Inducible Nitric Oxide Synthase mrna and Immunoreactivity in the Lungs of Rats Eight Hours after Antigen Challenge

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1 Inducible Nitric Oxide Synthase mrna and Immunoreactivity in the Lungs of Rats Eight Hours after Antigen Challenge Paolo M. Renzi, Noami Sebastiao, Ali S. Al Assaad, Adel Giaid, and Qutayba Hamid Department of Medicine and Pathology, Meakins Christie Labs, Royal Victoria and Montreal General Hospitals, McGill University and Pulmonary Unit, Notre Dame Hospital, University of Montreal, Montreal, Quebec, Canada We have previously shown that inducible nitric oxide (ino)-synthase immunoreactivity is expressed in bronchial epithelium and increased in asthma which suggests a possible role for NO in airway hyperresponsiveness. We tested the hypothesis that exposure of a sensitized animal to antigen could account for the increased expression of ino-synthase in the airways. We examined the expression of ino-synthase mrna and immunoreactivity in the lungs of ovalbumin (OA) sensitized Brown Norway (BN) rats 8 h after antigen challenge by in situ hybridization and immunocytochemistry. Sensitized and unchallenged or bovine serum albumin (BSA) challenged rats, or unsensitized and OA challenged rats served as controls. With the use of an ino-synthase probe we found a higher expression of ino-synthase mrna in BN rat airways after antigen challenge with OA but not after antigen challenge with BSA or in other controls. Most of the expression was in the epithelium of the airways with few cells positive in the subepithelial inflammatory infiltrate or in lung lavage. Very strong ino-synthase immunoreactivity was observed in the airway epithelium of sensitized and OA challenged rats. No significant immunoreactivity was observed in the inflammatory infiltrate of the airways or in lung parenchyma. In conclusion, ino-synthase increases in the airways of sensitized rats after exposure to antigen, the major source being from airway epithelial cells. NO may have a role in the development of the late airway response and bronchial hyperresponsiveness. Renzi, P. M., N. Sebastiao, A. S. Al Assaad, A. Giaid, and Q. Hamid Inducible nitric oxide synthase mrna and immunoreactivity in the lungs of rats eight hours after antigen challenge. Am. J. Respir. Cell Mol. Biol. 17: (Received in original form July 19, 1995 and in revised form December 2, 1996) Dr. Renzi and Dr. Hamid are Research Scholars from the Fonds de Recherche en Santé du Québec. Dr. Giaid is a Scholar of the Heart and Stroke Foundation of Canada. Address correspondence to: Dr. Qutayba Hamid, Meakins Christie Laboratories, 3626 St-Urbain Street, Montreal, PQ, H2X 2P2 Canada. Abbreviations: avidin biotin complex, ABC; Brown Norway, BN; bovine serum albumin, BSA; inducible nitric oxide, ino; nitric oxide, NO; ovalbumin, OA; phosphate-buffered saline, PBS. Am. J. Respir. Cell Mol. Biol. Vol. 17, pp , 1997 Nitric oxide (NO) is produced when NO synthases catalyze the conversion of L-arginine to L-citrulline. Although first regarded as an atmospheric pollutant, NO has been shown to be involved in vascular tone (as endothelium derived relaxing factor) (1), platelet aggregation, neurotransmission, and in immune responses (2, 3). In the respiratory tract, NO is produced by autonomic neurons, fibroblasts, endothelial cells, vascular and airway smooth muscle cells, skeletal muscle cells, inflammatory cells and in airway epithelial cells (4). Nitric oxide causes vasodilatation and bronchodilatation and has been implicated in the regulation of different pulmonary functions (3 8). We have recently reported reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension (9). These results suggest that NO may also be involved in the pathophysiology of lung diseases. Nitric oxide is detectable in exhaled air (10, 11). In asthma, a disease characterized by airway obstruction and inflammation (12), NO is increased in exhaled air (13). Although NO could have beneficial effects on the airways by relaxing airway smooth muscle (7), it may also have detrimental effects by causing edema and inflammation (14, 15). We have previously performed experiments to assess the site of expression of the enzymes that produce NO in asthma. Nitric oxide synthases are classified as either Ca 2 dependent (constitutive) or Ca 2 independent (inducible) (16). We found that the inducible form of NO synthase (ino) was present and increased in the airways of asthmatics (17). Most of the expression was localized to epithelial cells and NO synthase can be induced in normal epithelial cells by incubation with different cytokines (17). We have hypothesized that exposure of sensitized airways to an antigen leads to increased expression of inosynthases and could thus explain in part the increase in NO found in the exhaled air of asthmatics. To test this hypothesis we employed the Brown Norway (BN) rat. This rat strain is a high IgE producer after sensitization (18) and de-

