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Airway remodeling associated with cough hypersensitivity as a consequence of persistent cough: An experimental study
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Original article
Airway remodeling associated with cough hypersensitivity as a consequence of persistent cough: An experimental study Hitoshi Nakajia, Akio Niimi, MD, PhDa,b,n, Hirofumi Matsuokaa, Toshiyuki Iwataa, Shilei Cuia, Hisako Matsumotoa, Isao Itoa, Tsuyoshi Ogumaa, Kojiro Otsukaa, Tomoshi Takedaa, Hideki Inouea, Tomoko Tajiria, Tadao Nagasakia, Yoshihiro Kanemitsua,b, Kazuo Chinc, Michiaki Mishimaa a
Department of Respiratory Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan Department of Respiratory Medicine, Allergy and Clinical Immunology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-Cho, Muzuho-Ku, Nagoya 467-8601, Japan c Department of Respiratory Care and Sleep Control Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan b
art i cle i nfo
ab st rac t
Article history:
Background: Chronic cough involves airway remodeling associated with cough reflex
Received 16 September 2015
hypersensitivity. Whether cough itself induces these features remains unknown.
Received in revised form
Methods: Guinea pigs were assigned to receive treatment with citric acid (CA), saline (SA), or
31 May 2016
CAþdextromethorphan (DEX). All animals were exposed to 0.5 M CA on days 1 and 22. On days
Accepted 27 June 2016
4–20, the CA and CAþDEX groups were exposed to CA, and the SA group to saline thrice weekly, during which the CAþDEX group was administered DEX pretreatment to inhibit cough.
Keywords: Chronic cough Airway remodeling Cough reflex hypersensitivity Airway smooth muscle Mechanical stress
The number of coughs was counted during each 10-min CA or SA exposure. Terbutaline premedication was started to prevent bronchoconstriction. Bronchoalveolar lavage and pathology were examined on day 25. Average cough number for 10 CA exposures was examined as “cough index” in the CA group, which was divided into frequent (cough index45) and infrequent (o5) cough subgroups for lavage and pathology analysis. Results: The number of coughs significantly increased in the CA group from day 13 onwards. In the CAþDEX and SA groups, the number of coughs did not differ between days 1 and 22, while average number of coughs during days 4–20 was significantly lower than at days 1 and 22. Bronchoalveolar cell profiles were similar among the four groups. The smooth muscle area of small airways was significantly greater in the frequent-cough subgroup than in the other groups (in which it was similar), and highly correlated with cough index in CA group. Conclusion: Repeated cough induces airway smooth muscle remodeling associated with cough reflex hypersensitivity. & 2016 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.
n Corresponding author at: Department of Respiratory Medicine, Allergy and Clinical Immunology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-Cho, Muzuho-Ku, Nagoya 467-8601, Japan. Tel.: þ81 52 853 8214; fax: þ81 52 852 0849.
http://dx.doi.org/10.1016/j.resinv.2016.06.005 2212-5345/& 2016 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Nakaji H, et al. Airway remodeling associated with cough hypersensitivity as a consequence of persistent cough: An experimental study. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.06.005
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1.
Introduction
Chronic cough is an important clinical problem. Asthmarelated diagnoses such as cough-variant asthma involve eosinophilic airway inflammation [1,2]. Cough may also be ascribed to non-asthma-related diagnoses, such as gastroesophageal reflux disease, but in some patients, it remains unexplained despite thorough investigations. Such nonasthmatic chronic cough (NACC) also involves airway inflammation characterized by increased neutrophils, mast cells, or lymphocytes [2–4]. Airway remodeling is another feature of asthma [5]. The pathological changes of remodeling in cough-variant asthma include subepithelial thickening, goblet-cell hyperplasia, vascular proliferation, and airway-wall thickening [2,6,7]. Eosinophilic bronchitis also involves similar changes [8]. These changes may be ascribed to eosinophil-derived fibrogenic mediators such as transforming growth factor (TGF)-beta, and may result in accelerated lung function decline in intractable cases [1], as in asthma [5]. Interestingly, cases of NACC without airway eosinophilia also show features of remodeling, such as subepithelial thickening, increase of airway smooth muscle (ASM), goblet cells and vessels, epithelial shedding, airway-wall thickening [2,3,7], and irreversible airflow obstruction [9]. Especially noteworthy is the increase of ASM despite absence of airway hyperresponsiveness [10]. The pathogenesis and functional consequences of airway remodeling are not precisely known in chronic cough, especially for NACC [10]. Airway epithelium exposed to compressive stresses, an effect mimicking acute bronchoconstriction in asthma, increases its expression of genes relevant to remodeling, such as TGF-beta 2. Synthesis of extracellular matrix by cocultured fibroblasts is also increased [11]. Cough may also exert mechanical stress on the airway mucosa [12]. In patients with NACC, TGF-beta 1 levels and neutrophil counts in bronchoalveolar lavage fluid (BALF), as well as subepithelial thickness, were increased [13]. TGF-beta 1 expression in the epithelium and ASM were also increased, and TGF-beta 1 levels correlated with subepithelial thickness [13]. In NACC patients, the degree of remodeling (as indicated by goblet-cell hyperplasia, epithelial shedding, and airwaywall thickening) correlated with cough reflex sensitivity to capsaicin [2,7]. Persistent cough may thus result in airway remodeling, in the presence or absence of inflammation. This may lead to cough reflex hypersensitivity that might induce further cough, creating a vicious cycle [10]. A guinea pig model of airway collapse mimicking cough has been reported, induced by rapid repetition of negative pressure applied to the airways of artificially ventilated animals [14]. Capsaicin sensitivity and BALF neutrophils Abbreviations: CA,
citric acid; ASM,
airway smooth muscle; BAL,
transiently increased 6 hours after stimulus, and these features correlated with each other. However, this was a short-term experiment, failing to examine remodeling changes [14]. We investigated whether repeated induction of cough in awake guinea pigs is associated with airway remodeling and cough reflex hypersensitivity. Cough was induced by citric acid (CA) exposure, which is unlikely to induce tachyphylaxis and therefore useful for repeated cough induction and measurements [15–17].
