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Influence of cigarette smoke on the arginine pathway in asthmatic airways: Increased expression of arginase I
Influence of cigarette smoke on the arginine pathway in asthmatic airways: Increased expression of arginase I
Background: Up to 30% of asthmatic subjects are smokers, and smoking might be an important contributor to asthma pathology. Inducible nitric oxide synthase (iNOS), ornithine decarboxylase (ODC), and arginase I are involved in the arginine pathway. We have shown that arginase I and iNOS are upregulated in asthma. Smoking asthmatic subjects are reported to have low exhaled nitric oxide levels. The effect of cigarette smoking on the expression of arginase I in asthma is unknown. Objectives: The aims of this study were to investigate the expression of arginase I, ODC, and iNOS in asthmatic airways of smokers and nonsmokers and in vitro after nicotine stimulation. Methods: Endobronchial biopsies were performed on 24 steroid-naive subjects with mild asthma: 12 smokers and 12 nonsmokers. Arginase I, ODC, and iNOS levels were assessed by means of immunohistochemistry and in situ hybridization (arginase I). In vitro stimulation of airway cells with nicotine was performed, followed by real-time PCR. Results: Arginase I, ODC, and iNOS were expressed in the epithelium and smooth muscle bundles of both subgroups of asthmatic subjects. There was an increase of arginase I and ODC immunoreactivities in smoking compared with
From aMeakins-Christie Laboratories, McGill University, Montreal; bCentre de Recherche, Hoˆpital Laval, Institut Universitaire de Cardiologie et de Pneumologie; and cthe Division of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center. Supported by local funds and National Institutes of Health/National Institute of Allergy and Infectious Diseases R01 A153479. C. B. is a recipient of the Ann Woolcock fellowship. Disclosure of potential conflict of interest: L.-P. Boulet has consulting arrangements with Altana, AstraZeneca, GlaxoSmithKline, Novartis, and Merck Frost; has received grant support from 3M, Altana, Athmatx, AstraZeneca, Boehringer Ingelheim, Dynavax, Genentech, GlaxoSmithKline, IVAX, Merck Frost, Novartis, Pfizer, Roche, Schering, and Topingen; and is on the speakers’ bureau for 3M, Altana, AstraZeneca, GlaxoSmithKline, Merck Frost, and Novartis. M. Laviolette has received grant support from Merck Frost and is on the speakers’ bureau for 3M, AstraZeneca, and Glaxo Canada. N. Zimmermann has received grant support from the National Institutes of Health. M. E. Rothenberg has consulting arrangements with GlaxoSmithKline, Ception Therapeutics, Cambridge Antibody Technology, and MedaCorp; owns stock in Ception Therapeutics; has received grant support from Cambridge Antibody Technology; is on the speakers’ bureau for Merck and Tanox; and has received honorarium from GlaxoSmithKline, Ception Therapeutics, Merck, and Tanox. The rest of the authors have declared that they have no conflict of interest. Received for publication May 5, 2006; revised October 19, 2006; accepted for publication October 24, 2006. Reprint requests: Qutayba Hamid, MD, PhD, Meakins-Christie Laboratories, McGill University, 3626 St Urbain, Montreal, Quebec, Canada H2X 2P2. E-mail: [email protected]. 0091-6749/$32.00 Ó 2007 American Academy of Allergy, Asthma & Immunology doi:10.1016/j.jaci.2006.10.030
nonsmoking asthmatic subjects. There was no significant difference in immunoreactivity for iNOS between groups. Nicotine induced a 2-fold increase in arginase I and ODC expression in airway epithelial cells and fibroblasts. Conclusion: This study demonstrates that the expression of arginase I and ODC is increased in airways of smoking compared with nonsmoking asthmatic subjects and in vitro by nicotine. Clinical implications: Increased expression of arginase I might lead to low exhaled nitric oxide and chronic obstructive pulmonary disease–like airway remodeling in smoking asthmatic subjects. (J Allergy Clin Immunol 2007;119:391-7.) Key words: Arginase I, iNOS, ornithine decarboxylase, ornithine decarboxylase, asthma, cigarette smoke
The cigarette smoking habit is highly prevalent among asthmatic patients. Up to 30% of asthmatic patients are smokers.1 A number of epidemiologic studies have linked passive and active cigarette smoke exposure to asthma.2,3 Other studies have failed to show such an association.4-6 A recent study reported a significant increase in inflammatory cells and cytokines in bronchial biopsy specimens from asymptomatic smokers compared with nonsmokers.7 Sputum of smoking asthmatic subjects shows a higher number of neutrophils and higher IL-8 levels.8 Furthermore, clinical outcomes in smoking asthmatic subjects are worse than in nonsmoking asthmatic subjects.9 Characteristics of smoking asthmatic subjects are increased respiratory symptoms, a lower FEV1 over forced vital capacity, lower forced expiratory flow between 25% and 75% of forced vital capacity, lower lung diffusion capacity, increased functional residual capacity, a more acidic exhaled breath condensate, and more airway and parenchymal abnormalities on chest computed tomodensitometry.9 Recent microarray analysis has shown increased arginase expression to be associated with asthma in murine models.10 Arginase I mRNA and protein expression were also reported to be highly expressed in human asthmatic airways compared with in airways from healthy control subjects.10 Arginine metabolism takes place through the arginase and nitric oxide synthase (NOS) pathways. Arginase converts arginine to ornithine, which is the precursor of proline and polyamines. Proline is used in collagen and mucus production, whereas polyamines increase cell proliferation. High arginase activity might therefore contribute to airway remodeling by means of increased collagen deposition and cell proliferation. Moreover, it 391
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Ce´line Bergeron, MD, MSc,a Louis-Philippe Boulet, MD,b Nathalie Page, BSc,b Michel Laviolette, MD,b Nives Zimmermann, MD,c Marc E. Rothenberg, MD, PhD,c and Qutayba Hamid, MD, PhDa Montreal, Quebec, Canada, and Cincinnati, Ohio
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TABLE I. Characteristics of the subjects Abbreviations used iNOS: Inducible nitric oxide synthase NO: Nitric oxide NOS: Nitric oxide synthase ODC: Ornithine decarboxylase
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is proposed that substrate competition occurs between arginase and NOS. Enhanced arginase activity thus leads to less substrate for NOS and hence less nitric oxide (NO) production. High levels of exhaled NO and inducible NOS (iNOS) have been reported in patients with asthma.11-13 NO is produced in the airway epithelium by constitutive and inducible NOS. Excess NO and superoxide anion levels, both produced by iNOS, have adverse effects, including increased vascular permeability, mucus hypersecretion, and epithelial cell damage.14 Cigarette smoke decreases iNOS expression in vitro.15 Furthermore, exhaled NO levels are decreased by cigarette smoking in asthmatic patients.16 However, the exact clinical outcomes and physiopathological effects of cigarette smoking in asthma need to be studied further. This article is part of a study aiming to compare, at multiple levels, smoking and nonsmoking asthmatic patients. We know that high levels of iNOS and arginase I contribute to asthma pathology and that exhaled NO levels are decreased in smoking asthmatic subjects. However, the regulation of arginine pathways in smoking asthmatic subjects is unknown. The low exhaled NO levels observed in smoking asthmatic subjects can result from enhanced arginase expression, decreased iNOS expression, or both in the airway caused by cigarette smoke. This article aims to understand the influence of cigarette smoke on arginine pathways in airways of steroid-naive subjects with mild asthma.
