1400W is quite high.11 In the present study, using 1400W compound, we examined the role of iNOS-derived NO in the
INTRODUCTION Nitric oxide (NO) is a gas molecule synthesized by nitric oxide synthase (NOS) that has many physiological functions.1–3 Nitric Oxide Synthase has at least three isoforms which are composed from two types of constitutive NOS (nNOS) and an inducible NOS (iNOS). cNOS consists of neural NOS (nNOS) and endothelial NOS (eNOS). cNOS-derived NO may be important for physiological regulations such as vasodilation, neurotransmission and inhibition of platelet aggregation.3 In contrast, iNOS, presumably induced by inflammatory cytokines such as tumor necrosis factor (TNF)-, interleukin (IL)-1, and interferon (IFN)-,2 produces much larger amounts of NO than cNOS. iNOS has been reported to be expressed on airway epithelial cells and some inflammatory cells in asthma patients.4,5 It has also been ∗ Author for correspondence: First Department of Internal Medicine, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan. Fax: 81-22-717-7156. E-mail: [email protected] 1094-5539/00/060267+09 $35.00
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airway hyperresponsiveness as well as eosinophil accumulation into the airways in allergic mice model. We found that 1400W significantly inhibited both responses.
around the trachea and these numbers were corrected by dividing by the basement membrane’s length. Eosinophils were counted on each of five continuous sections and these numbers were averaged. Western blot analysis of iNOS
MATERIALS AND METHODS Mice Specific pathogen-free male Balb/c mice between 5 and 7 weeks of age purchased from the Institute for Experimental Animals (Tohoku University School of Medicine, Sendai, Japan) were used. Mice were maintained in conventional animal housing at a constant temperature and humidity with regular 12-h cycles of light and darkness. All of the experiments performed in this study were conducted with the consent of the Ethics Committee for the Use of Experimental Animals of the Tohoku University School of Medicine. Sensitization and allergen challenge Mice were sensitized and challenged as previously described.12 Briefly, mice were sensitized by an ip injection of 0.5 ml solution containing 8 g of ovalbumin (OVA) and 4 mg of aluminum hydroxide (alum) in saline on days 0 and 5. Twelve days later, mice were placed in a plexiglas chamber (10 cm×15 cm×25 cm) and challenged with aerosolized saline or 0.5% OVA in saline for 1 h on two occasions 4 h apart. The aerosolized OVA was produced by an ultrasonic nebulizer (NE-12, Omuron, Tokyo, Japan; output 0.8 ml/min). On day 17 at 2 h pre-challenge, mice were anesthetized with diethyl ether and implanted SC with osmotic mini-pumps (Alzet; ALZA Scientific Products, Palo Alto, California, USA), which provided a continuous infusion (1.0 l/h) of 1400W (10 mg/kg per h) dissolved in saline or saline alone until 24 h post-challenge.13 At 24 h post-challenge, mice were anesthetized by ip injection of pentobarbital (70 mg/kg body weight) and used for the following experiments. Quantification of eosinophil accumulation into the airways The trachea was perfused with 1% paraformaldehyde (PFA) in 50 mM phosphate buffered saline (PBS), immersed in 4% PFA fixative solution for 12 h at 4°C, and further immersed for 24 h at 4°C in 0.1 M PBS containing 15% sucrose. The tissues were then sectioned at a thickness of 6 m with a cryostat. The sections were stained using Hansel’s stain.14 Eosinophils were counted within the submucosal area all
The thorax was opened and the systemic circulation was perfused with ice-cold 50 mM PBS containing 1 mM ethylenediaminetetraacetic acid (EDTA) for 5 min at 100 mmHg using a 18 G-needle passed through a left ventriculoctomy into the ascending aorta. The pulmonary circulation was also perfused with 5 ml of the same solutions from the right ventricle. After that, lung tissue was isolated and its connective tissues, vasculatures and parenchyma were gently scraped off. The airways were homogenized in 0.8 ml of 50 mM Tris-HCl buffer (pH 7.8) containing 150 mM NaCl, 1 ml EDTA, 1 mM sodium vanadate, 1% NP-40, 1 mM para-amidino-phenylmethylsulfonyl fluoride (para-PMSF).10 The samples were centrifuged at 10 000 g for 15 min at 4°C and the supernatants were concentrated by a microcon 10 (Amicon, Inc., Beverly, USA) at 14 000 g for 50 min at 4°C. The protein content was determined using the method of Lowry15 with bovine serum albumin as the standard. Sample protein was added at one fourth the volume of a 250 mM Tris buffer containing 20% -mercaptoethanol, 0.01% bromophenol blue, 8% sodium dodecyl sulfate (SDS) and 40% glycerol and boiled for 5 min. Protein (90 g/lane) was separated on a 7.5% SDS polyacrylamide gel, transferred to a hydrophobic polyvinylidine difluoride membrane and probed with an anti-mouse iNOS rabbit antisera (Wako Pure Chemical Industries, Osaka, Japan). The secondary antibody used was anti-rabbit IgG from goat conjugated horseradished peroxidase (DAKO Japan Ltd., Kyoto, Japan). Antibody binding was detected using ECL-plus (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer’s recommendation. Samples of mouse macrophage lysates stimulated with interferon- (INF-) and lipopolysaccharide (LPS) were used as positive controls (Transduction Laboratories, Lexington, Kentucky, USA). The membrane was photographed by a midnight sun camera (C-1; Fuji Photo Film Co., Tokyo, Japan) and the intensity of the bands was quantified by densitometry (MCID image analyser; Imaging Inc. Research, St. Catherines, Ontario, Canada). iNOS immunostaining The trachea was perfused with 1% PFA, immersed in 4% PFA fixative solution for 12 h at 4°C, and further immersed for 24 h at 4°C in 0.1 M phosphate buffer containing 15% sucrose. The tissues were then sectioned at a thickness of 6 m with a cryostat. All
iNOS inhibitor and allergic responses
sections were mounted on chrom-alum gelatin-coated glass slides. Endogenous peroxidase activity was reduced by incubation in 3% hydrogen peroxide in 100% methanol for 5 min at room temperature. After washing in PBS, sections were incubated with primary antibody [polyclonal iNOS rabbit anti sera, 1:250 dilution (Wako Pure Chemical Industries). In order to reduce non-specific binding of the antibody, tissues were preincubated with 4% skim milk in PBS containing 0.3% Triton-X for 30 min and then incubated with 10% inactivated normal goat serum for 30 min at room temperature. The immunoreactions were visualized by the indirect immunoperoxidase method using ENVISION polymer reagent, which is anti-rabbit IgG from goat conjugated with peroxidase labeled dextran (DAKO Japan Ltd.), for 1 h at room temperature. The diaminobenzidine reaction was performed, followed by counterstaining with hematoxylin. Stained sections were viewed on a microscope (BX-40; Olympus, Tokyo, Japan) and captured with a 3-CCD color video camera (KY-F55MD; Olympus) using image analysis software (Mac SCOPE; Mitani Co., Fukui, Japan).