2.
Materials and methods
2.1.
Animals
Male Dunkin-Hartley guinea pigs were obtained and quarantined. All animal procedures conformed to Kyoto University's regulations on animal experimentation (Med Kyo 04449; approval date: Nov 25, 2004).
2.2.
Experimental design
Naïve guinea pigs were assigned to one of three treatment groups: CA group, saline (SA) group, or CAþdextromethorphan (DEX) group (Fig. 1). On days 1 and 22, guinea pigs in all groups were exposed to CA (0.5 M via nebulizer for 10 min) and the number of coughs was counted. From days 4 through 20, the CA and CAþDEX groups were exposed to 0.5 M CA, and the SA group to 0.9% saline for 10 min, three times weekly, at 2- or 3-day intervals (eight times in total). During this period, the CAþDEX group was treated with 30 mg/kg DEX intra-peritoneally (i.p.) 30 min prior to CA exposure, to inhibit cough. DEX was not administered on days 1 and 22. In the CA group, the mean number of coughs for 10 CA exposures was calculated as the “cough index” for each animal. The CA group was divided into a frequent cough subgroup (CA-F: Cough index Z5) and an infrequent cough subgroup (CA-I: Cough index o5) for analysis of BAL and pathology results. The median number of cough index (¼ 5) of the 18 animals in the CA group was used as the cut off level.
2.3.
System for cough measurement
Guinea pigs were placed in a chamber allowing free movement and equipped with an internal microphone and a pressure transducer [18]. They were connected to an Amplifier Interface Unit series pre-amplifier (EMMS, Bordon, UK). The chamber was provided airflow by a Basic Flow Supplier AIR 200 (EMMS) at 1500 mL/min. The changes in airflow induced by respiration and cough were recorded by a pneumotachograph. Cough sounds were amplified and recorded bronchoalveolar lavage; BALF,
BAL fluid; PAS,
periodic acid-
Schiff; TGF, transforming growth factor; NACC, non-asthmatic chronic cough E-mail addresses: [email protected] (H. Nakaji), [email protected] (A. Niimi), [email protected] (H. Matsuoka), [email protected] (T. Iwata), [email protected] (S. Cui), [email protected] (H. Matsumoto), [email protected] (I. Ito), [email protected] (T. Oguma), [email protected] (K. Otsuka), [email protected] (T. Takeda), [email protected] (H. Inoue), [email protected] (T. Tajiri), [email protected] (T. Nagasaki), [email protected] (Y. Kanemitsu), [email protected] (K. Chin), [email protected] (M. Mishima).
Please cite this article as: Nakaji H, et al. Airway remodeling associated with cough hypersensitivity as a consequence of persistent cough: An experimental study. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.06.005
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magnification of 400 in one section and examined. Each image was separated by 4200 mm. ASM area and submucosal area were measured with hematoxylin and eosin (H&E) staining. Epithelial area, goblet cell area, and length of basement membrane were measured using periodic acid-Schiff (PAS)-stained slices. Areas of goblet cells were measured as the total of PAS-positive areas. Each index was calculated (Fig. 2a) from an average of three images for each animal. Fig. 1 – Experimental protocol. Guinea pigs in all groups were exposed to CA (0.5 M) on days 1 and 22 for the evaluation of cough sensitivity. On days 4 through 20, the CA group (n ¼18) and CAþDEX group (n ¼9) were exposed to CA, and the SA group (n ¼9) to saline three times per week. During this period, the CAþDEX group was treated with 30 mg/kg DEX i.p. 30 min prior to CA exposure to inhibit cough. Terbutaline (0.05 mg/kg) was administered i.p. prior to each CA or SA exposure in all groups. CA: Citric acid, SA: Saline, DEX: Dextromethorphan.
by microphone. Guinea pig behavior was captured with an external camera. Data acquisition was performed with eDacq software (EMMS) [18]. After dosing with 0.05 mg/kg terbutaline i.p. to prevent bronchoconstriction [16,18], 0.5 M CA or 0.9% saline was delivered using a nebulizer with an output of 0.5 mL/min for 10 min (Fig. 1). The number of coughs over each 10-min exposure was counted, distinguished from sneezing by guinea pig posture, pressure changes, and sound.
2.4.
BAL
R specimens were cannulated with a catheter connected to a syringe, and washed thrice with 4 mL saline. After centrifugation of the collected BALF, total cells were counted and differentials (macrophages, neutrophils, eosinophils, lymphocytes, and epithelial cells) were analyzed.
2.6.
Histological measurements
Digitally photographed sections were analyzed using a quantification software (ImageJ, U.S. NIH, Bethesda, MD, USA). The large and small airways were examined.
2.6.1.
Small airways
In sections of L specimens, airways with internal diameters o1000 μm were examined. Two or three airways at a magnification of 200 were photographed, and the pathological changes were assessed (Fig. 2b). Goblet cells were unavailable because PAS staining was negative.
2.7.
Statistical analysis
Data are expressed as mean7SE. p valueso0.05 were considered statistically significant. Multiple groups were compared by one-way ANOVA, followed by Fisher's PLSD test. The number of coughs on days 1–22 among the three groups was analyzed by the Kruskal–Wallis test. Correlations were analyzed using Pearson's correlation coefficient. The difference in the number of coughs between days 1 and 22, and the difference in the mean number of coughs on days 1 and 22 from that on days 4–20 between the CAþDEX group and the SA group were analyzed using a paired t-test. Changes in cough numbers in the CA group were analyzed with repeated measures ANOVA.
Sacrifice and tissue processing
On day 25, all guinea pigs were anesthetized with sodium pentobarbital i.p. The trachea and lungs were removed together and ligated at the origin of the left main bronchus. The sample was divided into proximal trachea (T), distal trachea with right lung (R), and left lung (L), by cutting the trachea at 1 cm proximal from the bifurcation and left main bronchus just distal to the ligature. After fixation with 4% paraformaldehyde, 3-μm-thick sections were cut at the distal end of T and at 1 cm distal from the original cut end of L.
2.5.
2.6.2.