METHODS Study design and characteristic of subjects Groups of 12 nonsmoking and 12 smoking steroid-naive asthmatic subjects from Laval Hospital asthma clinic were recruited (Quebec, Canada). The nonsmoking group was defined as not having smoked in the last year, with less than 2 pack-years of smoking. The smoking group was defined as currently smoking more than 10 cigarettes per day, with more than 10 pack-years of smoking. Spirometry, methacholine challenge, allergy skin prick testing, and flexible bronchoscopy were performed. Inclusion criteria were as follows: patients 18 to 40 years of age and in good health apart from asthma, with a diagnosis of asthma for at least 2 years according to the criteria of the American Thoracic Society,17 defined on the basis of episodic or persistent chest tightness, wheeze, or cough with improvement in FEV1 of greater than 12% and greater than 180 mL from baseline FEV1 10 minutes after receiving 200 mg of salbutamol. On entry into the study, the FEV1 value was greater than 70% of predicted value, and medication was stable for at least 3 months. Patients had not used inhaled corticosteroids or any other treatment for their
Nonsmoking asthmatic subjects
Age (y) Sex (M/F) FEV1 (% predicted) PC20 (mg/mL) Positive skin prick test response (Y/N) Smoking history (pack-years) Current smoking (cigarettes/d)
25.8 6 2.3 3/9 92 6 5 1.7 6 0.9 10/2 None None
Smoking asthmatic subjects
32.7 6 4/8 88 6 1.5 6 8/4 16.7 6 19.5 6
2.2 4 1.3 2.2 3
M, Male; F, female; PC20, methacholine concentration provoking a decrease in FEV1 of 20% from baseline; Y, yes; N, no.
asthma, save only a short-acting b2-agonist on demand. No oral or inhaled corticosteroid had been taken in the previous 6 months. All subjects had provided informed consent, and the institutional ethics committee approved the study. Subjects were excluded from the study if they had been given diagnoses of any pulmonary disorders other than asthma, a coexisting illness was present that affected required testing, there was any history of lower respiratory tract infection in the previous 6 weeks, they were pregnant or lactating, contraindications to or an inability to perform proposed tests were present, or there was any history of upper respiratory tract infections in the previous month. Table I summarizes subjects’ characteristics.
Tissue sampling and preservation Bronchial biopsy specimens were obtained from all patients according to American Thoracic Society guidelines.18 Biopsy specimens were taken at bronchial segmental divisions. Tissues were fixed in acetone/methanol, blocked in optimal cutting temperature embedding medium (Sakura Finetechnical, Tokyo, Japan), and snap-frozen in liquid nitrogen-cooled isopentane (for in situ hybridization) or fixed in 4% paraformaldehyde and embedded in paraffin (for immunohistochemistry). Frozen tissues were kept at 2808C until use, and paraffin-embedded tissues were kept at room temperature.
Immunohistochemistry Immunohistochemistry was performed by using a peroxidase protocol on paraffin-embedded sections to detect arginase I, ornithine decarboxylase (ODC), and iNOS expression. First, slides were deparaffinized in xylene and, in decreasing concentrations of alcohol, permeabilized with Triton 0.2% and incubated with 5% peroxide. After blocking with Protein Block Serum Free solution for 30 minutes (Dako Canada Inc, Mississauga, Ontario, Canada), slides were incubated overnight with the following primary antibodies: specific mouse anti-human mAbs against arginase I (BD Biosciences, Mississauga, Ontario, Canada), ODC (Sigma-Aldrich, St Louis, Mo), and iNOS (R&D Systems, Minneapolis, Minn). After subsequent incubation with biotinylated secondary rabbit anti-mouse antibody (Dako) and with ABC complex (Dako), slides were stained with diaminobenzidine-chromogen (Dako) and counterstained with hematoxylin. Immunohistochemistry was performed with isotype antibodies as negative controls.
In situ hybridization Probe preparation. RNA probes coding for arginase I mRNA were prepared from cDNA, as previously described.19,20 Briefly, cDNA was inserted into pGEM vectors, linearized, and transcribed in vitro in the presence of a35S-UTP and either SP6 or T7 polymerases. Antisense (complementary to mRNA) and sense probes
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Quantification Two observers blinded as to group read all slides. In the nonsmoking group biopsy specimens from 3 subjects showed no epithelium and could not be used for quantification of epithelial immunoreactivities. Immunoreactivity for arginase I, ODC, and iNOS was evaluated in the epithelium and the smooth muscle bundles with a scoring technique, in which 0 represented absence of staining, 1 represented 12.5% of the epithelium with positive staining, 2 represented 25% with positive staining, and so on, up to 8, which represented 100% of the epithelium showing staining. The same scoring technique was used to quantify the positivity in the epithelium and smooth muscle bundles of arginase I mRNA detected by means of in situ hybridization. For immunohistochemistry, slides were observed under light microscopy, whereas for in situ hybridization, dark-field microscopy was used. Positive signals for immunohistochemistry appeared brown under light microscopy and for in situ hybridization as bright light under dark-field microscopy.
Cell culture and stimulation The human bronchial epithelial cell line BEAS-2B (ATCC, Manassas, Va) was cultured in bronchial epithelial growth media consisting of LHC basal medium (Biosource International, Camarillo, Calif), SAGM SingleQuots (Bio Science, Walkersville, Md), phosphorylethanolamine, ethanolamine, CaCl2, zinc, iron, magnesium, trace elements, 100 U/mL penicillin G, and 100 mg/mL streptomycin at 378C in the presence of 5% CO2. Airway fibroblasts were isolated from human nonallergic nasal polyps (n 5 3) by using collagenase H digestion. Isolated fibroblasts were characterized by means of immunofluorescence with mouse anti-human vimentin, anti-actin, anti-cytokeratin, and anti-human fibroblast antigen Ab-1 antibodies. This identification confirmed the purity of airway fibroblast cell cultures. Fibroblasts were used in passages 3 to 4. Cells were grown in Dulbecco modified Eagle medium (Invitrogen, Burlington, Ontario, Canada) supplemented with 10% heat-inactivated FCS (Medicorp, Montreal, Quebec, Canada), 100 U/mL penicillin G, and 100 mg/mL streptomycin at 378C in the presence of 5% CO2. Primary human airway smooth muscle cells were isolated from main bronchial airway segments (0.5-1.0 cm in diameter) and characterized as previously described.21 Cells were grown at 378C with 5% CO2 in Smooth Muscle Growth medium (Cambrex, Walkersville, Md). Subconfluent airway epithelial cells, fibroblasts, and smooth muscle cells were stimulated with increasing doses of nicotine (0.1-1000 mmol/L, Sigma-Aldrich). Time course up to 24 hours and dose-response experiments were conducted to choose the optimal condition. Nicotine stock was preserved at 1 mol/L in ethanol, and nonstimulated conditions were performed in medium supplemented with 0.1% ethanol. Stimulations were also done with preincubation of cells with IL-4 (0.1-10 ng/mL) or IL-5 (0.1-10 ng/mL) for 24 hours before nicotine stimulation.
RNA extraction and reverse transcriptase Airway structural cells were incubated with or without nicotine for 6 hours, and total RNA was extracted by using the RNeasy Mini Kit (Qiagen, Mississauga, Ontario, Canada), according to the manufacturer’s protocol. Concentrations of total RNA were estimated by
means of optic densitometry. RNA was analyzed by means of RTPCR amplification. Five hundred nanograms of total RNA per sample were denaturated at 658C for 10 minutes and incubated at 458C for 60 minutes, with the following mix: 25 mM deoxyribonucleoside triphosphate, 0.25 mg/mL polydT (Amersham Pharmacia Biotech, Piscataway, NJ), 220 U Superscript II (Invitrogen), 0.01 mol/L dithiothreitol (Invitrogen), RNAsin (Invitrogen), and first-strand buffer (Invitrogen).