Measurement of airway responsiveness Twenty-four hours after OVA challenge, mice were anesthetized with pentobarbital sodium (70 mg/kg) and pretreated with propranolol (4 mg/kg) ip 15 min before bronchoconstrictor challenges to avoid -adrenergic neural modifications. The trachea was canulated with a tracheal tube (0.6 mm outer diameter) and connected via two ports of a four-way connector to a rodent ventilator (model 683, Harvard Apparatus, South Natick, Massachusetts, USA). Airway opening pressure (Pao) was detected by a pressure transducer (model MP45-1, Validyne, Northridge, California, USA) with one side connected to the fourth port of a four-way connector. Mice were ventilated at 150 strokes/min with tidal volumes of 6 ml/kg. After establishing a stable baseline of Pao, acetyl--methylcholine chloride (methacholine, MCh) was cumulatively administered (10–1000 g/kg) intrajugularly with a 30G microsyringe and the changes of Pao were monitored.16
Drugs Ovalbumin (class V), paraformaldehyde, EDTA, sodium vanadate, Triton-X, and diaminobenzidine were obtained from Sigma Chemical Co. (St. Louis, Missouri, USA). -mercaptoethanol and SDS were purchased from Bio-Rad Laboratories. Pentobarbital was purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan), NP-40 from Nacalai Tesque, Inc. (Kyoto, Japan), and Hansel’s stain from Torii Pharmaceutical
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(Tokyo, Japan). Other chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan). Statistical analysis Data are expressed as mean±SEM. Multiple comparisons of mean data of iNOS Western blotting, airway hyperreactivity and eosinophil infiltrations among the groups were performed by one way analysis of variance (ANOVA) followed by Scheffe’s test as a post hoc test. Probability values <0.05 were considered statistically significant. RESULTS Microscopic examination and immunostaining of iNOS expression OVA inhalation challenge caused a significant eosinophil cell infiltration into the airways compared with saline inhalation challenged mice (Fig. 1). Tracheal eosinophil cell counts in saline- and OVA-challenged animals were 0.42±0.12/mm and 14.25±1.76/mm (P<0.0001), respectively (Fig. 2). Pretreatment with 1400W significantly inhibited the eosinophil accumulation into the airways after OVA challenge (P<0.05) (Fig. 2). iNOS expression was scanty within the tissues from saline challenged mice (Fig. 3A). In contrast, obvious iNOS expression was observed in the epithelial cells (Fig. 3B) and in infiltrated inflammatory cells (Fig. 3C) within the submucosa in OVA-challenged mice. iNOS Western blotting Basal iNOS protein levels in the airways were not significantly different between control (non-sensitized) and sensitized animals (data not shown). In sensitized animals, 24 h after OVA challenge, the iNOS protein level significantly increased compared with saline challenged mice (P<0.05) (Fig. 4). Airway hyperresponsiveness OVA inhalation challenge caused significant airway hyperresponsiveness to MCh compared with salinechallenged mice (Fig. 5). Pretreatment with 1400W had no effect on baseline airway caliber assessed by Pao. However, this compound completely inhibited the airway hyperresponsiveness to MCh in OVAchallenged mice (Fig. 5). DISCUSSION In this study, we demonstrated the presence of airway hyperresponsiveness and eosinophil infiltration 24 h
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Fig. 1 Microscopic examination of airway tissues at 24 h after saline- (A) and OVA- (B) challenge in OVA-sensitized mice. Eosinophil infiltration was shown in tracheal submucosal tissues in OVA-challenged mice but not in saline-challenged mice. Each section was stained with Hansel’s stain. Scale bars are 50 m.
after allergic reaction in allergic mice. At that time, iNOS overexpression was also observed. An iNOS specific inhibitor, 1400W, completely reduced the airway hyperresponsiveness and the lesser extent eosinophil infiltration, indicating that iNOS-derived NO may work as an inducer of allergic airway hyperresponsiveness and inflammation. 1400W is a newly developed selective iNOS inhibitor. All previously described selective inhibitors of iNOS such as isothioureas,17 aminoguanidine,18 cyclic amidines,19 and N-iminoethyl--lysine20 are at best 30-fold more potent against iNOS than eNOS. 1400W was more than 5000- and 200-fold more potent against purified human iNOS than eNOS and nNOS, respectively.11 In this study, we administered 1400W continuously using an osmotic pump at 10 mg/kg per
h, a dose that has been reported to be adequate to inhibit the effect of iNOS selectively and without nonspecific toxicity, such as loss of body weight and reduction of bone marrow formation in mice.13 The mechanisms of the exaggerated iNOS induction in allergic reaction are thought to involve inflammatory cytokines such as TNF-, IL-1, and IFN-,2 which are increased during allergic reaction.21 In rat, it has been reported that allergen inhalation in sensitized animals caused an enhancement of the iNOS expression in both messenger RNA and protein in the late asthmatic response.22 In the present study, iNOS immunoreactivity was observed both in airway epithelial cells and infiltrated inflammatory cells, which is compatible with previous studies.4,5,22,23 In the present study, infiltrated cells into the airways after
Eosinophils in submucosa per mm
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Fig. 2 Effect of 1400W (10 mg/kg per h) on eosinophil infiltration into the airways. For quantification, eosinophils were counted within the submucosal area all around the trachea and their numbers were divided by the basement membrane’s length. Each value indicates mean±SEM of 7–9 animals.
