Increased Expression of Transient Receptor Potential Vanilloid-1 in Airway Nerves of Chronic Cough

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1 Increased Expression of Transient Receptor Potential Vanilloid-1 in Airway Nerves of Chronic Cough David A. Groneberg, Akio Niimi, Q. Thai Dinh, Borja Cosio, Mark Hew, Axel Fischer, and K. Fan Chung Division of Allergy Research, Department of Pediatric Pneumology and Immunology, Charité Faculty of Medicine, Humboldt-Universität zu Berlin, Berlin, Germany; and Thoracic Medicine, National Heart and Lung Institute, Imperial College, London, United Kingdom Transient receptor potential vanniloid-1 (TRPV-1) mediates the cough response induced by the pepper extract capsaicin and is expressed in sensory nerves that innervate the airway wall. We determined the expression of TRPV-1 in the airways of patients with chronic persistent cough of diverse causes and with an enhanced capsaicin cough response. We obtained airway mucosal biopsies by fiberoptic bronchoscopy in 29 patients with chronic cough and 16 healthy volunteers without a cough. Immunostaining for nerve profiles with anti protein gene product (PGP)-9.5 antibody showed no increase in nerve profiles in the airway epithelium of patients with chronic cough; however, with an anti TRPV-1 antibody, there was a fivefold increase of TRPV-1 staining nerve profiles (p 0.001). There was a significant correlation between capsaicin tussive response and the number of TRPV-1 positive nerves within the patients with cough. Our findings indicate that TRPV-1 receptors may contribute to an enhanced cough reflex and the cough response in chronic persistent cough of diverse causes. Keywords: airway nerves; capsaicin; cough; transient receptor potential vanniloid-1 Chronic cough that persists over many months is a disorder that is often distressing and debilitating. In many patients, this may be associated with asthma or related conditions such as coughvariant asthma and eosinophilic bronchitis, gastroesophageal reflux disease, and rhinosinusitis (1, 2). Very often, no associated cause can be determined, as specific treatments do not control the cough (3). The cough reflex measured with inhalation of the pungent ingredient of chili peppers, capsaicin, is usually augmented in patients with chronic persistent cough (4). Little is known about the abnormalities of the cough receptor itself in these patients with chronic persistent cough. Nerve profiles in the airway submucosa of patients with chronic persistent cough are not increased, although the number of the neuropeptide calcitonin gene-related peptide (CGRP) containing nerve profiles were increased (5). It has been hypothesized that cough sensitization may occur either centrally within the brain stem or spinal cord afferents or peripherally in cough receptors. Cough itself is mediated by the activation of myelinated A fibers, as well as possibly unmyelinated C fibers (6). The cloned capsaicin receptor subtype termed transient receptor potential vanniloid-1 (TRPV-1) is a nonselective ion channel subunit of 838 amino acid sequence cloned in 1997 (7). Capsaicin and endogenous agonists, anandamide, eicosanoids, and bradykinin stimulate TRPV-1 (8, 9). TRPV-1 is expressed in sensory and afferent fibers innervating the airway wall emanating (Received in original form February 9, 2004; accepted in final form September 20, 2004) Supported by a research scholarship from the Deutsche Atemwegsliga. Correspondence and requests for reprints should be addressed to K. Fan Chung, M.D., D.Sc., National Heart Lung Institute, Imperial College, Dovehouse Street, London SW3 6LY, UK. f.chung@imperial.ac.uk Am J Respir Crit Care Med Vol 170. pp , 2004 Originally Published in Press as DOI: /rccm OC on September 24, 2004 Internet address: from vagal ganglia (10). Activation of TRPV-1 by agonists such as capsaicin induces Ca 2 influx resulting in cough (11). We postulated that airway nerves of patients with chronic persistent cough could express more TRPV-1 receptors, which could be the basis for the increased cough reflex and cough symptoms. The aim of this study was therefore to identify whether TRPV-1 immunoreactivity was augmented in the airways of patients with chronic cough. METHODS Patients We investigated 29 consecutive patients referred with chronic cough through a standard protocol to diagnose and treat the cause of the cough. The mean duration of cough was 6.7 years (SD, 1.2), and the causes were asthma (n 6), gastroesophageal reflux (n 4), rhinosinusitis (n 4), bronchiectasis (n 1), and unexplained (n 14). We also enrolled 16 healthy subjects with no history of cough (Table 1). Patients and volunteers underwent capsaicin cough challenge and fiberoptic bronchoscopy. The study was approved by the Royal Brompton and National Heart and Lung Institute