Real-time PCR Quantification of the cDNA message coding for ribosomal protein S9 (control), arginase I, ODC, and iNOS was performed by using Lightcycler technology (Roche Diagnostics, Laval, Quebec, Canada) after reverse transcriptase, as previously described.22 Primer sequences were as follows: S9 forward 59-TGCTGACGCTTGATGAGAAG-39, S9 reverse 59-CGCAGAGAGAAGTCGATGTG-39, arginase I forward 59-ATTGTTCCGTTCTTCTTGACTT-39, arginase I reverse 59-AGTG TGATGTGAAGGATTATG-39, iNOS forward 59-GCTGCCAAGCT GAAATTGA-39, iNOS reverse 59-TTCTTCGCCTCGTAAGGAAA39; ODC forward 59-GAGCACATCCCAAAGCAAAGT-39, ODC reverse 59-ATGAAAGGCAGTCTGAGACCT-39. PCR reactions were performed in a volume of 20 mL containing 1 mL of cDNA, 0.3 mmol/L of each primer, and 10 mL of QuantiTect SYBR Green PCR containing DNA polymerase, deoxyribonucleoside triphosphate mix, buffer, MgCl2, and fluorescent dyes. The PCR protocol consisted of 3 programs: denaturation, amplification, and melting-curve analysis for product identification. The denaturation and amplification conditions for S9, arginase I, ODC, and iNOS were 958C for 15 minutes, followed by 50 to 55 cycles of PCR. Each cycle included denaturation at 958C for 20 seconds, annealing for 20 seconds at 608C, and extension for 20 seconds at 728C. The temperature transition rate was 208C per second, except when heating at 728C (58C per second). Fluorescence was measured at the end of every cycle to allow quantification of cDNA. After amplification, a melting curve was obtained by slowly heating at 958C, with fluorescence detection every 0.28C after the normal cycle.
Statistics A paired 1-sample t test was used to test the null hypothesis that there was no difference in arginase I, ODC, and iNOS expression between nonsmoking and smoking asthmatic subjects. This test was set against the alternative hypothesis that there was a difference in the extent of arginase I, ODC, and iNOS expression and remodeling. Similar null and alternative hypotheses were used for analysis of PCR results. Data are presented as means 6 SEM, and the P value was considered significant at less than .05.
RESULTS Arginase I, ODC, and iNOS protein expression in airways of nonsmoking and smoking asthmatic subjects Immunoreactivity for arginase I, ODC, and iNOS was detected in the epithelium (Fig 1) and smooth muscle bundles in both nonsmoking and smoking asthmatic subjects. There was a significant difference between both groups in immunoreactivity for arginase I in the epithelium (score of 1.1 6 0.25 for nonsmokers vs 3 6 0.4 for smokers, P < .05) and in the smooth muscle bundles (score of 0.2 6 0.1 for nonsmokers vs 1.25 6 0.3 for smokers, P < .05; Fig 2, A). Significant differences were also observed for ODC immunoreactivities
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(identical to mRNA) were prepared. In situ hybridization with sense RNA was performed as negative controls. In situ hybridization. Sections of bronchial biopsy tissue were processed for in situ hybridization of arginase I mRNA with radiolabeled (a35S-UTP) sense and antisense probes hybridized and washed under high-stringency conditions, as previously described.19,20
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Mechanisms of asthma and allergic inflammation FIG 1. Representative pictures of arginase I (A), ODC (B), and iNOS (C) immunoreactivities in airways. Immunoreactive cells show a brown staining. Left column, Nonsmoking asthmatic subjects; right column, smoking asthmatic subjects. Magnification 3400.
(epithelium score of 1.3 6 0.3 for nonsmokers vs 4.1 6 0.5 for smokers, P < .05; smooth muscle bundles score for 1 6 0.3 for nonsmokers vs 3.2 6 0.2 for smokers, P < .05; Fig 2, B). iNOS-immunoreactive cells were detected to a similar extent in both groups (epithelium score of 2.3 6 0.4 for nonsmokers vs 2.1 6 0.6 for smokers, P 0.07; smooth muscle bundles score of 0.4 6 0.2 for nonsmokers vs 0.75 6 0.2 for smokers, P 5 .15; Fig 2, C). The balance between the expression of arginase I and iNOS is toward arginase I in smoking asthmatic subjects and in favor of iNOS in the nonsmoking asthmatic subjects in both the epithelium and the smooth muscle bundles (Fig 2, D).
Arginase I mRNA expression in nonsmoking and smoking asthmatic airways Arginase mRNA was also detected in the epithelium in both groups. The expression of arginase I mRNA was significantly higher in the epithelium of smoking asthmatic subjects than in nonsmoking asthmatic subjects (score of 1.28 6 0.28 for nonsmokers vs 4 6 0.3 for smokers, P < .05). Arginase I mRNA signals were detectable in smooth muscle bundles of smoking asthmatic subjects only.
Arginase I, ODC, and iNOS mRNA expression in airway structural cells on nicotine stimulation Nicotine significantly increased the mRNA expression of arginase I and ODC in the BEAS-2B–derived airway epithelial cell line (arginase I: 2-fold increase, P < .05; ODC: 1.8-fold increase, P < .05; Fig 3, A). iNOS mRNA was not detectable in our epithelial cell culture. In airway smooth muscle cells a trend toward increased expression of arginase I and ODC after nicotine stimulation was observed but did not reach statistical significance (Fig 3, B). In airway fibroblasts a 2-fold increase in the mRNA expression of ODC was observed (P < .05), and a 2.8fold increase for arginase I was detected (P > .05; Fig 3, C). We further analyzed the expression of arginase I, ODC, and iNOS in response to nicotine in cells previously stimulated with TH2 cytokines. In our 3 cell lines, IL-4 or IL-5 did not increase arginase I expression, and addition of nicotine was not synergetic (data not shown). Combination of TH2 cytokines and nicotine leads to a higher increase in ODC expression in fibroblasts and airway smooth muscle cells when compared with nicotine alone (data not shown). IL-4 and IL-5 also increase the expression of iNOS in fibroblasts and airway smooth muscle cells. Nicotine leads
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FIG 2. Arginase I (ARGI; A), ODC (B), and iNOS (C) immunoreactivity scores in the epithelium and smooth muscle bundles on airways of nonsmoking and smoking asthmatic subjects. D, Ratio of arginase I on iNOS in the epithelium and the smooth muscle bundles in both groups. Data are represented as means 6 SEM. *P < .05. Open boxes, Nonsmoking asthmatic subjects; solid boxes, smoking asthmatic subjects.
to an increased expression of iNOS in airway smooth muscle cells preincubated with IL-4 and IL-5 (data not shown).