2 Renzi, Sebastiao, Al Assaad, et al.: NO in Rat Lungs after Antigen Challenge 37 velops early and late airway obstructive responses as well as airway hyperresponsiveness after antigen challenge similar to those described in humans (19 21). We assessed ino-synthase mrna and immunoreactivity in the lungs of ovalbumin (OA) sensitized rats 8 h after antigen challenge by in situ hybridization and immunocytochemistry. Materials and Methods Animals and Sensitization Twenty male BN rats, 7 8 wk old, were actively sensitized by a single subcutaneous injection of 1 mg of OA and 200 mg of aluminum hydroxide. Simultaneously, 1 ml of Bordetella pertussis vaccine, containing heat-killed organisms, was given intraperitoneally as an adjuvant. An additional 5 male rats were not sensitized but challenged immediately with OA. Five sensitized rats were killed 14 days later by exsanguination after general anesthesia and served as unchallenged controls. Fifteen rats were challenged 14 days after sensitization with OA, five were challenged with bovine serum albumin (BSA). Antigen Challenge and Lung Preparation Twenty rats were studied 14 days after sensitization, 5 unsensitized rats were studied immediately. The animals were anesthetized with urethane (1.1 g kg 1 intraperitoneally). Blind orotracheal intubation was performed using a 6-cm long polyethylene catheter (PE 240; Commercial Plastics, Montreal, Canada). The end of the endotracheal tube was inserted into a small Plexiglas box (volume: 265 ml) for the delivery of aerosols. An aerosol of either OA (n 15) or BSA (n 5) was given at a concentration of 5 mg/ml, for 5 min using a Hudson nebulizer (Model 1400; Hudson, Inc., Temecula, CA) with an output of 0.18 ml/ min connected to one side port of the box. Animals were placed on a heating blanket and rectal temperature was maintained constant. The unchallenged animals were killed immediately (n 5), the challenged animals were killed 8 h later (n 20) by exsanguination under general anesthesia. In 5 sensitized and OA challenged rats, lung lavage was performed by injection and immediate retrieval of 5 5 ml aliquots of normal saline. The cells were washed and immediately spun onto poly-l-lysine coated cytocentrifuge slides with a cytospin 3 (Shandon, Cheshire, UK). In all other rats, the lungs were fixed for 4 h and 30 min in freshly prepared 4% paraformaldehyde in phosphate-buffered saline (PBS), ph 7.4, the first 30 min at a pressure of 25 cm H 2 O through the trachea; transferred to 15% (wt/vol) sucrose in PBS and left overnight. with 50% formamide in 2% standard saline citrate (SSC). Nonspecific binding was blocked by the use of N-ethylmaleimide, iodocacetamide, acetic anhydride and dithiotreitol. Hybridization was performed at 42 C for 12 h. The slides were washed in decreasing concentrations of SSC (4 SSC to 0.1 SSC) prior to autoradiography. Hybridization signal was visualized under light and dark field illumination by an observer blinded to the origin of the slides. As a control, sections were treated with sense probe or hybridized with antisense probe following RNase pretreatment. Immunocytochemistry For immunocytochemistry, 4 micron sections were cut from the cryostat blocks onto poly-l-lysine slides, postfixed in 1% paraformaldehyde for 1 h, washed in PBS, and processed for the avidin biotin complex (ABC) technique as previously described (24, 25). A rabbit polyclonal antibody which was raised against ino-synthase was used. The polyclonal antibody was produced by injecting a synthetic peptide fragment of ino-synthase. The sequence of the peptide that was used as an immunogen was: IQDDPK- SHQNGSC. This antibody was affinity purified, specific for ino-synthase (Figure 1) and does not recognize constitutive NO-synthase. We determined that the optimal dilution of the antibody was 1/400 by performing titration experiments with dilutions varying between 1/100 and 1/1,000. The second and third layer in the ABC technique was repeated to enhance the reaction. Immunostaining was assessed by a blinded observer according to a score which has been previously validated (9, 25). The score was from 0 to 4 where 0, 1, 2, 3, 4 represented no staining, weak, moderate, strong and very strong staining, respectively. Data Analysis The data are expressed throughout as the mean SE. The significance of differences in the scores prior to and after OA challenge and controls was assessed by analysis of variance followed by unpaired t tests. P values 0.05 were considered significant. In Situ Hybridization Cryostat blocks were made from the hilar part of the lung (maximum of 1 cm in diameter) and 10 micron sections were cut onto poly-l-lysine coated slides. Sections were incubated at 37 C for 12 h, processed immediately or stored at 80 C until used. In situ hybridization was performed as previously described (22, 23). In brief, rat inosynthase probes (antisense and sense) were labeled with 35 S. Prior to the application of the probes, the sections were permeabilized with proteinase K and then pre-hybridized Figure 1. Characterization of the polyclonal antibody against inducible nitric oxide synthase (inos). Western blot of 20 g of protein obtained from the molecular weight marker (MW), unstimulated macrophages (C), and macrophages stimulated with LPS (5 g/ml) and interferon gamma (100 U/ml) for 24 h. Notice the band for inos in stimulated macrophages.