Large airways
Sections from T specimens were examined. For each animal, three images (200 μm square) were photographed at a
3.
Results
3.1.
Change in number of coughs
The number of coughs in the CA group increased during the experimental period (po0.05). This was first recognized on day 13, and was seen consistently and significantly thereafter until day 22 (Fig. 3a). In the CAþDEX and SA groups, the number of coughs did not differ between days 1 and 22, while the mean number of coughs on days 4–20 was lower than that on days 1 and 22 (p¼ 0.025 and 0.011, respectively; Fig. 3b–c). Saline induced no coughs. The number of coughs differed significantly on days 4–20 except for day 8 among the three groups, while those on day 1 and on day 22 did not differ (Fig. 3, bottom). The mean number of coughs on days 4–20 differed among the three groups (7.871.7 for the CA group, 2.470.7 for the CAþDEX group, and 070 for the SA group, p ¼0.0067), and was higher in the CA group than in the CAþDEX group (p¼ 0.046) and the SA group (p¼ 0.0035). The difference between the latter two groups was also significant (p ¼0.0003).
3.2.
Inflammatory cells in BALF
The CA group was divided into two subgroups as above. The four groups (CA-F, CA-I, SA, and CAþDEX) did not differ in terms of neutrophil or eosinophil percentage or count (Figs. 4–5). The numbers of both cell types were unrelated to cough index in the
Please cite this article as: Nakaji H, et al. Airway remodeling associated with cough hypersensitivity as a consequence of persistent cough: An experimental study. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.06.005
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Goblet cells
Epithelium
Goblet cells
Basement membrane ASM
Submucosa
50 μm
Cartilage
Adventitia Smooth muscle
Basement membrane Epithelium 100 μm Fig. 2 – Images of large and small airways and their schema. (a) Large airway (trachea) airway smooth muscle (ASM) index (%): [ASM area/submucosal area] 100 Epithelial thickness (μm): epithelial area (including goblet cells)/length of basement membrane Goblet cell index (%): [PAS-positive area/epithelial area] 100. (b) Small airway ASM index (%): [ASM area/total wall area] 100 Epithelial thickness (μm): Epithelial area/length of basement membrane. CA group, or to the mean number of coughs on days 1 and 22 in the CAþDEX or SA group (data not shown). Total cell count, and percentage or count of macrophages, lymphocytes, and epithelial cells did not differ among the four groups (data not shown).
3.3.
Histology
3.3.1.
Large airways
Neither ASM index, epithelial thickness, or goblet cell index differed among the four groups (Fig. 6). Cough index was unrelated to any pathological parameters in the two CA subgroups combined (data not shown).
3.3.2.
Small airways
ASM index was higher in the CA-F subgroup than in the other groups, in which it was similar (Fig. 7a). Cough index correlated highly with ASM index in the two CA subgroups combined (Fig. 7b), but not in the other groups (data not shown). Epithelial thickness was similar among the groups (Fig. 7c) and was unrelated to cough index in the two CA subgroups combined (data not shown). Fig. 8 shows representative images.
4.
Discussion
We demonstrated that repeated cough induced by CA exposure was associated with heightened cough reflex sensitivity and airway remodeling (increased ASM) in small airways, without evidence of cellular inflammation, in guinea pigs.
Previous animal studies of cough mostly investigated the effects of allergen challenge or drug intervention on cough sensitivity. To our knowledge, this is the first study of the effect of repeated cough on cough reflex sensitivity and airway pathology. In asthma, airway remodeling has been ascribed to eosinophilic inflammation, but noninflammatory mechanisms have also been suggested [5]. An alteration of the relationships between the epithelium and myofibroblasts could lead to structural changes, as could mechanical stimuli. Bronchoconstriction produces folding of the airway wall, inducing stress on the epithelium and leading it to produce fibrogenic factors [11]. Bronchoconstriction without additional inflammation, elicited by inhaled methacholine, induces airway remodeling changes in asthmatics [19]. Likewise, the repetitive mechanical and physical effects of coughing on the airways may also result in airway remodeling. Cough consists of deep inspiration followed by forced rapid expiration [12]. The airways are compressed and narrowed during such expiration; the narrowing results from a transmural gradient between the extraluminal and intraluminal pressures [12]. Immediately before the expiration phase, both extraluminal and intraluminal pressures of the large airways become positive. In the expiration phase, intraluminal pressure suddenly decreases to ambient pressure, resulting in a large transmural pressure that compresses the airways [12,14]. Various remodeling changes observed in chronic cough patients [2,7] indicate the pathogenetic role of such nonspecific effects of coughing on remodeling, but it is also interesting that remodeling changes correlated with cough reflex sensitivity [2,7].
Please cite this article as: Nakaji H, et al. Airway remodeling associated with cough hypersensitivity as a consequence of persistent cough: An experimental study. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.06.005
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Fig. 3 – Changes in number of coughs during serial challenges in the three groups (a) CA group: the number of coughs significantly increased on day 13, and persisted until day 22. (b) CAþDEX group: there was no significant difference in the mean number of coughs between day 1 and day 22. (c) SA group: there was no significant difference in the number of coughs between day 1 and day 22. Data plots and bars are presented as the mean number of coughs7SE for each exposure. p values for differences between the three groups by ANOVA are presented at the bottom of Fig. 3. *po 0.05 by repeated measures ANOVA. †NS: not significant by paired t-test.