OVA-inhalation were mostly eosinophils. Therefore, the most plausible cell type which expresses iNOS is eosinophils,24 although other cells such as macrophages25 and neutrophils26 have been reported to express iNOS. In the present study, pretreatment with the iNOS selective inhibitor 1400W completely abolished the OVA-induced airway hyperresponsiveness, suggesting that iNOS-derived NO acts as an inducer of the airway hyperresponsiveness in the allergic reaction. There are some possible explanations for the airway hyperresponsiveness via iNOS-derived NO. First, an increased production of NO may cause airway vascular dilation and plasma exudation, resulting in airway wall edema which causes airway hyperresponsiveness.27,28 Actually, in the present study, airway wall thickening was observed after allergen inhalation challenge. We have recently reported that the non-selective NOS inhibitor -NAME reduces the plasma exudation in allergen-challenged guinea-pig airway during the late phase, which supports this hypothesis.29 Second, in the present study iNOS-derived NO suppression by 1400W reduced airway eosinophil infiltration as well as airway hyperresponsiveness. Because it has been reported that there is a relationship between airway eosinophilia and airway hyperresponsiveness,30 the effect of 1400W on the airway responsiveness may be due to the inhibition of eosinophil recruitment. In the present study, iNOS inhibition also caused a significant reduction in airway eosinophilia, indicating that iNOS-derived NO causes eosinophil infiltratin into the airways after the allergic response.31 NO may promote the chemotaxis of eosinophils, because the non-selective NOS inhibitor -NAME inhibits the eosinophil migration induced by several chemotactic
agents, such as platelet-activating factor (PAF), Nformyl-methionyl-leucyl-phenylalanine (fMLP) and leukotriene B4 in vitro and in vivo.32,33 Further, in allergic airway inflammation, NO may facilitate eosinophil infiltration into the airway via vasodilation and microvascular hyperpermeability.29 In contrast to our present study, Feder et al reported that the iNOS selective inhibitor -N6-(1-iminoethyl) lysine (-NIL) failed to inhibit allergen-induced eosinophil accumulation into the airways assessed by bronchoalveolar lavage.10 This discrepancy may be due to the degree of iNOS expression. Actually, we observed a significant increase in iNOS protein in the airway after allergen challenge. However, in the study by Feder et al, the iNOS immunoblot was not changed by allergen exposure. Compared to the complete inhibition on the airway hyperresponsiveness, the effect of 1400W on the airway eosinophilia was small. This fact may suggest that other factors besides iNOSderived NO may be involved in the airway eosinophil accumulation after allergic reaction. Recently, two experiments have been reported about the role of endogenous NO in allergic airway inflammation using iNOS deficient mice.34,35 These experiments showed that the degree of allergen-induced airway hyperresponsiveness was not different between iNOS deficient mice and wild mice, which conflicts with our present results. Concerning the allergeninduced eosinophil infiltration into airway, one study showed eosinophil accumulation in iNOS deficient mice decreased compared to wild mice,34 which is compatible with our results, although the other study did not.35 The reason for the discrepancy between the results for iNOS deficient mice and those of our present study is not clear. iNOS deficiency may cause some other enzyme induction or suppression which affects the allergic inflammation, but at the present
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Fig. 4 (A) Immunoblot analysis of the expression of iNOS in airway tissue isolated from sensitized mice. Positive control: sample of mouse macrophage lysates stimulated with interferon- (INF-) and lipopolysaccharide (LPS); OVA: 24 h after OVA challenge; saline: 24 h post saline challenge. (B) Quantification of the intensity of the bands by densitometry. Lanes are the same as in (A). Each value indicates mean±SEM of 6–9 animals.
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Pao (cmH2O)
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MCh (µg/ml) Fig. 5 Effect of 1400W on airway hyperresponsiveness after OVA challenge in sensitized mice. Airway responsiveness was assessed by means of airway opening pressure (Pao) measurement after intravenous MCh administration. ∗P<0.01 compared with the saline pretreated – saline challenged mice. †P<0.05 and ‡P<0.0001 compared with the 1400W pretreated – OVA challenged mice. Each value indicates mean±SEM of 7–9 animals. OVA=ovalbumin. Saline pretreated–saline challenged (Φ), saline pretreated–OVA challenged (∆), 1400W pretreated–OVA challenged (Ε).
time there is no evidence concerning this possibility. In summary, we have shown that allergen inhalation causes airway iNOS overexpression as well as airway hyperresponsiveness and eosinophil infiltration in an
allergic mice model. Pretreatment with a highly iNOS selective inhibitor, 1400W, completely inhibited the airway hyperresponsiveness and to a lesser extent eosinophil accumulation. These data indicate that
Fig. 3 Immunohistochemical localization of iNOS in airway tissues of OVA-sensitized mice. iNOS expression was scanty within the tissues from saline-challenged mice (A). In contrast, iNOS expression was shown in the epithelial cells (B) and some infiltrated inflammatory cells (arrow heads) (C) within the tissues in OVA-challenged mice. Scale bars are 50 m.
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iNOS-derived NO may work in allergic airway hyperresponsiveness and inflammation. Selectivee inhibitors for iNOS may be useful for future asthma therapy.
17.
18.
ACKNOWLEDGEMENT The writers thank Mr Brent Bell for reading the manuscript.
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REFERENCES 1. Moncada S, Palmer R M, Higgs E A. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991; 43: 109–142. 2. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J 1992; 6: 3051–3064. 3. Schmidt H H, Walter U. NO at work. Cell 1994; 78: 919–925. 4. Hamid Q, Springall D R, Riveros-Moreno V, Chanez P, Howarth P, Redington A, Bousquet J, Godard P, Holgate S, Polak J M. Induction of nitric oxide synthase in asthma. Lancet 1993; 342: 1510–1513. 5. Saleh D, Ernst P, Lim S, Barnes P J, Giaid A. Increased formation of the potent oxidant peroxynitrite in the airways of asthmatic patients is associated with induction of nitric oxide synthase: effect of inhaled glucocorticoid. FASEB J 1998; 12: 929–937. 6. Kharitonov S A, Yates D, Robbins R A, Logan-Sinclair R, Shinebourne E A, Barnes P J. Increased nitric oxide in exhaled air of asthmatic patients. Lancet 1994; 343: 133–135. 7. Jatakanon A, Lim S, Kharitonov S A, Chung C F, Barnes P J. Correlation between exhaled nitric oxide, sputum eosinophils, and methacholine responsiveness in patients with mild asthma. Thorax 1998; 53: 91–95. 8. Yates D H, Kharitonov S A, Thomas P S, Barnes P J. Endogenous nitric oxide is decreased in asthmatic patients by an inhibitor of inducible nitric oxide synthase. Am J Respir Crit Care Med 1996; 154: 247–250. 9. Schuiling M, Meurs H, Zuidhof A B, Venema N, Zaagsma J. Duel action of iNOS-derived nitric oxide in allergen-induced airway hyperreactivity in conscious, unrestrained guinea pigs. Am J Respir Crit Care Med 1998; 158: 1442–1449. 