DISCUSSION Arginase I is highly expressed in airways of asthmatic subjects.10,23 Airway hyperresponsiveness and remodeling are proposed as being directly linked to increased arginase I expression. In this study we demonstrated that cigarette smoke in airways of asthmatic subjects upregulates the expression of arginase I mRNA and protein. We detected arginase I protein not only in the epithelial cells but also in the smooth muscle bundles of airways of smoking and nonsmoking asthmatic subjects. In a previous study arginase I mRNA was found in epithelial cells of nonsmoking control subjects and asthmatic subjects.10 Similarly to that study, we detected arginase I mRNA in nonsmoking asthmatic subjects only in the epithelium, whereas in smoking asthmatic subjects mRNA was detectable in both the epithelium and the smooth muscle bundles. This observation suggests that cigarette smoke increases the expression level of arginase I over the detectable threshold of the in situ hybridization method. This effect might be attributable to different components of the cigarette smoke. We tested the effect of nicotine, which is known to have inflammatory, immunosuppressive, and addictive effects,24 in vitro. We found upregulation of arginase I in our cultured airway epithelial cell line, smooth muscle cells, and fibroblasts after stimulation with nicotine. Taken together, our in situ and in vitro results suggest that arginase I expression is upregulated in the airways of smoking asthmatic subjects. The potential roles of the increased expression of arginase I caused by cigarette smoke are discussed below.
FIG 3. Effect of nicotine on arginase I (ARGI), ODC, and iNOS mRNA expression in airway epithelial cell line (A), airway smooth muscle cells (B), and airway fibroblasts (C). Data are represented as mean 6 SEM of the fold increase after correction for the housekeeping gene S9. *P < .05. Open boxes, Nonstimulated cells; solid boxes, nicotine-stimulated cells.
This is the first report of detection of ODC in the airways. ODC is an effector enzyme in the arginase I pathway, converting ornithine to polyamines. ODC immunoreactivity presented a similar pattern to that of arginase I and was detected in the epithelium and smooth muscle bundles in both groups. Smoking asthmatic subjects have significantly more ODC expression than nonsmoking asthmatic subjects. Stimulation of structural cells with nicotine led to an increase in the ODC expression in our epithelial cell line, smooth muscle cells, and fibroblasts. These results strengthened our hypothesis that cigarette smoke stimulated cell proliferation by favoring the arginase I pathway and the production of polyamines. iNOS levels are known to be increased in patients with asthma,11 as are exhaled NO levels.12 We detected iNOS protein in the epithelium and smooth muscle cells in both groups. The level of expression was not significantly different between groups. Furthermore, iNOS expression was not increased after nicotine stimulation in smooth muscle cells and was not detectable in our epithelial cell line and fibroblasts. We can only conclude that the expression level of iNOS is very low in our cells and that nicotine did not upregulate its expression to a detectable threshold. Asthmatic patients who smoke cigarettes exhibit a balance between arginase I and iNOS in favor of the arginase I pathway. A previous study reported that these 2 enzymes compete for
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the same substrate, L-arginine.25 From our results, L-arginine, the common substrate of arginase I and iNOS, is proposed to be preferentially metabolized by the arginase I pathway in airways of smoking asthmatic patients. Less arginine availability for iNOS leads to less NO production. To support this hypothesis, previous studies have reported reduced exhaled NO levels in smoking asthmatic subjects compared with those in nonsmoking asthmatic subjects.16,26 Low NO levels might have negative outcomes. Bronchodilating and anti-inflammatory effects of NO might not be fully effective at lower levels and might lead to increased airway hyperresponsiveness.23 We propose that the low exhaled NO reported in smoking asthmatic subjects can be attributed to the reduced substrate availability resulting from increased arginase I expression and activity. We did not have a control group of nonsmoking and smoking ‘‘healthy’’ subjects to allow comparison. However, the aim of this study was to depict the differences between smoking and nonsmoking asthmatic subjects, thus having cigarette smoking exposure as the only variable between groups. Moreover, the expression of arginase I and iNOS was already reported between control and asthmatic subjects.10,11 In a concurrent study we demonstrated that airways of smoking and nonsmoking asthmatic subjects show extensive airway remodeling, including increased smooth muscle mass, subepithelial fibrosis, and basement membrane thickness (St Laurent et al, manuscript in preparation). An increase in arginase I expression should lead to a high rate of proline synthesis, a component of collagen. Despite the high level of arginase I expression, no significant difference was observed at the level of subepithelial fibrosis and the basement membrane (St Laurent et al, manuscript in preparation). The measurement of ornithine aminotransferase under the influence of cigarette smoke would have been interesting to clarify the effects on this pathway. Unfortunately, this intermediate enzyme, leading to proline, cannot be studied by means of immunohistochemistry. From another standpoint, airways of smoking asthmatic subjects show more epithelial metaplasia and goblet cell hyperplasia, as well as a trend toward higher submucosal gland area, than airways of nonsmoking asthmatic subjects (St Laurent et al, manuscript in preparation). These differences in the epithelium can be attributed to an increased proliferation rate of epithelial cells from polyamines produced by the arginase I and ODC pathways. Polyamines are well known to increase the proliferation rate in vitro27 and in vivo.28 Polyamines might also be effective on submucosal glands and connected to hyperplasia. Smoking asthmatic subjects have accelerated loss of lung function,29 which might be related to increased expression of arginase I, which can accelerate the process of airway remodeling. We did not record an enzyme activity measurement and can only suppose that an increased expression of enzymes would result in increased activity. A previous study demonstrated that both activity and mRNA expression of arginase I are increased after allergen challenge or TH2 cytokine stimulation.10 Furthermore, our in vitro experiments on the regulation of arginase I, ODC, and iNOS
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allowed us to conclude that nicotine, a major component of cigarette smoke, has at least the capability to upregulate arginase I and ODC expression in airway structural cells. A balance between arginase I and iNOS in favor of the arginase I pathway is seen in airways of smoking asthmatic subjects. This suggests that L-arginine will be preferentially metabolized through the arginase I pathway, leading to increased availability of proline and polyamines and decreased NO synthesis. The low exhaled NO level and the chronic obstructive pulmonary disease–like remodeling process observed in airways of smoking asthmatic subjects can be, at least in part, attributed to a high expression of arginase I induced by nicotine and other cigarette smoke components. We thank Andrea Mogas and Elsa Schotman for their technical help.
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15. Hoyt JC, Robbins RA, Habib M, Springall DR, Buttery LD, Polak JM, et al. Cigarette smoke decreases inducible nitric oxide synthase in lung epithelial cells. Exp Lung Res 2003;29:17-28. 16. Verleden GM, Dupont LJ, Verpeut AC, Demedts MG. The effect of cigarette smoking on exhaled nitric oxide in mild steroid-naive asthmatics. Chest 1999;116:59-64. 17. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease (COPD) and asthma. This official statement of the American Thoracic Society was adopted by the ATS Board of Directors, November 1986. Am Rev Respir Dis 1987;136:225-44. 18. Workshop summary and guidelines: investigative use of bronchoscopy, lavage, and bronchial biopsies in asthma and other airway diseases. J Allergy Clin Immunol 1991;88:808-14. 19. Hamid Q, Azzawi M, Ying S, Moqbel R, Wardlaw AJ, Corrigan CJ, et al. Expression of mRNA for interleukin-5 in mucosal bronchial biopsies from asthma. J Clin Invest 1991;87:1541-6. 20. Hamid Q, Wharton J, Terenghi G, Hassall CJ, Aimi J, Taylor KM, et al. Localization of atrial natriuretic peptide mRNA and immunoreactivity in the rat heart and human atrial appendage. Proc Natl Acad Sci U S A 1987;84:6760-4. 21. Gounni AS, Hamid Q, Rahman SM, Hoeck J, Yang J, Shan L. IL-9-mediated induction of eotaxin1/CCL11 in human airway smooth muscle cells. J Immunol 2004;173:2771-9.