In this study, increased cough reflex sensitivity after repeated CA exposure was associated with an increase in small airways ASM. Cough index correlated with ASM index. These are consistent with our clinical observations [2,7] and a biopsy study showing increased expression of TGF-beta 1 and transcription factor Smad 2/3 in ASM of NACC patients [13]. Chronic cough thus may induce the production of growth factors, leading to remodeling. An increase in ASM might also have resulted in cough hypersensitivity. Increased expression of capsaicin receptor TRPV-1 has been shown specifically in ASM of NACC patients [20]. ASM produces various mediators, including prostaglandin E2 (PGE2)[21], which has a tussive action and shows increased sputum levels in chronic cough patients [22]. TGF-beta 1 stimulates PGE2 production from ASM cells [23]. The series of changes observed in ASM of
NACC patients (increased volume [2] and expression of TGFbeta 1 [13] and TRPV-1 [20]) in conjunction with our results may imply novel mechanisms of cough involving ASM. The reason why ASM changes were confined to small airways in our study is uncertain, but in asthma, spirometry indices of small airway obstruction emerge earlier in the disease [24]. Mechanical and physical effects of coughing may have a greater effect on small airways, which lack cartilage and have thinner walls and smaller lumens, and are therefore more likely to collapse. Cellular inflammation was absent in our model. Hara and colleagues induced neutrophilic inflammation and cough reflex hypersensitivity by repeated pressure stress on airways, supporting an assumption that the trauma of coughing may induce non-specific inflammation [14]. However, BAL
Please cite this article as: Nakaji H, et al. Airway remodeling associated with cough hypersensitivity as a consequence of persistent cough: An experimental study. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.06.005
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Fig. 4 – Inflammatory cells in BALF in the four groups (a: neutrophils, b: eosinophils). There was no difference in the number of neutrophils or eosinophils among the groups. SA: saline group (n ¼8), CA: citric acid group; CA-I: infrequent cough subgroup (n¼ 9), CA-F: frequent cough subgroup (n ¼ 9), CAþDEX: CAþdextromethorphan group (n ¼7).
Fig. 5 – Inflammatory cells in BALF (a: neutrophils, b: eosinophils). The four groups did not differ in terms of neutrophil or eosinophil count. SA: saline group, CA: citric acid group; CA-I: infrequent cough subgroup, CA-F: frequent cough subgroup, CAþDEX: CAþdextromethorphan group.
neutrophilia disappeared 12 h after the pressure stress. We examined BAL 72 h after the final CA challenge, aiming to detect chronic rather than acute inflammation, which is more relevant to airway remodeling. We exposed animals to CA to induce cough and investigate the effect of repeated prolonged cough. Other methods of cough induction or mimicking (mechanical stimuli [25] or pressure changes [14]) were unsuitable, since they require anesthesia, surgical procedures, and mechanical ventilation, and thus cannot be repeated chronically. One might assume that repeated CA challenges could result in tachyphylaxis and decreased cough response, but this was unlikely to happen, because repeated challenges with 0.5 M CA at an interval of at least 2 days do not elicit tachyphylaxis [16]. Recently, guinea pigs repeatedly exposed to CA every 4 days until day 12 showed no change in the number of coughs induced [17]. These results were in broad agreement with ours, in which cough reflex hypersensitivity was first recognized on day 13 and persisted until day 22. Cough reflex sensitivity may thus increase when cough is induced for a longer period.
Other issues associated with the use of CA are its bronchoactive properties, and possible direct effects on airway inflammation/pathology. In our study, guinea pigs were treated with 0.05 mg/kg terbutaline prior to CA exposure, which effectively inhibits bronchoconstriction without affecting cough [16]. In the CAþDEX group, pathological changes were similar to those in the SA and CA-I groups, and cough reflex sensitivity was unchanged. Given that DEX is a centrally acting antitussive with no recognized anti-inflammatory effect, potential effects of CA on airways must have remained in the CAþDEX group, while cough was significantly suppressed. The increased cough reflex sensitivity and ASM induced in the CA group should therefore be ascribed to repeated coughs rather than other effects of CA.
5.
Conclusion
Repeated cough itself may induce airway remodeling associated with cough reflex hypersensitivity. Cough may lead to
Please cite this article as: Nakaji H, et al. Airway remodeling associated with cough hypersensitivity as a consequence of persistent cough: An experimental study. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.06.005
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Fig. 6 – Histological indices in the trachea (a: ASM index, b: epithelial thickness, c: goblet cell index). There were no differences in any of the pathological indices among the groups. ASM: airway smooth muscle. See Fig. 4 for other abbreviations.
Fig. 7 – Indices of airway remodeling in small airways (a) ASM index (%): ASM index was higher in the CA-F subgroup than in the other groups. (b) Relationship between ASM index and cough index in the CA group: Cough index highly correlated with ASM index in the two CA subgroups combined. (c) Epithelial thickness (lm): Epithelial thickness was similar among the groups. *p: statistically significant by Fisher's PLSD post-hoc test. See Figs. 4 and 6 for abbreviations.
Please cite this article as: Nakaji H, et al. Airway remodeling associated with cough hypersensitivity as a consequence of persistent cough: An experimental study. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.06.005
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Fig. 8 – Representative photomicrographs of small airways of animals. The photomicrographs from the (a) SA group, (b) CA-I subgroup, and (c) CA-F subgroup are shown. A marked increase in ASM is noted in the CA-F subgroup (arrows). airway remodeling, and remodeling may be a cause of cough reflex hypersensitivity or chronic cough. The concomitance of both mechanisms may lead to a positive feedback mechanism for cough persistence, possibly involved in chronic intractable cough [10]. Another consequence of persistent coughing and resultant airway remodeling may be irreversible airflow obstruction, as suggested by a study of patients with intractable nonasthmatic chronic cough [9]. Despite the often-difficult treatment of chronic cough, clinicians should make every effort to resolve coughing, not only for the sake of patients’ quality of life, but also to avoid these undesirable clinical and functional consequences.
Conflict of interest Akio Niimi received lecture fees from AstraZeneca, Astellas and Kyorin, and received research funding from AstraZeneca and Astellas. Isao Ito received a research grant. Tsuyoshi Oguma received research funding from Olympus Corporation. Kazuo Chin received lecture fees from Teijin HomeMedical belongs to endowed departments sponsored by Teijin Pharma, Philips & Respironics, Fukuda Denshi and Fukuda Lifetec Keiji.