10. Feder L S, Stelts D, Chapman R W, Manfra D, Crawley Y, Jones H, Minnicozzi M, Fernandez X, Pater T, Egan R W, Kreutner W, Kung T T. Role of nitric oxide on eosinophilic lung inflammation in allergic mice. Am J Respir Cell Mol 1997; 17: 436–442. 11. Garvey E P, Oplinger J A, Furfine E S, Kiff R J, Laszlo F, Whittle B J, Knowles R G. 1400W is a slow, tight binding, and highly selective inhibitor of inducible nitric-oxide synthase in vitro and in vivo. J Biol Chem 1997; 272: 4959–4963. 12. Ohkawara Y, Lei X F, Stampfli M R, Marshall J S, Xing Z, Jordana M. Cytokine and eosinophil responses in the lung, peripheral blood, and bone marrow compartments in a murine model of allergen-induced airways inflammation. Am J Respir Cell Mol Biol 1997; 16: 510–520. 13. Thomsen L L, Scott J M, Topley P, Knowles R G, Keerie A J, Frend A J. Selective inhibition of inducible nitric oxide synthase inhibits tumor growth in vivo: studies with 1400W, a novel inhibitor. Cancer Res 1997; 57: 3300–3304. 14. Hansel F K. The cytology of the secretions in allergy. In: Hansel F K, ed. Clinical Allergy. St Louis: The C. V. Mosby Company, 1953; 409–419. 15. Lowry H, Rosebrough N J, Farr A L, Randall R J. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 265–275. 16. Martin T R, Gerard N P, Galli S J, Drazen J M. Pulmonary
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
responses to bronchoconstrictor agonists in the mouse. J Appl Physiol 1988; 64: 2318–2323. Nakane M, Klinghofer V, Kuk J E, Donnelly J L, Budzik G P, Pollock J S, Basha F, Carter G W. Novel potent and selective inhibitors of inducible nitric oxide synthase. Mol Pharmacol 1995; 47: 831–834. Hasan K, Heesen B J, Corbett J A, McDaniel M L, Chang K, Allison W, Williamson J R, Tilton R G. Inhibition of nitric oxide formation by guanidines. Eur J Pharmacol 1993; 249: 101–106. Moore W M, Webber R K, Fok K F, Jerome G M, Connor J R, Manning P T, Wyatt P S, Misko T P, Tjoeng F S, Currie M G. 2-Iminopiperidine and other 2-iminoazaheterocycles as potent inhibitors of human nitric oxide synthase isoforms. J Med Chem 1996; 39: 669–672. Connor J R, Manning P T, Settle S L, Moore W M, Jerome G M, Webber R K, Tjoeng F S, Currie M G. Suppression of adjuvant-induced arthritis by selective inhibition of inducible nitric oxide synthase. Eur J Pharmacol 1995; 273: 15–24. Lim K G, Weller P F. Lymphokines. In: Barnes P J, Grunstein M M, Leff A R, Woolcock A J, eds. Asthma. Vol 1. Philadelphia: Lippincott-Raven Publishers, 1997; 663–672. Renzi P M, Sebastiao N, al Assaad A S, Giaid A, Hamid Q. Inducible nitric oxide synthase mRNA and immunoreactivity in the lungs of rats eight hours after antigen challenge. Am J Respir Cell Mol Biol 1997; 17: 36–40. Liu S F, Haddad E B, Adcock I, Salmon M, Koto H, Gilbey T, Barnes P J, Chung K F. Inducible nitric oxide synthase after sensitization and allergen challenge of Brown Norway rat lung. Br J Pharmacol 1997; 121: 1241–1246. del Pozo V, de Arruda-Chaves E, de Andres B, Cardaba B, Lopez-Farre A, Gallardo S, Cortegano I, Vidarte L, Jurado A, Sastre J, Palomino P, Lahoz C. Eosinophils transcribe and translate messenger RNA for inducible nitric oxide synthase. J Immunol 1997; 158: 859–864. Xie Q W, Cho H J, Calaycay J, Mumford R A, Swiderek K M, Lee T D, Ding A, Troso T, Nathan C. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science 1992; 256: 225–228. Yui Y, Hattori R, Kosuga K, Eizawa H, Hiki K, Ohkawa S, Ohnishi K, Terao S, Kawai C. Calmodulin-independent nitric oxide synthase from rat polymorphonuclear neutrophils. J Biol Chem 1991; 266: 3369–3371. Hogg J C, Pare P D, Moreno R. The effect of submucosal edema on airways resistance. Am Rev Respir Dis 1987; 135: S54–S56. Kimura K, Inoue H, Ichinose M, Miura M, Katsumata U, Takahashi T, Takishima T. Bradykinin causes airway hyperresponsiveness and enhances maximal airway narrowing. Role of microvascular leakage and airway edema. Am Rev Respir Dis 1992; 146: 1301–1305. Sugiura H, Ichinose M, Oyake T, Mashito Y, Ohuchi Y, Endoh N, Kiura M, Yamagata S, Koarai A, Akaike T, Maeda H, Shirato K. Role of peroxynitrite in airway microvascular hyperpermeability during late allergic phase in guinea pigs. Am J Respir Crit Care Med 1999; 160: 663–671. Foster P S, Hogan S P, Ramsay A J, Matthaei K I, Young I G. Interleukin 5 deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model. J Exp Med 1996; 183: 195–201. Ferreira H H, Bevilacqua E, Gagioti S M, De Luca I M, Zanardo R C, Teixeira C E, Sannomiya M, Antunes P, De Nucci G. Nitric oxide modulates eosinophil infiltration in antigen-induced airway inflammation in rats. Eur J Pharmacol 1998; 358: 253–259. Ferreira H H, Medeiros M V, Lima C S, Flores C A, Sannomiya P, Autunes E, De Nucci G. Inhibition of eosinophil chemotaxis by chronic blockade of nitric oxide biosynthesis. Eur J Pharmacol 1996; 310: 201–207. Zanardo RC, Costa E, Ferreira HH, Antunes E, Martins AR, Murad F, De Nucci G. Pharmacological and immunohistochemical evidence for a functional nitric oxide synthase system in rat peritoneal eosinophils. Proc Natl Acad Sci 1997; 94: 14111–14114.
iNOS inhibitor and allergic responses 34. Xiong Y, Karupiah G, Hogan S P, Foster P S, Ramsay A J. Inhibition of allergic airway inflammation in mice lacking nitric oxide synthase 2. J Immunol 1999; 162: 445–452. 35. De Sanctis GT, MacLean J A, Hamada K, Mehta S, Scott J A, Jiao A, Yandava C N, Kobzik L, Wolyniec W W, Fabian A J, Venugopal C S, Grasemann H, Huang P L, Drazen J M. Contribution of nitric oxide synthases 1, 2, and
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3 to airway hyperresponsiveness and inflammation in a murine model of asthma. J Exp Med 1999; 189: 1621–1630. Date received: 19 May 2000. Date revised: 21 August 2000. Date accepted: 1 September 2000. Published electronically: 6 October 2000.
PULMONARY PHARMACOLOGY & THERAPEUTICS
Allergic Airway Hyperresponsiveness and Eosinophil Infiltration is Reduced by a Selective iNOS Inhibitor, 1400W, in Mice Akira Koarai, Masakazu Ichinose∗, Hisatoshi Sugiura, Shunsuke Yamagata, Toshio Hattori, Kunio Shirato First Department of Internal Medicine, Tohoku University School of Medicine, Sendai, Japan
SUMMARY: Nitric oxide (NO) hyperproduction has been reported in asthmatic airways and may contribute to airway inflammatory responses. The purpose of this study was to examine the role of NO via inducible NO synthase (iNOS) in allergic airway inflammation using a selective iNOS inhibitor, N-[3-(aminomethyl)benzyl] acetamidine (1400W), in ovalbumin (OVA)-sensitized Balb/c mice. Sensitized animals were challenged with aerosolized 0.5% OVA for 1 h on two occasions 4 h apart. 1400W or the vehicle was administered by osmotic mini-pump from 2 h before to 24 h after OVA challenge. Twenty-four hours after OVA challenge, the vehicletreated mice showed a significant airway hyperresponsiveness to intravenous methacholine (P<0.05) as well as an influx of eosinophils into the airways (P<0.05). iNOS immunoreactivity was obvious in the epithelial and, to a lesser extent, the infiltrated inflammatory cells. iNOS protein in the airway assessed by Western blotting also increased. Pretreatment with 1400W almost completely abolished the OVA-induced airway hyperresponsiveness and to a lesser extent eosinophil accumulation into the airways. These results suggest that NO synthesized by iNOS may participate in airway hyperresponsiveness and eosinophil infiltration into the airways after allergic reaction. 2000 Academic Press KEY WORDS: Asthma, Airway resposiveness, Nitric oxide, iNOS protein, iNOS immunoreactivity, Eosinophil infiltration.