3 38 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL Results Expression of ino-synthase mrna in the Lungs after Antigen Challenge With in situ hybridization we demonstrated a strong signal for ino-synthase mrna in the lungs of OA challenged rats (Figure 2, top left). Messenger RNA was mostly localized to the epithelium of the airways. Occasional cells expressing mrna for ino-synthase were also demonstrated in the subepithelial inflammatory infiltrate. No significant mrna expression for ino-synthase was found in the airways of OA sensitized rats that were unchallenged, challenged with BSA, or unsensitized and challenged with OA (neither in the epithelium nor the subepithelial tissue). Sections from all the preparations did not show any signal when they were hybridized with sense ino-synthase probe nor when they were hybridized with an antisense probe following RNase pre-treatment confirming the specificity of the hybridization. Figure 2. In situ hybridization and immunocytochemistry for inducible NO synthase in the lungs of Brown Norway rats. Dark field illumination of a cryostat section of the lung of an ovalbumin challenged Brown Norway rat hybridized with 35 S labeled inducible NO synthase probe (top left). Strong hybridization (arrows) was observed mainly in bronchial epithelium. Immunocytochemistry of inducible NO synthase after ovalbumin challenge (top right) and bovine serum albumin challenge (bottom left). Note the difference in the staining. Negative control for immunostaining of rat lung after ovalbumin challenge (bottom right).

4 Renzi, Sebastiao, Al Assaad, et al.: NO in Rat Lungs after Antigen Challenge 39 Expression of ino-synthase Immunoreactivity in the Lungs after Antigen Challenge Strong ino-synthase immunoreactivity was observed in the bronchial epithelia of OA challenged rats (Figure 2, top right; Table 1). Scattered immunoreactive cells were observed in the subepithelial inflammatory infiltrate or in the lung parenchyma. Weak immunostaining was obtained in the epithelial cells of airways of OA sensitized rats unchallenged, challenged with BSA, or unsensitized rats challenged with OA. There was a significant difference between the intensity of immunostaining in the epithelium of OA challenged lungs compared with BSA challenged lungs (P 0.05; Table 1, Figure 2, bottom left). No immunoreactivity for ino-synthase was demonstrated in the subepithelial tissue of OA sensitized rats challenged with BSA. There was no immunostaining when the antibody was omitted from the immunocytochemical reaction as a control (Figure 2, bottom right). Expression of ino-synthase mrna and Immunoreactivity in Lung Lavage after Antigen Challenge Lung lavage contained 52 7% macrophages, % neutrophils, 2 2% lymphocytes and 3.5 1% eosinophils. We found very little mrna expression or immunoreactivity for ino-synthase in the cells obtained from lung lavage 8 h after OA challenge. The amount of cells staining positive was always less than 3% of cells for ino-synthase mrna and less than 5% of cells for immunoreactivity. Discussion In this study we demonstrated increased expression of mrna and immunoreactivity for ino-synthase in the lungs of BN rats 8 h after antigen challenge, the predominant site of expression being in the epithelium. Nitric oxide is produced by several cells that are present in the lungs of animals and humans (4) and has been shown to have different physiological effects. Endogenous nitrogen oxide regulates pulmonary vascular function (26), leukocyte adhesion to post-capillary venules (27) and macrophage mediated suppression of T-cell proliferation (28). Several recent findings suggest that NO may be involved in the pathophysiology of asthma, a disease characterized by airways obstruction, inflammation, and increased airway responsiveness (12). Nitric oxide is the principal neurotransmitter of the inhibitory response of the non-adrenergic, non-cholinergic TABLE 1 Inducible nitric oxide synthase immunoreactivity score of the airways after antigen challenge Treatment Mean ino Synthase Immunoreactivity Score SEM Unsensitized, challenged with OA Sensitized with OA, unchallenged Sensitized with OA, challenged with BSA Sensitized with OA, challenged with OA * OA ovalbumin; BSA bovine serum albumin. * P 0.05 versus all 3 control groups. Lungs were analyzed from 5 rats in each group. neural