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Please cite this article as: Nakaji H, et al. Airway remodeling associated with cough hypersensitivity as a consequence of persistent cough: An experimental study. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.06.005
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Please cite this article as: Nakaji H, et al. Airway remodeling associated with cough hypersensitivity as a consequence of persistent cough: An experimental study. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.06.005
Contents lists available at ScienceDirect
Respiratory Investigation journal homepage: www.elsevier.com/locate/resinv
Original article
Airway remodeling associated with cough hypersensitivity as a consequence of persistent cough: An experimental study Hitoshi Nakajia, Akio Niimi, MD, PhDa,b,n, Hirofumi Matsuokaa, Toshiyuki Iwataa, Shilei Cuia, Hisako Matsumotoa, Isao Itoa, Tsuyoshi Ogumaa, Kojiro Otsukaa, Tomoshi Takedaa, Hideki Inouea, Tomoko Tajiria, Tadao Nagasakia, Yoshihiro Kanemitsua,b, Kazuo Chinc, Michiaki Mishimaa a
Department of Respiratory Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan Department of Respiratory Medicine, Allergy and Clinical Immunology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-Cho, Muzuho-Ku, Nagoya 467-8601, Japan c Department of Respiratory Care and Sleep Control Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan b
art i cle i nfo
ab st rac t
Article history:
Background: Chronic cough involves airway remodeling associated with cough reflex
Received 16 September 2015
hypersensitivity. Whether cough itself induces these features remains unknown.
Received in revised form
Methods: Guinea pigs were assigned to receive treatment with citric acid (CA), saline (SA), or
31 May 2016
CAþdextromethorphan (DEX). All animals were exposed to 0.5 M CA on days 1 and 22. On days
Accepted 27 June 2016
4–20, the CA and CAþDEX groups were exposed to CA, and the SA group to saline thrice weekly, during which the CAþDEX group was administered DEX pretreatment to inhibit cough.
Keywords: Chronic cough Airway remodeling Cough reflex hypersensitivity Airway smooth muscle Mechanical stress
The number of coughs was counted during each 10-min CA or SA exposure. Terbutaline premedication was started to prevent bronchoconstriction. Bronchoalveolar lavage and pathology were examined on day 25. Average cough number for 10 CA exposures was examined as “cough index” in the CA group, which was divided into frequent (cough index45) and infrequent (o5) cough subgroups for lavage and pathology analysis. Results: The number of coughs significantly increased in the CA group from day 13 onwards. In the CAþDEX and SA groups, the number of coughs did not differ between days 1 and 22, while average number of coughs during days 4–20 was significantly lower than at days 1 and 22. Bronchoalveolar cell profiles were similar among the four groups. The smooth muscle area of small airways was significantly greater in the frequent-cough subgroup than in the other groups (in which it was similar), and highly correlated with cough index in CA group. Conclusion: Repeated cough induces airway smooth muscle remodeling associated with cough reflex hypersensitivity. & 2016 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.
n Corresponding author at: Department of Respiratory Medicine, Allergy and Clinical Immunology, Nagoya City University Graduate School of Medical Sciences, 1 Kawasumi, Mizuho-Cho, Muzuho-Ku, Nagoya 467-8601, Japan. Tel.: þ81 52 853 8214; fax: þ81 52 852 0849.
http://dx.doi.org/10.1016/j.resinv.2016.06.005 2212-5345/& 2016 The Japanese Respiratory Society. Published by Elsevier B.V. All rights reserved.
Please cite this article as: Nakaji H, et al. Airway remodeling associated with cough hypersensitivity as a consequence of persistent cough: An experimental study. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.06.005
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1.
Introduction
Chronic cough is an important clinical problem. Asthmarelated diagnoses such as cough-variant asthma involve eosinophilic airway inflammation [1,2]. Cough may also be ascribed to non-asthma-related diagnoses, such as gastroesophageal reflux disease, but in some patients, it remains unexplained despite thorough investigations. Such nonasthmatic chronic cough (NACC) also involves airway inflammation characterized by increased neutrophils, mast cells, or lymphocytes [2–4]. Airway remodeling is another feature of asthma [5]. The pathological changes of remodeling in cough-variant asthma include subepithelial thickening, goblet-cell hyperplasia, vascular proliferation, and airway-wall thickening [2,6,7]. Eosinophilic bronchitis also involves similar changes [8]. These changes may be ascribed to eosinophil-derived fibrogenic mediators such as transforming growth factor (TGF)-beta, and may result in accelerated lung function decline in intractable cases [1], as in asthma [5]. Interestingly, cases of NACC without airway eosinophilia also show features of remodeling, such as subepithelial thickening, increase of airway smooth muscle (ASM), goblet cells and vessels, epithelial shedding, airway-wall thickening [2,3,7], and irreversible airflow obstruction [9]. Especially noteworthy is the increase of ASM despite absence of airway hyperresponsiveness [10]. The pathogenesis and functional consequences of airway remodeling are not precisely known in chronic cough, especially for NACC [10]. Airway epithelium exposed to compressive stresses, an effect mimicking acute bronchoconstriction in asthma, increases its expression of genes relevant to remodeling, such as TGF-beta 2. Synthesis of extracellular matrix by cocultured fibroblasts is also increased [11]. Cough may also exert mechanical stress on the airway mucosa [12]. In patients with NACC, TGF-beta 1 levels and neutrophil counts in bronchoalveolar lavage fluid (BALF), as well as subepithelial thickness, were increased [13]. TGF-beta 1 expression in the epithelium and ASM were also increased, and TGF-beta 1 levels correlated with subepithelial thickness [13]. In NACC patients, the degree of remodeling (as indicated by goblet-cell hyperplasia, epithelial shedding, and airwaywall thickening) correlated with cough reflex sensitivity to capsaicin [2,7]. Persistent cough may thus result in airway remodeling, in the presence or absence of inflammation. This may lead to cough reflex hypersensitivity that might induce further cough, creating a vicious cycle [10]. A guinea pig model of airway collapse mimicking cough has been reported, induced by rapid repetition of negative pressure applied to the airways of artificially ventilated animals [14]. Capsaicin sensitivity and BALF neutrophils Abbreviations: CA,
citric acid; ASM,
airway smooth muscle; BAL,
transiently increased 6 hours after stimulus, and these features correlated with each other. However, this was a short-term experiment, failing to examine remodeling changes [14]. We investigated whether repeated induction of cough in awake guinea pigs is associated with airway remodeling and cough reflex hypersensitivity. Cough was induced by citric acid (CA) exposure, which is unlikely to induce tachyphylaxis and therefore useful for repeated cough induction and measurements [15–17].