Ethics Committee, and patients gave informed consent. Capsaicin Challenge Coughs were counted for 1 minute after single-breath inhalation of 0.9% NaCl and capsaicin solutions of increasing concentrations ( M). Aerosols were generated from a dosimeter attached to a nebulizer set at a dosing period of 1 minute. Increasing concentrations of capsaicin were inhaled until five or more coughs were counted. The concentration at which this occurs was recorded as the concentration that causes five or more coughs (PC 5 ). Fiberoptic Bronchoscopy Fiberoptic bronchoscopy was performed according to established guidelines. Oxygen (3 L/minute) was administered via nasal prongs, and oxygen saturation was monitored with a digital oximeter. Topical anesthesia of the upper airways and larynx was obtained using lidocaine (2%). Bronchial biopsies were taken from the segmental and subsegmental carinae in the right lung and were immediately placed in optimal cutting temperature embedding media, snap frozen in isopentane precooled with liquid nitrogen, and stored at 70 C, before sectioning and immunostaining. Immunohistochemistry Immunohistochemistry was performed on cryostat-cut, 8- m sections obtained from one subsegmental biopsy from each subject. Sections were preincubated with 0.1-M phosphate buffer containing 1% bovine serum albumin and 10% normal swine serum for 1 hour to block nonspecific binding and incubated with polyclonal rabbit antibody against the pan-neuronal marker protein gene product (PGP)-9.5 (1/400; Biotrend, Cologne, Germany). To assess epithelial TRPV-1 expression, alternate sections were incubated with a previously described polyclonal rabbit antibody against TRPV-1 (1/15,000; GlaxoSmithKline, Harlow, UK) (12). Signaling was detected by incubation with biotinylated goat anti-rabbit IgG (1/200; Amersham, Braunschweig, Germany) in combination with a Streptavidin-Texas Red conjugate (1/50; Amersham) or with a fluorescein isothiocyanate conjugated goat anti rabbit-igg (1/400; Cappel, OH). Fluorescence signaling was analyzed using an epifluorescence microscope and the combination of an

2 Groneberg, Niimi, Dinh, et al.: TRPV-1 Expression in Chronic Cough 1277 TABLE 1. CHARACTERISTICS OF NORMAL VOLUNTEERS WITHOUT COUGH AND OF PATIENTS WITH COUGH PC 5 Age Sex FEV 1 Capsaicin* Smoking n (yr) Male:Female (% Predicted) ( mol) (Ex-smokers) Normal volunteers (15.4) 9: (13.3) 125 ( ) 0 Patients with cough (14.6) 9:20 97 (15) 3.9 ( ) 7 Definition of abbreviation: PC 5 concentration of capsaicin causing five coughs or more. * Median (range). Participants were current nonsmokers, and ex-smokers were defined as those that stopped smoking for 3 or more years at the time of study with less than 5 pack-years of smoking. Mean (SD). p excitation filter with a band-pass of 546/10 nm and a barrier filter with a long-pass of 590 nm. Images of epithelium were captured using an image system and computerized by SPOT Advanced software (SPOT Insight QE version 3.5.1; Visitron Systems, Puchheim, Germany). The observers were unaware of the clinical details of the participating subjects. Images of the epithelium were captured, and the area of specific immunostaining and the total area were measured. The PGP-9.5 or TRPV-1 positive nerve densities were expressed as the percentage of the epithelial area (5, 12). The immunoreactive nerves were distinguished from any background staining. Data Analysis For statistical analysis of the immunoreactive percentage of nerve fibers, the Mann-Whitney U test was applied as the data were not normally distributed. Pearson rank correlation was used to determine correlations. A p value of less than 0.05 was taken as significant. RESULTS Both normal volunteers and patients with cough showed no evidence of airflow obstruction, but the patients with cough were on average 30-fold more sensitive to the tussive effects of capsaicin (Table 1). Staining for PGP-9.5 revealed specific staining of nerve profiles in the biopsies. TRPV-1 immunohistochemistry also led to specific staining of nerve profiles in the subepithelial and epithelial layers of the biopsies. Occasional staining of epithelial cells was present and consisted of less than 1% of epithelial cells; there were no differences between normal and patients with cough. Nerve fiber profiles were measured only in the epithelium. Nerve fibers immunostained for the general nerve marker, PGP-9.5, and for TRPV-1 varied in their density among cases and between groups. The median (range) total nerve density (PGP-9.5 positive fibers) was 1.68% (0 to 4.05) in the patients and was not significantly different at 1.40% (0 to 2.94) in the control group (Figure 1), in agreement with an earlier study (5). However, significant differences were found for TRPV-1 positive nerve fibers, which were higher in cough biopsies with values of 