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22. Bergeron C, Page N, Joubert P, Barbeau B, Hamid Q, Chakir J. Regulation of procollagen I (alpha1) by interleukin-4 in human bronchial fibroblasts: a possible role in airway remodelling in asthma. Clin Exp Allergy 2003;33:1389-97. 23. Morris CR, Poljakovic M, Lavrisha L, Machado L, Kuypers FA, Morris SM Jr. Decreased arginine bioavailability and increased serum arginase activity in asthma. Am J Respir Crit Care Med 2004;170:148-53. 24. Bartal M. Health effects of tobacco use and exposure. Monaldi Arch Chest Dis 2001;56:545-54. 25. Bruch-Gerharz D, Schnorr O, Suschek C, Beck KF, Pfeilschifter J, Ruzicka T, et al. Arginase 1 overexpression in psoriasis: limitation of inducible nitric oxide synthase activity as a molecular mechanism for keratinocyte hyperproliferation. Am J Pathol 2003;162:203-11. 26. Ichinose M, Sugiura H, Yamagata S, Koarai A, Shirato K. Increase in reactive nitrogen species production in chronic obstructive pulmonary disease airways. Am J Respir Crit Care Med 2000;162:701-6. 27. Herbst EJ, Elliot QD. Role of polyamines in HeLa cell proliferation. Med Biol 1981;59:410-6. 28. Pegg AE. Polyamine metabolism and its importance in neoplastic growth and a target for chemotherapy. Cancer Res 1988;48:759-74. 29. James AL, Palmer LJ, Kicic E, Maxwell PS, Lagan SE, Ryan GF, Musk AW. Decline in lung function in the Busselton Health Study: the effects of asthma and cigarette smoking. Am J Respir Crit Care Med 2005;171:109-14.
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Background: Up to 30% of asthmatic subjects are smokers, and smoking might be an important contributor to asthma pathology. Inducible nitric oxide synthase (iNOS), ornithine decarboxylase (ODC), and arginase I are involved in the arginine pathway. We have shown that arginase I and iNOS are upregulated in asthma. Smoking asthmatic subjects are reported to have low exhaled nitric oxide levels. The effect of cigarette smoking on the expression of arginase I in asthma is unknown. Objectives: The aims of this study were to investigate the expression of arginase I, ODC, and iNOS in asthmatic airways of smokers and nonsmokers and in vitro after nicotine stimulation. Methods: Endobronchial biopsies were performed on 24 steroid-naive subjects with mild asthma: 12 smokers and 12 nonsmokers. Arginase I, ODC, and iNOS levels were assessed by means of immunohistochemistry and in situ hybridization (arginase I). In vitro stimulation of airway cells with nicotine was performed, followed by real-time PCR. Results: Arginase I, ODC, and iNOS were expressed in the epithelium and smooth muscle bundles of both subgroups of asthmatic subjects. There was an increase of arginase I and ODC immunoreactivities in smoking compared with
From aMeakins-Christie Laboratories, McGill University, Montreal; bCentre de Recherche, Hoˆpital Laval, Institut Universitaire de Cardiologie et de Pneumologie; and cthe Division of Allergy and Immunology, Cincinnati Children’s Hospital Medical Center. Supported by local funds and National Institutes of Health/National Institute of Allergy and Infectious Diseases R01 A153479. C. B. is a recipient of the Ann Woolcock fellowship. Disclosure of potential conflict of interest: L.-P. Boulet has consulting arrangements with Altana, AstraZeneca, GlaxoSmithKline, Novartis, and Merck Frost; has received grant support from 3M, Altana, Athmatx, AstraZeneca, Boehringer Ingelheim, Dynavax, Genentech, GlaxoSmithKline, IVAX, Merck Frost, Novartis, Pfizer, Roche, Schering, and Topingen; and is on the speakers’ bureau for 3M, Altana, AstraZeneca, GlaxoSmithKline, Merck Frost, and Novartis. M. Laviolette has received grant support from Merck Frost and is on the speakers’ bureau for 3M, AstraZeneca, and Glaxo Canada. N. Zimmermann has received grant support from the National Institutes of Health. M. E. Rothenberg has consulting arrangements with GlaxoSmithKline, Ception Therapeutics, Cambridge Antibody Technology, and MedaCorp; owns stock in Ception Therapeutics; has received grant support from Cambridge Antibody Technology; is on the speakers’ bureau for Merck and Tanox; and has received honorarium from GlaxoSmithKline, Ception Therapeutics, Merck, and Tanox. The rest of the authors have declared that they have no conflict of interest. Received for publication May 5, 2006; revised October 19, 2006; accepted for publication October 24, 2006. Reprint requests: Qutayba Hamid, MD, PhD, Meakins-Christie Laboratories, McGill University, 3626 St Urbain, Montreal, Quebec, Canada H2X 2P2. E-mail: [email protected]. 0091-6749/$32.00 Ó 2007 American Academy of Allergy, Asthma & Immunology doi:10.1016/j.jaci.2006.10.030
nonsmoking asthmatic subjects. There was no significant difference in immunoreactivity for iNOS between groups. Nicotine induced a 2-fold increase in arginase I and ODC expression in airway epithelial cells and fibroblasts. Conclusion: This study demonstrates that the expression of arginase I and ODC is increased in airways of smoking compared with nonsmoking asthmatic subjects and in vitro by nicotine. Clinical implications: Increased expression of arginase I might lead to low exhaled nitric oxide and chronic obstructive pulmonary disease–like airway remodeling in smoking asthmatic subjects. (J Allergy Clin Immunol 2007;119:391-7.) Key words: Arginase I, iNOS, ornithine decarboxylase, ornithine decarboxylase, asthma, cigarette smoke
The cigarette smoking habit is highly prevalent among asthmatic patients. Up to 30% of asthmatic patients are smokers.1 A number of epidemiologic studies have linked passive and active cigarette smoke exposure to asthma.2,3 Other studies have failed to show such an association.4-6 A recent study reported a significant increase in inflammatory cells and cytokines in bronchial biopsy specimens from asymptomatic smokers compared with nonsmokers.7 Sputum of smoking asthmatic subjects shows a higher number of neutrophils and higher IL-8 levels.8 Furthermore, clinical outcomes in smoking asthmatic subjects are worse than in nonsmoking asthmatic subjects.9 Characteristics of smoking asthmatic subjects are increased respiratory symptoms, a lower FEV1 over forced vital capacity, lower forced expiratory flow between 25% and 75% of forced vital capacity, lower lung diffusion capacity, increased functional residual capacity, a more acidic exhaled breath condensate, and more airway and parenchymal abnormalities on chest computed tomodensitometry.9 Recent microarray analysis has shown increased arginase expression to be associated with asthma in murine models.10 Arginase I mRNA and protein expression were also reported to be highly expressed in human asthmatic airways compared with in airways from healthy control subjects.10 Arginine metabolism takes place through the arginase and nitric oxide synthase (NOS) pathways. Arginase converts arginine to ornithine, which is the precursor of proline and polyamines. Proline is used in collagen and mucus production, whereas polyamines increase cell proliferation. High arginase activity might therefore contribute to airway remodeling by means of increased collagen deposition and cell proliferation. Moreover, it 391
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Ce´line Bergeron, MD, MSc,a Louis-Philippe Boulet, MD,b Nathalie Page, BSc,b Michel Laviolette, MD,b Nives Zimmermann, MD,c Marc E. Rothenberg, MD, PhD,c and Qutayba Hamid, MD, PhDa Montreal, Quebec, Canada, and Cincinnati, Ohio
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TABLE I. Characteristics of the subjects Abbreviations used iNOS: Inducible nitric oxide synthase NO: Nitric oxide NOS: Nitric oxide synthase ODC: Ornithine decarboxylase
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is proposed that substrate competition occurs between arginase and NOS. Enhanced arginase activity thus leads to less substrate for NOS and hence less nitric oxide (NO) production. High levels of exhaled NO and inducible NOS (iNOS) have been reported in patients with asthma.11-13 NO is produced in the airway epithelium by constitutive and inducible NOS. Excess NO and superoxide anion levels, both produced by iNOS, have adverse effects, including increased vascular permeability, mucus hypersecretion, and epithelial cell damage.14 Cigarette smoke decreases iNOS expression in vitro.15 Furthermore, exhaled NO levels are decreased by cigarette smoking in asthmatic patients.16 However, the exact clinical outcomes and physiopathological effects of cigarette smoking in asthma need to be studied further. This article is part of a study aiming to compare, at multiple levels, smoking and nonsmoking asthmatic patients. We know that high levels of iNOS and arginase I contribute to asthma pathology and that exhaled NO levels are decreased in smoking asthmatic subjects. However, the regulation of arginine pathways in smoking asthmatic subjects is unknown. The low exhaled NO levels observed in smoking asthmatic subjects can result from enhanced arginase expression, decreased iNOS expression, or both in the airway caused by cigarette smoke. This article aims to understand the influence of cigarette smoke on arginine pathways in airways of steroid-naive subjects with mild asthma.