system and activation of this pathway will lead to bronchodilatation (29). Nitric oxide is also a bronchodilator (7, 30, 31). Although NO has a weak direct bronchodilatory effect in humans, it does modulate the response of airways to cholinergic agonists toward bronchodilatation in guinea pigs, rabbits and humans. However, NO production may have detrimental effects on the airways in asthma (3). High levels of NO produced by ino-synthase may lead to downregulation of NO synthase or actually desensitize the enzyme guanyl cyclase involved in cgmp production and lead to airway constriction. In addition, in inflamed tissue NO may be transformed into free radicals which are strong oxidants and may be involved in edema and inflammation (14, 15). We have previously reported that immunoreactivity to ino-synthase was present in bronchial biopsies of asthmatics (17). The presence of this enzyme in the airways of asthmatics suggests that NO may be involved in the pathophysiology of asthma. In this study, we questioned whether exposure to antigen may result in the expression of NO synthase in the airways of sensitized rats at the time of the late airway response. We looked specifically for expression of ino-synthase in the lungs of BN rats not only since we have previously found it to be increased in asthma but also because it is regulated by cytokines, mediators that are involved in the inflammatory changes that occur in the airways of asthmatics (12, 23). Other factors suggesting that inducible but not constitutive NO synthase is important in asthma are the fact that ino-synthase is produced in much larger amounts than constitutive NO synthase after activation of cells (3) and the fact that only ino-synthase is inhibited by corticosteroids, effective medications in the treatment of asthma (12). We chose the BN rat because this animal has several characteristics in common with atopic asthmatics. This rat strain produces high serum levels of IgE 14 to 21 days after sensitization (18) and develops early and late airway obstructive responses 4 to 8 h after antigen challenge as described in atopic asthmatics (19, 20). In addition, lymphocyte subset changes in the blood and lungs of these animals are the same as those described in humans, suggesting that the same immune response is operative in the BN rat and in human asthmatics after antigen challenge. It has been previously shown that NO increases in the exhaled air of guinea pigs after antigen challenge and rapidly returns to baseline levels (32). We studied the expression of ino-synthase in the lungs of rats 8 h after antigen challenge. We chose to measure expression of ino-synthase at 8 h since this time point is at the end of the late airway response, precedes increased airway responsiveness (21), and is also a time when cytokine expression increases in the lungs (33). We found that ino-synthase was expressed in large amounts in the airways 8 h after antigen challenge. Recently, NO has been reported to be increased in the exhaled air of asthmatics during late responses (34, 35). These results suggest that NO may be involved in increased airway responsiveness. Expression of NO synthase could have occurred in epithelial, endothelial, or inflammatory lung cells (4). Since an inflammatory response occurs in the lungs of BN rats 8 h after antigen challenge (20) and since NO synthase can

5 40 AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL be expressed in inflammatory cells (36, 37), it is surprising that we found little expression of mrna and practically no protein expression for NO synthase in inflammatory cells in the airways or in lung lavage after antigen challenge. Our results are in accordance with those that we previously reported in human asthmatics (17, 38) and confirm Nijkamp s hypothesis (39) that epithelial cells are the source of increased expression of ino synthase. In conclusion, we have shown that exposure to antigen leads to the expression of inducible NO synthase in the epithelium of the airways of sensitized rats 8 h after antigen challenge. We suggest that this response is the result of exposure of epithelial cells to cytokines. Acknowledgments: This study was supported in part by Inspiraplex, the J. T. Costello Memorial Research Fund, and the Montreal Chest Institute. 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