reported that the exhaled NO levels are increased in bronchial asthma patients compared with those of healthy subjects,6 and that the increase of exhaled NO is correlated with eosinophilic inflammation,7 which is a prominent feature of bronchial asthma. Therefore, although NO produced by iNOS appears to be involved in the inflammatory process of asthma, the precise role of NO in asthmatic airway inflammation has not yet been fully elucidated. Until now, the effect of iNOS-derived NO has been examined using selective iNOS inhibitors such as aminoguanidine8,9 and -N6-(1-iminoethyl) lysine (NIL).10 However, the selectivity of aminoguanidine and -NIL for iNOS is not very high. Therefore, some results obtained using these compounds may be misleading. Recently, a new, highly selective inhibitor for iNOS, N-[3-(aminomethyl)benzyl] acetamidine (1400W), has been developed by Garvey et al.11 Its inhibitory potency to iNOS/eNOS is at least 1000, indicating the selectivity for iNOS by 1400W is quite high.11 In the present study, using 1400W compound, we examined the role of iNOS-derived NO in the
INTRODUCTION Nitric oxide (NO) is a gas molecule synthesized by nitric oxide synthase (NOS) that has many physiological functions.1–3 Nitric Oxide Synthase has at least three isoforms which are composed from two types of constitutive NOS (nNOS) and an inducible NOS (iNOS). cNOS consists of neural NOS (nNOS) and endothelial NOS (eNOS). cNOS-derived NO may be important for physiological regulations such as vasodilation, neurotransmission and inhibition of platelet aggregation.3 In contrast, iNOS, presumably induced by inflammatory cytokines such as tumor necrosis factor (TNF)-, interleukin (IL)-1, and interferon (IFN)-,2 produces much larger amounts of NO than cNOS. iNOS has been reported to be expressed on airway epithelial cells and some inflammatory cells in asthma patients.4,5 It has also been ∗ Author for correspondence: First Department of Internal Medicine, Tohoku University School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan. Fax: 81-22-717-7156. E-mail: [email protected] 1094-5539/00/060267+09 $35.00
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airway hyperresponsiveness as well as eosinophil accumulation into the airways in allergic mice model. We found that 1400W significantly inhibited both responses.
around the trachea and these numbers were corrected by dividing by the basement membrane’s length. Eosinophils were counted on each of five continuous sections and these numbers were averaged. Western blot analysis of iNOS
MATERIALS AND METHODS Mice Specific pathogen-free male Balb/c mice between 5 and 7 weeks of age purchased from the Institute for Experimental Animals (Tohoku University School of Medicine, Sendai, Japan) were used. Mice were maintained in conventional animal housing at a constant temperature and humidity with regular 12-h cycles of light and darkness. All of the experiments performed in this study were conducted with the consent of the Ethics Committee for the Use of Experimental Animals of the Tohoku University School of Medicine. Sensitization and allergen challenge Mice were sensitized and challenged as previously described.12 Briefly, mice were sensitized by an ip injection of 0.5 ml solution containing 8 g of ovalbumin (OVA) and 4 mg of aluminum hydroxide (alum) in saline on days 0 and 5. Twelve days later, mice were placed in a plexiglas chamber (10 cm×15 cm×25 cm) and challenged with aerosolized saline or 0.5% OVA in saline for 1 h on two occasions 4 h apart. The aerosolized OVA was produced by an ultrasonic nebulizer (NE-12, Omuron, Tokyo, Japan; output 0.8 ml/min). On day 17 at 2 h pre-challenge, mice were anesthetized with diethyl ether and implanted SC with osmotic mini-pumps (Alzet; ALZA Scientific Products, Palo Alto, California, USA), which provided a continuous infusion (1.0 l/h) of 1400W (10 mg/kg per h) dissolved in saline or saline alone until 24 h post-challenge.13 At 24 h post-challenge, mice were anesthetized by ip injection of pentobarbital (70 mg/kg body weight) and used for the following experiments. Quantification of eosinophil accumulation into the airways The trachea was perfused with 1% paraformaldehyde (PFA) in 50 mM phosphate buffered saline (PBS), immersed in 4% PFA fixative solution for 12 h at 4°C, and further immersed for 24 h at 4°C in 0.1 M PBS containing 15% sucrose. The tissues were then sectioned at a thickness of 6 m with a cryostat. The sections were stained using Hansel’s stain.14 Eosinophils were counted within the submucosal area all
The thorax was opened and the systemic circulation was perfused with ice-cold 50 mM PBS containing 1 mM ethylenediaminetetraacetic acid (EDTA) for 5 min at 100 mmHg using a 18 G-needle passed through a left ventriculoctomy into the ascending aorta. The pulmonary circulation was also perfused with 5 ml of the same solutions from the right ventricle. After that, lung tissue was isolated and its connective tissues, vasculatures and parenchyma were gently scraped off. The airways were homogenized in 0.8 ml of 50 mM Tris-HCl buffer (pH 7.8) containing 150 mM NaCl, 1 ml EDTA, 1 mM sodium vanadate, 1% NP-40, 1 mM para-amidino-phenylmethylsulfonyl fluoride (para-PMSF).10 The samples were centrifuged at 10 000 g for 15 min at 4°C and the supernatants were concentrated by a microcon 10 (Amicon, Inc., Beverly, USA) at 14 000 g for 50 min at 4°C. The protein content was determined using the method of Lowry15 with bovine serum albumin as the standard. Sample protein was added at one fourth the volume of a 250 mM Tris buffer containing 20% -mercaptoethanol, 0.01% bromophenol blue, 8% sodium dodecyl sulfate (SDS) and 40% glycerol and boiled for 5 min. Protein (90 g/lane) was separated on a 7.5% SDS polyacrylamide gel, transferred to a hydrophobic polyvinylidine difluoride membrane and probed with an anti-mouse iNOS rabbit antisera (Wako Pure Chemical Industries, Osaka, Japan). The secondary antibody used was anti-rabbit IgG from goat conjugated horseradished peroxidase (DAKO Japan Ltd., Kyoto, Japan). Antibody binding was detected using ECL-plus (Amersham Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer’s recommendation. Samples of mouse macrophage lysates stimulated with interferon- (INF-) and lipopolysaccharide (LPS) were used as positive controls (Transduction Laboratories, Lexington, Kentucky, USA). The membrane was photographed by a midnight sun camera (C-1; Fuji Photo Film Co., Tokyo, Japan) and the intensity of the bands was quantified by densitometry (MCID image analyser; Imaging Inc. Research, St. Catherines, Ontario, Canada). iNOS immunostaining The trachea was perfused with 1% PFA, immersed in 4% PFA fixative solution for 12 h at 4°C, and further immersed for 24 h at 4°C in 0.1 M phosphate buffer containing 15% sucrose. The tissues were then sectioned at a thickness of 6 m with a cryostat. All
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sections were mounted on chrom-alum gelatin-coated glass slides. Endogenous peroxidase activity was reduced by incubation in 3% hydrogen peroxide in 100% methanol for 5 min at room temperature. After washing in PBS, sections were incubated with primary antibody [polyclonal iNOS rabbit anti sera, 1:250 dilution (Wako Pure Chemical Industries). In order to reduce non-specific binding of the antibody, tissues were preincubated with 4% skim milk in PBS containing 0.3% Triton-X for 30 min and then incubated with 10% inactivated normal goat serum for 30 min at room temperature. The immunoreactions were visualized by the indirect immunoperoxidase method using ENVISION polymer reagent, which is anti-rabbit IgG from goat conjugated with peroxidase labeled dextran (DAKO Japan Ltd.), for 1 h at room temperature. The diaminobenzidine reaction was performed, followed by counterstaining with hematoxylin. Stained sections were viewed on a microscope (BX-40; Olympus, Tokyo, Japan) and captured with a 3-CCD color video camera (KY-F55MD; Olympus) using image analysis software (Mac SCOPE; Mitani Co., Fukui, Japan).