2.
Materials and methods
2.1.
Animals
Male Dunkin-Hartley guinea pigs were obtained and quarantined. All animal procedures conformed to Kyoto University's regulations on animal experimentation (Med Kyo 04449; approval date: Nov 25, 2004).
2.2.
Experimental design
Naïve guinea pigs were assigned to one of three treatment groups: CA group, saline (SA) group, or CAþdextromethorphan (DEX) group (Fig. 1). On days 1 and 22, guinea pigs in all groups were exposed to CA (0.5 M via nebulizer for 10 min) and the number of coughs was counted. From days 4 through 20, the CA and CAþDEX groups were exposed to 0.5 M CA, and the SA group to 0.9% saline for 10 min, three times weekly, at 2- or 3-day intervals (eight times in total). During this period, the CAþDEX group was treated with 30 mg/kg DEX intra-peritoneally (i.p.) 30 min prior to CA exposure, to inhibit cough. DEX was not administered on days 1 and 22. In the CA group, the mean number of coughs for 10 CA exposures was calculated as the “cough index” for each animal. The CA group was divided into a frequent cough subgroup (CA-F: Cough index Z5) and an infrequent cough subgroup (CA-I: Cough index o5) for analysis of BAL and pathology results. The median number of cough index (¼ 5) of the 18 animals in the CA group was used as the cut off level.
2.3.
System for cough measurement
Guinea pigs were placed in a chamber allowing free movement and equipped with an internal microphone and a pressure transducer [18]. They were connected to an Amplifier Interface Unit series pre-amplifier (EMMS, Bordon, UK). The chamber was provided airflow by a Basic Flow Supplier AIR 200 (EMMS) at 1500 mL/min. The changes in airflow induced by respiration and cough were recorded by a pneumotachograph. Cough sounds were amplified and recorded bronchoalveolar lavage; BALF,
BAL fluid; PAS,
periodic acid-
Schiff; TGF, transforming growth factor; NACC, non-asthmatic chronic cough E-mail addresses: [email protected] (H. Nakaji), [email protected] (A. Niimi), [email protected] (H. Matsuoka), [email protected] (T. Iwata), [email protected] (S. Cui), [email protected] (H. Matsumoto), [email protected] (I. Ito), [email protected] (T. Oguma), [email protected] (K. Otsuka), [email protected] (T. Takeda), [email protected] (H. Inoue), [email protected] (T. Tajiri), [email protected] (T. Nagasaki), [email protected] (Y. Kanemitsu), [email protected] (K. Chin), [email protected] (M. Mishima).
Please cite this article as: Nakaji H, et al. Airway remodeling associated with cough hypersensitivity as a consequence of persistent cough: An experimental study. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.06.005
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magnification of 400 in one section and examined. Each image was separated by 4200 mm. ASM area and submucosal area were measured with hematoxylin and eosin (H&E) staining. Epithelial area, goblet cell area, and length of basement membrane were measured using periodic acid-Schiff (PAS)-stained slices. Areas of goblet cells were measured as the total of PAS-positive areas. Each index was calculated (Fig. 2a) from an average of three images for each animal. Fig. 1 – Experimental protocol. Guinea pigs in all groups were exposed to CA (0.5 M) on days 1 and 22 for the evaluation of cough sensitivity. On days 4 through 20, the CA group (n ¼18) and CAþDEX group (n ¼9) were exposed to CA, and the SA group (n ¼9) to saline three times per week. During this period, the CAþDEX group was treated with 30 mg/kg DEX i.p. 30 min prior to CA exposure to inhibit cough. Terbutaline (0.05 mg/kg) was administered i.p. prior to each CA or SA exposure in all groups. CA: Citric acid, SA: Saline, DEX: Dextromethorphan.
by microphone. Guinea pig behavior was captured with an external camera. Data acquisition was performed with eDacq software (EMMS) [18]. After dosing with 0.05 mg/kg terbutaline i.p. to prevent bronchoconstriction [16,18], 0.5 M CA or 0.9% saline was delivered using a nebulizer with an output of 0.5 mL/min for 10 min (Fig. 1). The number of coughs over each 10-min exposure was counted, distinguished from sneezing by guinea pig posture, pressure changes, and sound.
2.4.
BAL
R specimens were cannulated with a catheter connected to a syringe, and washed thrice with 4 mL saline. After centrifugation of the collected BALF, total cells were counted and differentials (macrophages, neutrophils, eosinophils, lymphocytes, and epithelial cells) were analyzed.
2.6.
Histological measurements
Digitally photographed sections were analyzed using a quantification software (ImageJ, U.S. NIH, Bethesda, MD, USA). The large and small airways were examined.
2.6.1.
Small airways
In sections of L specimens, airways with internal diameters o1000 μm were examined. Two or three airways at a magnification of 200 were photographed, and the pathological changes were assessed (Fig. 2b). Goblet cells were unavailable because PAS staining was negative.
2.7.
Statistical analysis
Data are expressed as mean7SE. p valueso0.05 were considered statistically significant. Multiple groups were compared by one-way ANOVA, followed by Fisher's PLSD test. The number of coughs on days 1–22 among the three groups was analyzed by the Kruskal–Wallis test. Correlations were analyzed using Pearson's correlation coefficient. The difference in the number of coughs between days 1 and 22, and the difference in the mean number of coughs on days 1 and 22 from that on days 4–20 between the CAþDEX group and the SA group were analyzed using a paired t-test. Changes in cough numbers in the CA group were analyzed with repeated measures ANOVA.
Sacrifice and tissue processing
On day 25, all guinea pigs were anesthetized with sodium pentobarbital i.p. The trachea and lungs were removed together and ligated at the origin of the left main bronchus. The sample was divided into proximal trachea (T), distal trachea with right lung (R), and left lung (L), by cutting the trachea at 1 cm proximal from the bifurcation and left main bronchus just distal to the ligature. After fixation with 4% paraformaldehyde, 3-μm-thick sections were cut at the distal end of T and at 1 cm distal from the original cut end of L.