METHODS Study design and characteristic of subjects Groups of 12 nonsmoking and 12 smoking steroid-naive asthmatic subjects from Laval Hospital asthma clinic were recruited (Quebec, Canada). The nonsmoking group was defined as not having smoked in the last year, with less than 2 pack-years of smoking. The smoking group was defined as currently smoking more than 10 cigarettes per day, with more than 10 pack-years of smoking. Spirometry, methacholine challenge, allergy skin prick testing, and flexible bronchoscopy were performed. Inclusion criteria were as follows: patients 18 to 40 years of age and in good health apart from asthma, with a diagnosis of asthma for at least 2 years according to the criteria of the American Thoracic Society,17 defined on the basis of episodic or persistent chest tightness, wheeze, or cough with improvement in FEV1 of greater than 12% and greater than 180 mL from baseline FEV1 10 minutes after receiving 200 mg of salbutamol. On entry into the study, the FEV1 value was greater than 70% of predicted value, and medication was stable for at least 3 months. Patients had not used inhaled corticosteroids or any other treatment for their
Nonsmoking asthmatic subjects
Age (y) Sex (M/F) FEV1 (% predicted) PC20 (mg/mL) Positive skin prick test response (Y/N) Smoking history (pack-years) Current smoking (cigarettes/d)
25.8 6 2.3 3/9 92 6 5 1.7 6 0.9 10/2 None None
Smoking asthmatic subjects
32.7 6 4/8 88 6 1.5 6 8/4 16.7 6 19.5 6
2.2 4 1.3 2.2 3
M, Male; F, female; PC20, methacholine concentration provoking a decrease in FEV1 of 20% from baseline; Y, yes; N, no.
asthma, save only a short-acting b2-agonist on demand. No oral or inhaled corticosteroid had been taken in the previous 6 months. All subjects had provided informed consent, and the institutional ethics committee approved the study. Subjects were excluded from the study if they had been given diagnoses of any pulmonary disorders other than asthma, a coexisting illness was present that affected required testing, there was any history of lower respiratory tract infection in the previous 6 weeks, they were pregnant or lactating, contraindications to or an inability to perform proposed tests were present, or there was any history of upper respiratory tract infections in the previous month. Table I summarizes subjects’ characteristics.
Tissue sampling and preservation Bronchial biopsy specimens were obtained from all patients according to American Thoracic Society guidelines.18 Biopsy specimens were taken at bronchial segmental divisions. Tissues were fixed in acetone/methanol, blocked in optimal cutting temperature embedding medium (Sakura Finetechnical, Tokyo, Japan), and snap-frozen in liquid nitrogen-cooled isopentane (for in situ hybridization) or fixed in 4% paraformaldehyde and embedded in paraffin (for immunohistochemistry). Frozen tissues were kept at 2808C until use, and paraffin-embedded tissues were kept at room temperature.
Immunohistochemistry Immunohistochemistry was performed by using a peroxidase protocol on paraffin-embedded sections to detect arginase I, ornithine decarboxylase (ODC), and iNOS expression. First, slides were deparaffinized in xylene and, in decreasing concentrations of alcohol, permeabilized with Triton 0.2% and incubated with 5% peroxide. After blocking with Protein Block Serum Free solution for 30 minutes (Dako Canada Inc, Mississauga, Ontario, Canada), slides were incubated overnight with the following primary antibodies: specific mouse anti-human mAbs against arginase I (BD Biosciences, Mississauga, Ontario, Canada), ODC (Sigma-Aldrich, St Louis, Mo), and iNOS (R&D Systems, Minneapolis, Minn). After subsequent incubation with biotinylated secondary rabbit anti-mouse antibody (Dako) and with ABC complex (Dako), slides were stained with diaminobenzidine-chromogen (Dako) and counterstained with hematoxylin. Immunohistochemistry was performed with isotype antibodies as negative controls.
In situ hybridization Probe preparation. RNA probes coding for arginase I mRNA were prepared from cDNA, as previously described.19,20 Briefly, cDNA was inserted into pGEM vectors, linearized, and transcribed in vitro in the presence of a35S-UTP and either SP6 or T7 polymerases. Antisense (complementary to mRNA) and sense probes
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Quantification Two observers blinded as to group read all slides. In the nonsmoking group biopsy specimens from 3 subjects showed no epithelium and could not be used for quantification of epithelial immunoreactivities. Immunoreactivity for arginase I, ODC, and iNOS was evaluated in the epithelium and the smooth muscle bundles with a scoring technique, in which 0 represented absence of staining, 1 represented 12.5% of the epithelium with positive staining, 2 represented 25% with positive staining, and so on, up to 8, which represented 100% of the epithelium showing staining. The same scoring technique was used to quantify the positivity in the epithelium and smooth muscle bundles of arginase I mRNA detected by means of in situ hybridization. For immunohistochemistry, slides were observed under light microscopy, whereas for in situ hybridization, dark-field microscopy was used. Positive signals for immunohistochemistry appeared brown under light microscopy and for in situ hybridization as bright light under dark-field microscopy.