Measurement of airway responsiveness Twenty-four hours after OVA challenge, mice were anesthetized with pentobarbital sodium (70 mg/kg) and pretreated with propranolol (4 mg/kg) ip 15 min before bronchoconstrictor challenges to avoid -adrenergic neural modifications. The trachea was canulated with a tracheal tube (0.6 mm outer diameter) and connected via two ports of a four-way connector to a rodent ventilator (model 683, Harvard Apparatus, South Natick, Massachusetts, USA). Airway opening pressure (Pao) was detected by a pressure transducer (model MP45-1, Validyne, Northridge, California, USA) with one side connected to the fourth port of a four-way connector. Mice were ventilated at 150 strokes/min with tidal volumes of 6 ml/kg. After establishing a stable baseline of Pao, acetyl--methylcholine chloride (methacholine, MCh) was cumulatively administered (10–1000 g/kg) intrajugularly with a 30G microsyringe and the changes of Pao were monitored.16
Drugs Ovalbumin (class V), paraformaldehyde, EDTA, sodium vanadate, Triton-X, and diaminobenzidine were obtained from Sigma Chemical Co. (St. Louis, Missouri, USA). -mercaptoethanol and SDS were purchased from Bio-Rad Laboratories. Pentobarbital was purchased from Tokyo Kasei Kogyo Co. (Tokyo, Japan), NP-40 from Nacalai Tesque, Inc. (Kyoto, Japan), and Hansel’s stain from Torii Pharmaceutical
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(Tokyo, Japan). Other chemicals were purchased from Wako Pure Chemical Industries (Osaka, Japan). Statistical analysis Data are expressed as mean±SEM. Multiple comparisons of mean data of iNOS Western blotting, airway hyperreactivity and eosinophil infiltrations among the groups were performed by one way analysis of variance (ANOVA) followed by Scheffe’s test as a post hoc test. Probability values <0.05 were considered statistically significant. RESULTS Microscopic examination and immunostaining of iNOS expression OVA inhalation challenge caused a significant eosinophil cell infiltration into the airways compared with saline inhalation challenged mice (Fig. 1). Tracheal eosinophil cell counts in saline- and OVA-challenged animals were 0.42±0.12/mm and 14.25±1.76/mm (P<0.0001), respectively (Fig. 2). Pretreatment with 1400W significantly inhibited the eosinophil accumulation into the airways after OVA challenge (P<0.05) (Fig. 2). iNOS expression was scanty within the tissues from saline challenged mice (Fig. 3A). In contrast, obvious iNOS expression was observed in the epithelial cells (Fig. 3B) and in infiltrated inflammatory cells (Fig. 3C) within the submucosa in OVA-challenged mice. iNOS Western blotting Basal iNOS protein levels in the airways were not significantly different between control (non-sensitized) and sensitized animals (data not shown). In sensitized animals, 24 h after OVA challenge, the iNOS protein level significantly increased compared with saline challenged mice (P<0.05) (Fig. 4). Airway hyperresponsiveness OVA inhalation challenge caused significant airway hyperresponsiveness to MCh compared with salinechallenged mice (Fig. 5). Pretreatment with 1400W had no effect on baseline airway caliber assessed by Pao. However, this compound completely inhibited the airway hyperresponsiveness to MCh in OVAchallenged mice (Fig. 5). DISCUSSION In this study, we demonstrated the presence of airway hyperresponsiveness and eosinophil infiltration 24 h
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Fig. 1 Microscopic examination of airway tissues at 24 h after saline- (A) and OVA- (B) challenge in OVA-sensitized mice. Eosinophil infiltration was shown in tracheal submucosal tissues in OVA-challenged mice but not in saline-challenged mice. Each section was stained with Hansel’s stain. Scale bars are 50 m.
after allergic reaction in allergic mice. At that time, iNOS overexpression was also observed. An iNOS specific inhibitor, 1400W, completely reduced the airway hyperresponsiveness and the lesser extent eosinophil infiltration, indicating that iNOS-derived NO may work as an inducer of allergic airway hyperresponsiveness and inflammation. 1400W is a newly developed selective iNOS inhibitor. All previously described selective inhibitors of iNOS such as isothioureas,17 aminoguanidine,18 cyclic amidines,19 and N-iminoethyl--lysine20 are at best 30-fold more potent against iNOS than eNOS. 1400W was more than 5000- and 200-fold more potent against purified human iNOS than eNOS and nNOS, respectively.11 In this study, we administered 1400W continuously using an osmotic pump at 10 mg/kg per
h, a dose that has been reported to be adequate to inhibit the effect of iNOS selectively and without nonspecific toxicity, such as loss of body weight and reduction of bone marrow formation in mice.13 The mechanisms of the exaggerated iNOS induction in allergic reaction are thought to involve inflammatory cytokines such as TNF-, IL-1, and IFN-,2 which are increased during allergic reaction.21 In rat, it has been reported that allergen inhalation in sensitized animals caused an enhancement of the iNOS expression in both messenger RNA and protein in the late asthmatic response.22 In the present study, iNOS immunoreactivity was observed both in airway epithelial cells and infiltrated inflammatory cells, which is compatible with previous studies.4,5,22,23 In the present study, infiltrated cells into the airways after
Eosinophils in submucosa per mm
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Fig. 2 Effect of 1400W (10 mg/kg per h) on eosinophil infiltration into the airways. For quantification, eosinophils were counted within the submucosal area all around the trachea and their numbers were divided by the basement membrane’s length. Each value indicates mean±SEM of 7–9 animals.