1.15% (0 to 3.39) in the patients versus 0.23% (0 1.23) in the control group (p ) (Figure 2). We have also quantified the expression of TRPV-1 in the biopsies as a ratio of the PGP-9.5 expression measured in the adjacent section for each subject. Thus, the TRPV-1 to PGP-9.5 ratio was 0.17 (0 0.65) in normal volunteers and 0.75 (0 0.96) in the patients with chronic cough (p ). This indicates a 4.4-fold increase in the staining of epithelial nerves in patients with chronic cough. There were no significant differences in the expression of PGP-9.5 or of TRPV-1 between the patients with unexplained cough and those in which the cough was associated with a cause. Within the 29 patients with cough, the number of TRPV-1 positive fibers were inversely correlated to PC 5 (r 0.41, p 0.05); there was no significant correlation between PGP-9.5 expression and PC 5 (Figure 3). DISCUSSION We have shown in a cohort of patients with chronic cough an increase in the nerve profiles expressing TRPV-1, although the nerve profiles stained with the neuronal marker PGP-9.5 were not increased as compared with healthy volunteers who do not suffer from chronic cough. The area of positive staining with the anti TRPV-1 antibody was 75% of the PGP-9.5 positive staining, indicating that 75% of the nerve profiles detected in the epithelium expressed TRPV-1 compared with only 17% in the normal control subjects. We found an inverse correlation between the capsaicin cough responsiveness and the nerve profiles stained with those expressing TRPV-1 within the group of patients with a cough. These results indicate that TRPV-1 may be important in the pathogenesis of chronic cough. The cohort of patients with a chronic cough that we studied had a wide spectrum of associated causes that included asthma, gastroesophageal reflux, and rhinosinusitis, but the majority of these cases had unexplained cough in that none of the putative causes of cough was found to be causing the cough. These patients were referred to our cough clinic from a wide area of southern United Kingdom and have often been seen by other colleagues and received treatment. In nearly half of the patients (48%) in this small cohort, we could not identify a cause, in contrast to previous series (13). All patients with cough had a sensitive cough response to capsaicin. In addition, we found that there was no difference in the expression of either PGP-9.5 or of TRPV-1 between the patients with unexplained cough and those in whom the cough was associated with a cause. The comparative group of normal control subjects that we recruited was not equally balanced with the group of patients with cough in terms of sex and age. The predominance of females in the patients with a chronic cough is as one may expect. The influence of sex and age on the degree of expression of PGP-9.5 and TRPV-1 positive neural fibers in the airway epithelium is unknown. Within the group of normal volunteers and the those with a cough, we found no significant differences between men and women in terms of the PGP-9.5 and TRPV-1 expression, and there was no significant correlation between age and the capsaicin response and TRPV-1 expression within the normal volunteers. The lack of relationship between these factors in our study would suggest that these may not have influenced the results we found. The phenotypic expression of airway nerves in patients with chronic cough was changed in that there was a fivefold greater expression of TRPV-1. TRPV-1 gene expression is found predominantly in nociceptive-like primary afferent neurones whose

3 1278 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL Figure 1. Airway staining for protein gene product (PGP)-9.5. Immunofluorescence staining of airway nerves in a bronchial biopsy with the pan-neuronal marker PGP-9.5 in a normal noncoughing volunteer (norm; panel 1) and in a patient with cough (cough; panel 2). Panel 3 shows no staining with the negative control when the primary antibody was not added. Individual percentages of bronchial epithelial area staining positive for PGP-9.5 are shown between patients with a chronic cough and healthy control subjects (panel 4). The arrows denote positive neuronal staining. Original magnification 250. Figure 2. Airway staining for transient receptor potential vanniloid-1 (TRPV-1). Immunofluorescence staining of airway nerves in a bronchial biopsy with an anti TRPV-1 antibody in a normal noncoughing volunteer (norm; panel 1) and in a patient with cough (cough; panel 2). Panel 3 shows no staining with the negative control when the primary antibody was not added. Individual percentages of bronchial epithelial area stained positive for TRPV-1 was significantly greater in patients with chronic cough than in the control group (panel 4). Arrows denote positive neuronal staining. Original magnification 250.