2.5.
2.6.2.
Large airways
Sections from T specimens were examined. For each animal, three images (200 μm square) were photographed at a
3.
Results
3.1.
Change in number of coughs
The number of coughs in the CA group increased during the experimental period (po0.05). This was first recognized on day 13, and was seen consistently and significantly thereafter until day 22 (Fig. 3a). In the CAþDEX and SA groups, the number of coughs did not differ between days 1 and 22, while the mean number of coughs on days 4–20 was lower than that on days 1 and 22 (p¼ 0.025 and 0.011, respectively; Fig. 3b–c). Saline induced no coughs. The number of coughs differed significantly on days 4–20 except for day 8 among the three groups, while those on day 1 and on day 22 did not differ (Fig. 3, bottom). The mean number of coughs on days 4–20 differed among the three groups (7.871.7 for the CA group, 2.470.7 for the CAþDEX group, and 070 for the SA group, p ¼0.0067), and was higher in the CA group than in the CAþDEX group (p¼ 0.046) and the SA group (p¼ 0.0035). The difference between the latter two groups was also significant (p ¼0.0003).
3.2.
Inflammatory cells in BALF
The CA group was divided into two subgroups as above. The four groups (CA-F, CA-I, SA, and CAþDEX) did not differ in terms of neutrophil or eosinophil percentage or count (Figs. 4–5). The numbers of both cell types were unrelated to cough index in the
Please cite this article as: Nakaji H, et al. Airway remodeling associated with cough hypersensitivity as a consequence of persistent cough: An experimental study. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.06.005
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Goblet cells
Epithelium
Goblet cells
Basement membrane ASM
Submucosa
50 μm
Cartilage
Adventitia Smooth muscle
Basement membrane Epithelium 100 μm Fig. 2 – Images of large and small airways and their schema. (a) Large airway (trachea) airway smooth muscle (ASM) index (%): [ASM area/submucosal area] 100 Epithelial thickness (μm): epithelial area (including goblet cells)/length of basement membrane Goblet cell index (%): [PAS-positive area/epithelial area] 100. (b) Small airway ASM index (%): [ASM area/total wall area] 100 Epithelial thickness (μm): Epithelial area/length of basement membrane. CA group, or to the mean number of coughs on days 1 and 22 in the CAþDEX or SA group (data not shown). Total cell count, and percentage or count of macrophages, lymphocytes, and epithelial cells did not differ among the four groups (data not shown).
3.3.
Histology
3.3.1.
Large airways
Neither ASM index, epithelial thickness, or goblet cell index differed among the four groups (Fig. 6). Cough index was unrelated to any pathological parameters in the two CA subgroups combined (data not shown).
3.3.2.
Small airways
ASM index was higher in the CA-F subgroup than in the other groups, in which it was similar (Fig. 7a). Cough index correlated highly with ASM index in the two CA subgroups combined (Fig. 7b), but not in the other groups (data not shown). Epithelial thickness was similar among the groups (Fig. 7c) and was unrelated to cough index in the two CA subgroups combined (data not shown). Fig. 8 shows representative images.
4.
Discussion
We demonstrated that repeated cough induced by CA exposure was associated with heightened cough reflex sensitivity and airway remodeling (increased ASM) in small airways, without evidence of cellular inflammation, in guinea pigs.
Previous animal studies of cough mostly investigated the effects of allergen challenge or drug intervention on cough sensitivity. To our knowledge, this is the first study of the effect of repeated cough on cough reflex sensitivity and airway pathology. In asthma, airway remodeling has been ascribed to eosinophilic inflammation, but noninflammatory mechanisms have also been suggested [5]. An alteration of the relationships between the epithelium and myofibroblasts could lead to structural changes, as could mechanical stimuli. Bronchoconstriction produces folding of the airway wall, inducing stress on the epithelium and leading it to produce fibrogenic factors [11]. Bronchoconstriction without additional inflammation, elicited by inhaled methacholine, induces airway remodeling changes in asthmatics [19]. Likewise, the repetitive mechanical and physical effects of coughing on the airways may also result in airway remodeling. Cough consists of deep inspiration followed by forced rapid expiration [12]. The airways are compressed and narrowed during such expiration; the narrowing results from a transmural gradient between the extraluminal and intraluminal pressures [12]. Immediately before the expiration phase, both extraluminal and intraluminal pressures of the large airways become positive. In the expiration phase, intraluminal pressure suddenly decreases to ambient pressure, resulting in a large transmural pressure that compresses the airways [12,14]. Various remodeling changes observed in chronic cough patients [2,7] indicate the pathogenetic role of such nonspecific effects of coughing on remodeling, but it is also interesting that remodeling changes correlated with cough reflex sensitivity [2,7].
Please cite this article as: Nakaji H, et al. Airway remodeling associated with cough hypersensitivity as a consequence of persistent cough: An experimental study. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.06.005
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Fig. 3 – Changes in number of coughs during serial challenges in the three groups (a) CA group: the number of coughs significantly increased on day 13, and persisted until day 22. (b) CAþDEX group: there was no significant difference in the mean number of coughs between day 1 and day 22. (c) SA group: there was no significant difference in the number of coughs between day 1 and day 22. Data plots and bars are presented as the mean number of coughs7SE for each exposure. p values for differences between the three groups by ANOVA are presented at the bottom of Fig. 3. *po 0.05 by repeated measures ANOVA. †NS: not significant by paired t-test.