OVA-inhalation were mostly eosinophils. Therefore, the most plausible cell type which expresses iNOS is eosinophils,24 although other cells such as macrophages25 and neutrophils26 have been reported to express iNOS. In the present study, pretreatment with the iNOS selective inhibitor 1400W completely abolished the OVA-induced airway hyperresponsiveness, suggesting that iNOS-derived NO acts as an inducer of the airway hyperresponsiveness in the allergic reaction. There are some possible explanations for the airway hyperresponsiveness via iNOS-derived NO. First, an increased production of NO may cause airway vascular dilation and plasma exudation, resulting in airway wall edema which causes airway hyperresponsiveness.27,28 Actually, in the present study, airway wall thickening was observed after allergen inhalation challenge. We have recently reported that the non-selective NOS inhibitor -NAME reduces the plasma exudation in allergen-challenged guinea-pig airway during the late phase, which supports this hypothesis.29 Second, in the present study iNOS-derived NO suppression by 1400W reduced airway eosinophil infiltration as well as airway hyperresponsiveness. Because it has been reported that there is a relationship between airway eosinophilia and airway hyperresponsiveness,30 the effect of 1400W on the airway responsiveness may be due to the inhibition of eosinophil recruitment. In the present study, iNOS inhibition also caused a significant reduction in airway eosinophilia, indicating that iNOS-derived NO causes eosinophil infiltratin into the airways after the allergic response.31 NO may promote the chemotaxis of eosinophils, because the non-selective NOS inhibitor -NAME inhibits the eosinophil migration induced by several chemotactic
agents, such as platelet-activating factor (PAF), Nformyl-methionyl-leucyl-phenylalanine (fMLP) and leukotriene B4 in vitro and in vivo.32,33 Further, in allergic airway inflammation, NO may facilitate eosinophil infiltration into the airway via vasodilation and microvascular hyperpermeability.29 In contrast to our present study, Feder et al reported that the iNOS selective inhibitor -N6-(1-iminoethyl) lysine (-NIL) failed to inhibit allergen-induced eosinophil accumulation into the airways assessed by bronchoalveolar lavage.10 This discrepancy may be due to the degree of iNOS expression. Actually, we observed a significant increase in iNOS protein in the airway after allergen challenge. However, in the study by Feder et al, the iNOS immunoblot was not changed by allergen exposure. Compared to the complete inhibition on the airway hyperresponsiveness, the effect of 1400W on the airway eosinophilia was small. This fact may suggest that other factors besides iNOSderived NO may be involved in the airway eosinophil accumulation after allergic reaction. Recently, two experiments have been reported about the role of endogenous NO in allergic airway inflammation using iNOS deficient mice.34,35 These experiments showed that the degree of allergen-induced airway hyperresponsiveness was not different between iNOS deficient mice and wild mice, which conflicts with our present results. Concerning the allergeninduced eosinophil infiltration into airway, one study showed eosinophil accumulation in iNOS deficient mice decreased compared to wild mice,34 which is compatible with our results, although the other study did not.35 The reason for the discrepancy between the results for iNOS deficient mice and those of our present study is not clear. iNOS deficiency may cause some other enzyme induction or suppression which affects the allergic inflammation, but at the present
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Fig. 4 (A) Immunoblot analysis of the expression of iNOS in airway tissue isolated from sensitized mice. Positive control: sample of mouse macrophage lysates stimulated with interferon- (INF-) and lipopolysaccharide (LPS); OVA: 24 h after OVA challenge; saline: 24 h post saline challenge. (B) Quantification of the intensity of the bands by densitometry. Lanes are the same as in (A). Each value indicates mean±SEM of 6–9 animals.
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MCh (µg/ml) Fig. 5 Effect of 1400W on airway hyperresponsiveness after OVA challenge in sensitized mice. Airway responsiveness was assessed by means of airway opening pressure (Pao) measurement after intravenous MCh administration. ∗P<0.01 compared with the saline pretreated – saline challenged mice. †P<0.05 and ‡P<0.0001 compared with the 1400W pretreated – OVA challenged mice. Each value indicates mean±SEM of 7–9 animals. OVA=ovalbumin. Saline pretreated–saline challenged (Φ), saline pretreated–OVA challenged (∆), 1400W pretreated–OVA challenged (Ε).
time there is no evidence concerning this possibility. In summary, we have shown that allergen inhalation causes airway iNOS overexpression as well as airway hyperresponsiveness and eosinophil infiltration in an
allergic mice model. Pretreatment with a highly iNOS selective inhibitor, 1400W, completely inhibited the airway hyperresponsiveness and to a lesser extent eosinophil accumulation. These data indicate that
Fig. 3 Immunohistochemical localization of iNOS in airway tissues of OVA-sensitized mice. iNOS expression was scanty within the tissues from saline-challenged mice (A). In contrast, iNOS expression was shown in the epithelial cells (B) and some infiltrated inflammatory cells (arrow heads) (C) within the tissues in OVA-challenged mice. Scale bars are 50 m.
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iNOS-derived NO may work in allergic airway hyperresponsiveness and inflammation. Selectivee inhibitors for iNOS may be useful for future asthma therapy.
17.
18.
ACKNOWLEDGEMENT The writers thank Mr Brent Bell for reading the manuscript.