4 Groneberg, Niimi, Dinh, et al.: TRPV-1 Expression in Chronic Cough 1279 nonasthmatic cough (20), to release lipoxygenase products that could activate TRPV-1 receptors (21). In summary, we found increased expression of TRPV-1 in airway epithelial nerves of patients with chronic cough and a significant correlation of this expression with the capsaicin cough sensitivity. Thus, TRPV-1 expression may be one of the determinants of the enhanced cough reflex found in patients with chronic cough. TRPV-1 antagonists have been described (22), and these could be effective in the treatment of chronic cough, irrespective of the type or cause of chronic cough. Figure 3. Correlation between PGP-9.5 expression (percentage of epithelial area) and the concentration of capsaicin causing five coughs or more (PC 5 response) ( M) (upper panel), and between TRPV-1 expression (percentage of epithelial area) and the capsaicin PC 5 response ( M) (lower panel) in the 29 patients with chronic cough. There was a significant correlation between TRPV-1 expression and the PC 5 response but not between PGP-9.5 expression and the PC 5 response. cell bodies reside in the dorsal root, trigeminal, and nodose ganglia. Antibodies directed against TRPV-1 have revealed the cellular distribution of TRPV-1 in sensory neurones (14). It is not excluded that there may also be upregulation in airway ganglia, which would not be accessible from the mucosal biopsy method. The cause of the increased number of TRPV-1 positive nerve fibers in patients with chronic cough is unknown. Expression of TRPV-1 receptors is known to be regulated by growth factors such as nerve growth factor and glial cell line derived neurotrophic factor (15). Immunoreactivity to CGRP, which is regulated by nerve growth factor, has been reported to be increased in epithelial nerves in chronic cough (5), making the role for nerve growth factor and other growth factors more likely. Such CGRP-positive nerve profiles may also be expressing TRPV-1. TRPV-1 mediates the cough induced by capsaicin as, in studies in guinea pigs, the capsaicin antagonist capsazepine inhibits capsaicin-induced cough and the endogenous TRPV-1 ligand anandamide causes cough, an effect inhibited by capsazepine and resinoferatoxin, which are both TRPV-1 antagonists (11). Although an increase in the number of TRPV-1 receptors may contribute to the enhanced cough reflex to capsaicin, other factors may also be involved. The activation of A fibers and C fibers in the airways of guinea pigs or rats induced by decreasing ph involves TRPV-1 because protons can increase the TRPV-1 ion channel opening (16). Heat-activated currents in TRPV-1 can be potentiated by relatively small changes in ph (17), and this would indicate the potential for low ph to augment capsaicin cough sensitivity in situations such as gastroesophageal reflux of gastric acid. An increase in the content of protons in exhaled breath condensate of the order of half-log in chronic cough has been reported (18). In addition, the reported increase in CGRPimmunoreactive nerves in this condition (5) also indicates the presence of a neuropeptide that could sensitize visceral afferents (19). Nerve growth factor could also act on mast cells, which we have found to be increased in biopsies from patients with Conflict of Interest Statement : D.A.G. obtained research scholarships funded by Aventis, Merck, Sharpe & Dohme, GlaxoSmithKline companies; A.N. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; Q.T.D. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; B.C. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; M.H. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.F. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; K.F.C. has been a member of scientific Advisory Boards for Novartis, GlaxoSmithKline, Astra Zeneca, Altana, and Fujisawa and has received lecture fees from Novartis, GlaxoSmithKline, Altana, and Celgene and has been reimbursed by Novartis and Boehringer Ingelheim for attending scientific conferences. 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