In this study, increased cough reflex sensitivity after repeated CA exposure was associated with an increase in small airways ASM. Cough index correlated with ASM index. These are consistent with our clinical observations [2,7] and a biopsy study showing increased expression of TGF-beta 1 and transcription factor Smad 2/3 in ASM of NACC patients [13]. Chronic cough thus may induce the production of growth factors, leading to remodeling. An increase in ASM might also have resulted in cough hypersensitivity. Increased expression of capsaicin receptor TRPV-1 has been shown specifically in ASM of NACC patients [20]. ASM produces various mediators, including prostaglandin E2 (PGE2)[21], which has a tussive action and shows increased sputum levels in chronic cough patients [22]. TGF-beta 1 stimulates PGE2 production from ASM cells [23]. The series of changes observed in ASM of
NACC patients (increased volume [2] and expression of TGFbeta 1 [13] and TRPV-1 [20]) in conjunction with our results may imply novel mechanisms of cough involving ASM. The reason why ASM changes were confined to small airways in our study is uncertain, but in asthma, spirometry indices of small airway obstruction emerge earlier in the disease [24]. Mechanical and physical effects of coughing may have a greater effect on small airways, which lack cartilage and have thinner walls and smaller lumens, and are therefore more likely to collapse. Cellular inflammation was absent in our model. Hara and colleagues induced neutrophilic inflammation and cough reflex hypersensitivity by repeated pressure stress on airways, supporting an assumption that the trauma of coughing may induce non-specific inflammation [14]. However, BAL
Please cite this article as: Nakaji H, et al. Airway remodeling associated with cough hypersensitivity as a consequence of persistent cough: An experimental study. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.06.005
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Fig. 4 – Inflammatory cells in BALF in the four groups (a: neutrophils, b: eosinophils). There was no difference in the number of neutrophils or eosinophils among the groups. SA: saline group (n ¼8), CA: citric acid group; CA-I: infrequent cough subgroup (n¼ 9), CA-F: frequent cough subgroup (n ¼ 9), CAþDEX: CAþdextromethorphan group (n ¼7).
Fig. 5 – Inflammatory cells in BALF (a: neutrophils, b: eosinophils). The four groups did not differ in terms of neutrophil or eosinophil count. SA: saline group, CA: citric acid group; CA-I: infrequent cough subgroup, CA-F: frequent cough subgroup, CAþDEX: CAþdextromethorphan group.
neutrophilia disappeared 12 h after the pressure stress. We examined BAL 72 h after the final CA challenge, aiming to detect chronic rather than acute inflammation, which is more relevant to airway remodeling. We exposed animals to CA to induce cough and investigate the effect of repeated prolonged cough. Other methods of cough induction or mimicking (mechanical stimuli [25] or pressure changes [14]) were unsuitable, since they require anesthesia, surgical procedures, and mechanical ventilation, and thus cannot be repeated chronically. One might assume that repeated CA challenges could result in tachyphylaxis and decreased cough response, but this was unlikely to happen, because repeated challenges with 0.5 M CA at an interval of at least 2 days do not elicit tachyphylaxis [16]. Recently, guinea pigs repeatedly exposed to CA every 4 days until day 12 showed no change in the number of coughs induced [17]. These results were in broad agreement with ours, in which cough reflex hypersensitivity was first recognized on day 13 and persisted until day 22. Cough reflex sensitivity may thus increase when cough is induced for a longer period.
Other issues associated with the use of CA are its bronchoactive properties, and possible direct effects on airway inflammation/pathology. In our study, guinea pigs were treated with 0.05 mg/kg terbutaline prior to CA exposure, which effectively inhibits bronchoconstriction without affecting cough [16]. In the CAþDEX group, pathological changes were similar to those in the SA and CA-I groups, and cough reflex sensitivity was unchanged. Given that DEX is a centrally acting antitussive with no recognized anti-inflammatory effect, potential effects of CA on airways must have remained in the CAþDEX group, while cough was significantly suppressed. The increased cough reflex sensitivity and ASM induced in the CA group should therefore be ascribed to repeated coughs rather than other effects of CA.
5.
Conclusion
Repeated cough itself may induce airway remodeling associated with cough reflex hypersensitivity. Cough may lead to
Please cite this article as: Nakaji H, et al. Airway remodeling associated with cough hypersensitivity as a consequence of persistent cough: An experimental study. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.06.005
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Fig. 6 – Histological indices in the trachea (a: ASM index, b: epithelial thickness, c: goblet cell index). There were no differences in any of the pathological indices among the groups. ASM: airway smooth muscle. See Fig. 4 for other abbreviations.
Fig. 7 – Indices of airway remodeling in small airways (a) ASM index (%): ASM index was higher in the CA-F subgroup than in the other groups. (b) Relationship between ASM index and cough index in the CA group: Cough index highly correlated with ASM index in the two CA subgroups combined. (c) Epithelial thickness (lm): Epithelial thickness was similar among the groups. *p: statistically significant by Fisher's PLSD post-hoc test. See Figs. 4 and 6 for abbreviations.
Please cite this article as: Nakaji H, et al. Airway remodeling associated with cough hypersensitivity as a consequence of persistent cough: An experimental study. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.06.005
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Fig. 8 – Representative photomicrographs of small airways of animals. The photomicrographs from the (a) SA group, (b) CA-I subgroup, and (c) CA-F subgroup are shown. A marked increase in ASM is noted in the CA-F subgroup (arrows). airway remodeling, and remodeling may be a cause of cough reflex hypersensitivity or chronic cough. The concomitance of both mechanisms may lead to a positive feedback mechanism for cough persistence, possibly involved in chronic intractable cough [10]. Another consequence of persistent coughing and resultant airway remodeling may be irreversible airflow obstruction, as suggested by a study of patients with intractable nonasthmatic chronic cough [9]. Despite the often-difficult treatment of chronic cough, clinicians should make every effort to resolve coughing, not only for the sake of patients’ quality of life, but also to avoid these undesirable clinical and functional consequences.
Conflict of interest Akio Niimi received lecture fees from AstraZeneca, Astellas and Kyorin, and received research funding from AstraZeneca and Astellas. Isao Ito received a research grant. Tsuyoshi Oguma received research funding from Olympus Corporation. Kazuo Chin received lecture fees from Teijin HomeMedical belongs to endowed departments sponsored by Teijin Pharma, Philips & Respironics, Fukuda Denshi and Fukuda Lifetec Keiji.
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Please cite this article as: Nakaji H, et al. Airway remodeling associated with cough hypersensitivity as a consequence of persistent cough: An experimental study. Respiratory Investigation (2016), http://dx.doi.org/10.1016/j.resinv.2016.06.005