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REFERENCES 1. Moncada S, Palmer R M, Higgs E A. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharmacol Rev 1991; 43: 109–142. 2. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J 1992; 6: 3051–3064. 3. Schmidt H H, Walter U. NO at work. Cell 1994; 78: 919–925. 4. Hamid Q, Springall D R, Riveros-Moreno V, Chanez P, Howarth P, Redington A, Bousquet J, Godard P, Holgate S, Polak J M. Induction of nitric oxide synthase in asthma. Lancet 1993; 342: 1510–1513. 5. Saleh D, Ernst P, Lim S, Barnes P J, Giaid A. Increased formation of the potent oxidant peroxynitrite in the airways of asthmatic patients is associated with induction of nitric oxide synthase: effect of inhaled glucocorticoid. FASEB J 1998; 12: 929–937. 6. Kharitonov S A, Yates D, Robbins R A, Logan-Sinclair R, Shinebourne E A, Barnes P J. Increased nitric oxide in exhaled air of asthmatic patients. Lancet 1994; 343: 133–135. 7. Jatakanon A, Lim S, Kharitonov S A, Chung C F, Barnes P J. Correlation between exhaled nitric oxide, sputum eosinophils, and methacholine responsiveness in patients with mild asthma. Thorax 1998; 53: 91–95. 8. Yates D H, Kharitonov S A, Thomas P S, Barnes P J. Endogenous nitric oxide is decreased in asthmatic patients by an inhibitor of inducible nitric oxide synthase. Am J Respir Crit Care Med 1996; 154: 247–250. 9. Schuiling M, Meurs H, Zuidhof A B, Venema N, Zaagsma J. Duel action of iNOS-derived nitric oxide in allergen-induced airway hyperreactivity in conscious, unrestrained guinea pigs. Am J Respir Crit Care Med 1998; 158: 1442–1449. 10. Feder L S, Stelts D, Chapman R W, Manfra D, Crawley Y, Jones H, Minnicozzi M, Fernandez X, Pater T, Egan R W, Kreutner W, Kung T T. Role of nitric oxide on eosinophilic lung inflammation in allergic mice. Am J Respir Cell Mol 1997; 17: 436–442. 11. Garvey E P, Oplinger J A, Furfine E S, Kiff R J, Laszlo F, Whittle B J, Knowles R G. 1400W is a slow, tight binding, and highly selective inhibitor of inducible nitric-oxide synthase in vitro and in vivo. J Biol Chem 1997; 272: 4959–4963. 12. Ohkawara Y, Lei X F, Stampfli M R, Marshall J S, Xing Z, Jordana M. Cytokine and eosinophil responses in the lung, peripheral blood, and bone marrow compartments in a murine model of allergen-induced airways inflammation. Am J Respir Cell Mol Biol 1997; 16: 510–520. 13. Thomsen L L, Scott J M, Topley P, Knowles R G, Keerie A J, Frend A J. Selective inhibition of inducible nitric oxide synthase inhibits tumor growth in vivo: studies with 1400W, a novel inhibitor. Cancer Res 1997; 57: 3300–3304. 14. Hansel F K. The cytology of the secretions in allergy. In: Hansel F K, ed. Clinical Allergy. St Louis: The C. V. Mosby Company, 1953; 409–419. 15. Lowry H, Rosebrough N J, Farr A L, Randall R J. Protein measurement with the folin phenol reagent. J Biol Chem 1951; 193: 265–275. 16. Martin T R, Gerard N P, Galli S J, Drazen J M. Pulmonary
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
responses to bronchoconstrictor agonists in the mouse. J Appl Physiol 1988; 64: 2318–2323. Nakane M, Klinghofer V, Kuk J E, Donnelly J L, Budzik G P, Pollock J S, Basha F, Carter G W. Novel potent and selective inhibitors of inducible nitric oxide synthase. Mol Pharmacol 1995; 47: 831–834. Hasan K, Heesen B J, Corbett J A, McDaniel M L, Chang K, Allison W, Williamson J R, Tilton R G. Inhibition of nitric oxide formation by guanidines. Eur J Pharmacol 1993; 249: 101–106. Moore W M, Webber R K, Fok K F, Jerome G M, Connor J R, Manning P T, Wyatt P S, Misko T P, Tjoeng F S, Currie M G. 2-Iminopiperidine and other 2-iminoazaheterocycles as potent inhibitors of human nitric oxide synthase isoforms. J Med Chem 1996; 39: 669–672. Connor J R, Manning P T, Settle S L, Moore W M, Jerome G M, Webber R K, Tjoeng F S, Currie M G. Suppression of adjuvant-induced arthritis by selective inhibition of inducible nitric oxide synthase. Eur J Pharmacol 1995; 273: 15–24. Lim K G, Weller P F. Lymphokines. In: Barnes P J, Grunstein M M, Leff A R, Woolcock A J, eds. Asthma. Vol 1. Philadelphia: Lippincott-Raven Publishers, 1997; 663–672. Renzi P M, Sebastiao N, al Assaad A S, Giaid A, Hamid Q. Inducible nitric oxide synthase mRNA and immunoreactivity in the lungs of rats eight hours after antigen challenge. Am J Respir Cell Mol Biol 1997; 17: 36–40. Liu S F, Haddad E B, Adcock I, Salmon M, Koto H, Gilbey T, Barnes P J, Chung K F. Inducible nitric oxide synthase after sensitization and allergen challenge of Brown Norway rat lung. Br J Pharmacol 1997; 121: 1241–1246. del Pozo V, de Arruda-Chaves E, de Andres B, Cardaba B, Lopez-Farre A, Gallardo S, Cortegano I, Vidarte L, Jurado A, Sastre J, Palomino P, Lahoz C. Eosinophils transcribe and translate messenger RNA for inducible nitric oxide synthase. J Immunol 1997; 158: 859–864. Xie Q W, Cho H J, Calaycay J, Mumford R A, Swiderek K M, Lee T D, Ding A, Troso T, Nathan C. Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. Science 1992; 256: 225–228. Yui Y, Hattori R, Kosuga K, Eizawa H, Hiki K, Ohkawa S, Ohnishi K, Terao S, Kawai C. Calmodulin-independent nitric oxide synthase from rat polymorphonuclear neutrophils. J Biol Chem 1991; 266: 3369–3371. Hogg J C, Pare P D, Moreno R. The effect of submucosal edema on airways resistance. Am Rev Respir Dis 1987; 135: S54–S56. Kimura K, Inoue H, Ichinose M, Miura M, Katsumata U, Takahashi T, Takishima T. Bradykinin causes airway hyperresponsiveness and enhances maximal airway narrowing. Role of microvascular leakage and airway edema. Am Rev Respir Dis 1992; 146: 1301–1305. Sugiura H, Ichinose M, Oyake T, Mashito Y, Ohuchi Y, Endoh N, Kiura M, Yamagata S, Koarai A, Akaike T, Maeda H, Shirato K. Role of peroxynitrite in airway microvascular hyperpermeability during late allergic phase in guinea pigs. Am J Respir Crit Care Med 1999; 160: 663–671. Foster P S, Hogan S P, Ramsay A J, Matthaei K I, Young I G. Interleukin 5 deficiency abolishes eosinophilia, airways hyperreactivity, and lung damage in a mouse asthma model. J Exp Med 1996; 183: 195–201. Ferreira H H, Bevilacqua E, Gagioti S M, De Luca I M, Zanardo R C, Teixeira C E, Sannomiya M, Antunes P, De Nucci G. Nitric oxide modulates eosinophil infiltration in antigen-induced airway inflammation in rats. Eur J Pharmacol 1998; 358: 253–259. Ferreira H H, Medeiros M V, Lima C S, Flores C A, Sannomiya P, Autunes E, De Nucci G. Inhibition of eosinophil chemotaxis by chronic blockade of nitric oxide biosynthesis. Eur J Pharmacol 1996; 310: 201–207. Zanardo RC, Costa E, Ferreira HH, Antunes E, Martins AR, Murad F, De Nucci G. Pharmacological and immunohistochemical evidence for a functional nitric oxide synthase system in rat peritoneal eosinophils. Proc Natl Acad Sci 1997; 94: 14111–14114.
iNOS inhibitor and allergic responses 34. Xiong Y, Karupiah G, Hogan S P, Foster P S, Ramsay A J. Inhibition of allergic airway inflammation in mice lacking nitric oxide synthase 2. J Immunol 1999; 162: 445–452. 35. De Sanctis GT, MacLean J A, Hamada K, Mehta S, Scott J A, Jiao A, Yandava C N, Kobzik L, Wolyniec W W, Fabian A J, Venugopal C S, Grasemann H, Huang P L, Drazen J M. Contribution of nitric oxide synthases 1, 2, and
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3 to airway hyperresponsiveness and inflammation in a murine model of asthma. J Exp Med 1999; 189: 1621–1630. Date received: 19 May 2000. Date revised: 21 August 2000. Date accepted: 1 September 2000. Published electronically: 6 October 2000.