Cell culture and stimulation The human bronchial epithelial cell line BEAS-2B (ATCC, Manassas, Va) was cultured in bronchial epithelial growth media consisting of LHC basal medium (Biosource International, Camarillo, Calif), SAGM SingleQuots (Bio Science, Walkersville, Md), phosphorylethanolamine, ethanolamine, CaCl2, zinc, iron, magnesium, trace elements, 100 U/mL penicillin G, and 100 mg/mL streptomycin at 378C in the presence of 5% CO2. Airway fibroblasts were isolated from human nonallergic nasal polyps (n 5 3) by using collagenase H digestion. Isolated fibroblasts were characterized by means of immunofluorescence with mouse anti-human vimentin, anti-actin, anti-cytokeratin, and anti-human fibroblast antigen Ab-1 antibodies. This identification confirmed the purity of airway fibroblast cell cultures. Fibroblasts were used in passages 3 to 4. Cells were grown in Dulbecco modified Eagle medium (Invitrogen, Burlington, Ontario, Canada) supplemented with 10% heat-inactivated FCS (Medicorp, Montreal, Quebec, Canada), 100 U/mL penicillin G, and 100 mg/mL streptomycin at 378C in the presence of 5% CO2. Primary human airway smooth muscle cells were isolated from main bronchial airway segments (0.5-1.0 cm in diameter) and characterized as previously described.21 Cells were grown at 378C with 5% CO2 in Smooth Muscle Growth medium (Cambrex, Walkersville, Md). Subconfluent airway epithelial cells, fibroblasts, and smooth muscle cells were stimulated with increasing doses of nicotine (0.1-1000 mmol/L, Sigma-Aldrich). Time course up to 24 hours and dose-response experiments were conducted to choose the optimal condition. Nicotine stock was preserved at 1 mol/L in ethanol, and nonstimulated conditions were performed in medium supplemented with 0.1% ethanol. Stimulations were also done with preincubation of cells with IL-4 (0.1-10 ng/mL) or IL-5 (0.1-10 ng/mL) for 24 hours before nicotine stimulation.
RNA extraction and reverse transcriptase Airway structural cells were incubated with or without nicotine for 6 hours, and total RNA was extracted by using the RNeasy Mini Kit (Qiagen, Mississauga, Ontario, Canada), according to the manufacturer’s protocol. Concentrations of total RNA were estimated by
means of optic densitometry. RNA was analyzed by means of RTPCR amplification. Five hundred nanograms of total RNA per sample were denaturated at 658C for 10 minutes and incubated at 458C for 60 minutes, with the following mix: 25 mM deoxyribonucleoside triphosphate, 0.25 mg/mL polydT (Amersham Pharmacia Biotech, Piscataway, NJ), 220 U Superscript II (Invitrogen), 0.01 mol/L dithiothreitol (Invitrogen), RNAsin (Invitrogen), and first-strand buffer (Invitrogen).
Real-time PCR Quantification of the cDNA message coding for ribosomal protein S9 (control), arginase I, ODC, and iNOS was performed by using Lightcycler technology (Roche Diagnostics, Laval, Quebec, Canada) after reverse transcriptase, as previously described.22 Primer sequences were as follows: S9 forward 59-TGCTGACGCTTGATGAGAAG-39, S9 reverse 59-CGCAGAGAGAAGTCGATGTG-39, arginase I forward 59-ATTGTTCCGTTCTTCTTGACTT-39, arginase I reverse 59-AGTG TGATGTGAAGGATTATG-39, iNOS forward 59-GCTGCCAAGCT GAAATTGA-39, iNOS reverse 59-TTCTTCGCCTCGTAAGGAAA39; ODC forward 59-GAGCACATCCCAAAGCAAAGT-39, ODC reverse 59-ATGAAAGGCAGTCTGAGACCT-39. PCR reactions were performed in a volume of 20 mL containing 1 mL of cDNA, 0.3 mmol/L of each primer, and 10 mL of QuantiTect SYBR Green PCR containing DNA polymerase, deoxyribonucleoside triphosphate mix, buffer, MgCl2, and fluorescent dyes. The PCR protocol consisted of 3 programs: denaturation, amplification, and melting-curve analysis for product identification. The denaturation and amplification conditions for S9, arginase I, ODC, and iNOS were 958C for 15 minutes, followed by 50 to 55 cycles of PCR. Each cycle included denaturation at 958C for 20 seconds, annealing for 20 seconds at 608C, and extension for 20 seconds at 728C. The temperature transition rate was 208C per second, except when heating at 728C (58C per second). Fluorescence was measured at the end of every cycle to allow quantification of cDNA. After amplification, a melting curve was obtained by slowly heating at 958C, with fluorescence detection every 0.28C after the normal cycle.
Statistics A paired 1-sample t test was used to test the null hypothesis that there was no difference in arginase I, ODC, and iNOS expression between nonsmoking and smoking asthmatic subjects. This test was set against the alternative hypothesis that there was a difference in the extent of arginase I, ODC, and iNOS expression and remodeling. Similar null and alternative hypotheses were used for analysis of PCR results. Data are presented as means 6 SEM, and the P value was considered significant at less than .05.
RESULTS Arginase I, ODC, and iNOS protein expression in airways of nonsmoking and smoking asthmatic subjects Immunoreactivity for arginase I, ODC, and iNOS was detected in the epithelium (Fig 1) and smooth muscle bundles in both nonsmoking and smoking asthmatic subjects. There was a significant difference between both groups in immunoreactivity for arginase I in the epithelium (score of 1.1 6 0.25 for nonsmokers vs 3 6 0.4 for smokers, P < .05) and in the smooth muscle bundles (score of 0.2 6 0.1 for nonsmokers vs 1.25 6 0.3 for smokers, P < .05; Fig 2, A). Significant differences were also observed for ODC immunoreactivities
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(identical to mRNA) were prepared. In situ hybridization with sense RNA was performed as negative controls. In situ hybridization. Sections of bronchial biopsy tissue were processed for in situ hybridization of arginase I mRNA with radiolabeled (a35S-UTP) sense and antisense probes hybridized and washed under high-stringency conditions, as previously described.19,20
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Mechanisms of asthma and allergic inflammation FIG 1. Representative pictures of arginase I (A), ODC (B), and iNOS (C) immunoreactivities in airways. Immunoreactive cells show a brown staining. Left column, Nonsmoking asthmatic subjects; right column, smoking asthmatic subjects. Magnification 3400.
(epithelium score of 1.3 6 0.3 for nonsmokers vs 4.1 6 0.5 for smokers, P < .05; smooth muscle bundles score for 1 6 0.3 for nonsmokers vs 3.2 6 0.2 for smokers, P < .05; Fig 2, B). iNOS-immunoreactive cells were detected to a similar extent in both groups (epithelium score of 2.3 6 0.4 for nonsmokers vs 2.1 6 0.6 for smokers, P 0.07; smooth muscle bundles score of 0.4 6 0.2 for nonsmokers vs 0.75 6 0.2 for smokers, P 5 .15; Fig 2, C). The balance between the expression of arginase I and iNOS is toward arginase I in smoking asthmatic subjects and in favor of iNOS in the nonsmoking asthmatic subjects in both the epithelium and the smooth muscle bundles (Fig 2, D).
Arginase I mRNA expression in nonsmoking and smoking asthmatic airways Arginase mRNA was also detected in the epithelium in both groups. The expression of arginase I mRNA was significantly higher in the epithelium of smoking asthmatic subjects than in nonsmoking asthmatic subjects (score of 1.28 6 0.28 for nonsmokers vs 4 6 0.3 for smokers, P < .05). Arginase I mRNA signals were detectable in smooth muscle bundles of smoking asthmatic subjects only.
Arginase I, ODC, and iNOS mRNA expression in airway structural cells on nicotine stimulation Nicotine significantly increased the mRNA expression of arginase I and ODC in the BEAS-2B–derived airway epithelial cell line (arginase I: 2-fold increase, P < .05; ODC: 1.8-fold increase, P < .05; Fig 3, A). iNOS mRNA was not detectable in our epithelial cell culture. In airway smooth muscle cells a trend toward increased expression of arginase I and ODC after nicotine stimulation was observed but did not reach statistical significance (Fig 3, B). In airway fibroblasts a 2-fold increase in the mRNA expression of ODC was observed (P < .05), and a 2.8fold increase for arginase I was detected (P > .05; Fig 3, C). We further analyzed the expression of arginase I, ODC, and iNOS in response to nicotine in cells previously stimulated with TH2 cytokines. In our 3 cell lines, IL-4 or IL-5 did not increase arginase I expression, and addition of nicotine was not synergetic (data not shown). Combination of TH2 cytokines and nicotine leads to a higher increase in ODC expression in fibroblasts and airway smooth muscle cells when compared with nicotine alone (data not shown). IL-4 and IL-5 also increase the expression of iNOS in fibroblasts and airway smooth muscle cells. Nicotine leads
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FIG 2. Arginase I (ARGI; A), ODC (B), and iNOS (C) immunoreactivity scores in the epithelium and smooth muscle bundles on airways of nonsmoking and smoking asthmatic subjects. D, Ratio of arginase I on iNOS in the epithelium and the smooth muscle bundles in both groups. Data are represented as means 6 SEM. *P < .05. Open boxes, Nonsmoking asthmatic subjects; solid boxes, smoking asthmatic subjects.
to an increased expression of iNOS in airway smooth muscle cells preincubated with IL-4 and IL-5 (data not shown).
DISCUSSION Arginase I is highly expressed in airways of asthmatic subjects.10,23 Airway hyperresponsiveness and remodeling are proposed as being directly linked to increased arginase I expression. In this study we demonstrated that cigarette smoke in airways of asthmatic subjects upregulates the expression of arginase I mRNA and protein. We detected arginase I protein not only in the epithelial cells but also in the smooth muscle bundles of airways of smoking and nonsmoking asthmatic subjects. In a previous study arginase I mRNA was found in epithelial cells of nonsmoking control subjects and asthmatic subjects.10 Similarly to that study, we detected arginase I mRNA in nonsmoking asthmatic subjects only in the epithelium, whereas in smoking asthmatic subjects mRNA was detectable in both the epithelium and the smooth muscle bundles. This observation suggests that cigarette smoke increases the expression level of arginase I over the detectable threshold of the in situ hybridization method. This effect might be attributable to different components of the cigarette smoke. We tested the effect of nicotine, which is known to have inflammatory, immunosuppressive, and addictive effects,24 in vitro. We found upregulation of arginase I in our cultured airway epithelial cell line, smooth muscle cells, and fibroblasts after stimulation with nicotine. Taken together, our in situ and in vitro results suggest that arginase I expression is upregulated in the airways of smoking asthmatic subjects. The potential roles of the increased expression of arginase I caused by cigarette smoke are discussed below.
FIG 3. Effect of nicotine on arginase I (ARGI), ODC, and iNOS mRNA expression in airway epithelial cell line (A), airway smooth muscle cells (B), and airway fibroblasts (C). Data are represented as mean 6 SEM of the fold increase after correction for the housekeeping gene S9. *P < .05. Open boxes, Nonstimulated cells; solid boxes, nicotine-stimulated cells.
This is the first report of detection of ODC in the airways. ODC is an effector enzyme in the arginase I pathway, converting ornithine to polyamines. ODC immunoreactivity presented a similar pattern to that of arginase I and was detected in the epithelium and smooth muscle bundles in both groups. Smoking asthmatic subjects have significantly more ODC expression than nonsmoking asthmatic subjects. Stimulation of structural cells with nicotine led to an increase in the ODC expression in our epithelial cell line, smooth muscle cells, and fibroblasts. These results strengthened our hypothesis that cigarette smoke stimulated cell proliferation by favoring the arginase I pathway and the production of polyamines. iNOS levels are known to be increased in patients with asthma,11 as are exhaled NO levels.12 We detected iNOS protein in the epithelium and smooth muscle cells in both groups. The level of expression was not significantly different between groups. Furthermore, iNOS expression was not increased after nicotine stimulation in smooth muscle cells and was not detectable in our epithelial cell line and fibroblasts. We can only conclude that the expression level of iNOS is very low in our cells and that nicotine did not upregulate its expression to a detectable threshold. Asthmatic patients who smoke cigarettes exhibit a balance between arginase I and iNOS in favor of the arginase I pathway. A previous study reported that these 2 enzymes compete for
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the same substrate, L-arginine.25 From our results, L-arginine, the common substrate of arginase I and iNOS, is proposed to be preferentially metabolized by the arginase I pathway in airways of smoking asthmatic patients. Less arginine availability for iNOS leads to less NO production. To support this hypothesis, previous studies have reported reduced exhaled NO levels in smoking asthmatic subjects compared with those in nonsmoking asthmatic subjects.16,26 Low NO levels might have negative outcomes. Bronchodilating and anti-inflammatory effects of NO might not be fully effective at lower levels and might lead to increased airway hyperresponsiveness.23 We propose that the low exhaled NO reported in smoking asthmatic subjects can be attributed to the reduced substrate availability resulting from increased arginase I expression and activity. We did not have a control group of nonsmoking and smoking ‘‘healthy’’ subjects to allow comparison. However, the aim of this study was to depict the differences between smoking and nonsmoking asthmatic subjects, thus having cigarette smoking exposure as the only variable between groups. Moreover, the expression of arginase I and iNOS was already reported between control and asthmatic subjects.10,11 In a concurrent study we demonstrated that airways of smoking and nonsmoking asthmatic subjects show extensive airway remodeling, including increased smooth muscle mass, subepithelial fibrosis, and basement membrane thickness (St Laurent et al, manuscript in preparation). An increase in arginase I expression should lead to a high rate of proline synthesis, a component of collagen. Despite the high level of arginase I expression, no significant difference was observed at the level of subepithelial fibrosis and the basement membrane (St Laurent et al, manuscript in preparation). The measurement of ornithine aminotransferase under the influence of cigarette smoke would have been interesting to clarify the effects on this pathway. Unfortunately, this intermediate enzyme, leading to proline, cannot be studied by means of immunohistochemistry. From another standpoint, airways of smoking asthmatic subjects show more epithelial metaplasia and goblet cell hyperplasia, as well as a trend toward higher submucosal gland area, than airways of nonsmoking asthmatic subjects (St Laurent et al, manuscript in preparation). These differences in the epithelium can be attributed to an increased proliferation rate of epithelial cells from polyamines produced by the arginase I and ODC pathways. Polyamines are well known to increase the proliferation rate in vitro27 and in vivo.28 Polyamines might also be effective on submucosal glands and connected to hyperplasia. Smoking asthmatic subjects have accelerated loss of lung function,29 which might be related to increased expression of arginase I, which can accelerate the process of airway remodeling. We did not record an enzyme activity measurement and can only suppose that an increased expression of enzymes would result in increased activity. A previous study demonstrated that both activity and mRNA expression of arginase I are increased after allergen challenge or TH2 cytokine stimulation.10 Furthermore, our in vitro experiments on the regulation of arginase I, ODC, and iNOS
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allowed us to conclude that nicotine, a major component of cigarette smoke, has at least the capability to upregulate arginase I and ODC expression in airway structural cells. A balance between arginase I and iNOS in favor of the arginase I pathway is seen in airways of smoking asthmatic subjects. This suggests that L-arginine will be preferentially metabolized through the arginase I pathway, leading to increased availability of proline and polyamines and decreased NO synthesis. The low exhaled NO level and the chronic obstructive pulmonary disease–like remodeling process observed in airways of smoking asthmatic subjects can be, at least in part, attributed to a high expression of arginase I induced by nicotine and other cigarette smoke components. We thank Andrea Mogas and Elsa Schotman for their technical help.
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Mechanisms of asthma and allergic inflammation
J ALLERGY CLIN IMMUNOL VOLUME 119, NUMBER 2