Rhinovirus upregulates transient receptor potential channels in a human neuronal cell line: implications for respiratory virus-induced cough reflex sensitivity.

BACKGROUND: The mechanism underlying respiratory virus-induced cough hypersensitivity is unknown. Upregulation of airway neuronal receptors responsible for sensing physical and chemical stimuli is one possibility, and the transient receptor potential (TRP) channel family are potential candidates. We have used an in vitro model of sensory neurons and human rhinovirus (HRV-16) to study the effect of virus infection on TRP expression. METHODS: IMR-32 neuroblastoma cells were differentiated in culture to express three TRP channels: TRPV1, TRPA1 and TRPM8. Flow cytometry and qRT-PCR were used to measure TRP channel protein and mRNA levels following inoculation with live virus, inactivated virus, virus-induced soluble factors or pelleted virus particles. Multiplex bioassay was used to determine nerve growth factor (NGF), interleukin (IL)-1ß, IL-6 and IL-8 levels in response to infection. RESULTS: Early upregulation of TRPA1 and TRPV1 expression occurred 2-4 h post infection. This was independent of replicating virus as virus-induced soluble factors alone were sufficient to increase channel expression 50-fold and 15-fold, respectively. NGF, IL-6 and IL-8 levels, increased in infected cell supernatants, represent possible candidates. In contrast, TRPM8 expression was maximal at 48 h (9.6-fold) and required virus replication rather than soluble factors. CONCLUSIONS: We show for the first time that rhinovirus can infect neuronal cells. Furthermore, infection causes upregulation of TRP channels by channel-specific mechanisms. The increase in TRPA1 and TRPV1 levels can be mediated by soluble factors induced by infection whereas TRPM8 requires replicating virus. TRP channels may be novel therapeutic targets for controlling virus-induced cough.

Eosinophil peroxidase induces the expression and function of acid-sensing ion channel-3 in allergic rhinitis: in vitro evidence in cultured epithelial cells.

BACKGROUND: Acid-sensing ion channels (ASIC) are a family of acid-activated ligand-gated cation channels. As tissue acidosis is a feature of inflammatory conditions, such as allergic rhinitis (AR), we investigated the expression and function of these channels in AR. OBJECTIVES: The aim of the study was to assess expression and function of ASIC channels in the nasal mucosa of control and AR subjects. METHODS: Immunohistochemical localization of ASIC receptors and functional responses to lactic acid application were investigated. In vitro studies on cultured epithelial cells were performed to assess underlying mechanisms of ASIC function. RESULTS: Lactic acid at pH 7.03 induced a significant rise in nasal fluid secretion that was inhibited by pre-treatment with the ASIC inhibitor amiloride in AR subjects (n = 19). Quantitative PCR on cDNA isolated from nasal biopsies from control and AR subjects demonstrated that ASIC-1 was equally expressed in both populations, but ASIC-3 was significantly more highly expressed in AR (P < 0.02). Immunohistochemistry confirmed significantly higher ASIC-3 protein expression on nasal epithelial cells in AR patients than controls (P < 0.01). Immunoreactivity for EPO+ eosinophils in both nasal epithelium and submucosa was more prominent in AR compared with controls. A mechanism of induction of ASIC-3 expression relevant to AR was suggested by the finding that eosinophil peroxidase (EPO), acting via ERK1/2, induced the expression of ASIC-3 in epithelial cells. Furthermore, using a quantitative functional measure of epithelial cell secretory function in vitro, EPO increased the air-surface liquid depth via an ASIC-dependent chloride secretory pathway. CONCLUSIONS: This data suggests a possible mechanism for the observed association of eosinophils and rhinorrhoea in AR and is manifested through enhanced ASIC-3 expression.

Patterns of cough in the clinic.

Current guidelines for the management of cough highlight the value of a taking a careful history to establish specific features of the cough in particular its duration, typical triggers or aggravants and associated symptoms. Unfortunately the diagnostic yield from a history alone is poor and there is a need to understand the pattern of clinical cough in a more precise way. As the technology to record cough in ambulatory settings becomes more sophisticated so the possibility that precise measurement of the cough frequency, intensity and acoustic characteristics may offer diagnostically valuable information in individual patients becomes a reality. In this article the current knowledge of the clinical patterns of cough is discussed and the potential for new technology to record cough patterns in a meaningful way is considered.

Clinical cough and its mechanisms.

Cough is the commonest symptom of clinical importance and the most frequent reason for new consultations with a doctor. Although therapy directed at any underlying cause for cough can be effective there is a clinical need for new treatments specifically directed at the cough itself. A major obstacle to the development of such therapy has been an imprecise understanding of the pathophysiological mechanisms responsible for cough. In this article, we review the important clinical aspects of both acute and chronic cough, offer practical insight into the existing treatment options, highlight the current understanding of cough pathophysiology and identify important areas for future research effort.

Is there an association between angiotensin-converting enzyme gene variants and chronic nonproductive cough?

BACKGROUND: It is unclear why some patients develop a chronic nonproductive cough. Angiotensin-converting enzyme (ACE) inactivates tussive peptides in the airways such as bradykinin and tachykinins. An insertion/deletion polymorphism in the ACE gene accounts for variation in ACE levels, and patients with the II genotype have lowest serum ACE levels compared with ID and DD genotypes. We hypothesized that the II genotype would be associated with increased risk of developing a chronic cough. MATERIALS AND METHODS: We recruited 47 patients (33 women), referred for evaluation of cough (median cough duration, 24 months; range, 2 to 240 months). Cough patients were evaluated using a comprehensive diagnostic protocol, and cough reflex sensitivity was measured using a capsaicin inhalation challenge. ACE genotyping was performed on DNA samples from patients using the polymerase chain reaction followed by agarose gel electrophoresis. ACE genotypes in patients with chronic cough were compared with those in 199 healthy control subjects. Serum ACE levels were determined using a colorimetric assay. RESULTS: Genotype frequencies for the ACE gene were similar between patients and control subjects. There was no correlation between capsaicin sensitivity and ACE genotypes or serum ACE levels. CONCLUSION: Susceptibility to develop chronic cough is not associated with ACE genotype.

Cytokine concentrations and neutrophil elastase activity in bronchoalveolar lavage and induced sputum from patients with cystic fibrosis, mild asthma and healthy volunteers.

BACKGROUND: Induced sputum (IS) has been proposed as a non-invasive alternative to bronchoalveolar lavage (BAL) for the assessment and monitoring of airways inflammation. The aim of this study was to compare both methods in patients with cystic fibrosis (CF). The possible differences between subjects with CF, mild asthma and healthy volunteers (HV) was also assessed. METHOD: In a single centre, randomised, two way crossover study, 11 patients with CF, 9 mild asthmatics (MA) and 11 HV underwent BAL and hypertonic saline induction on consecutive days. Free neutrophil elastase (NE), neutrophil elastase/alpha(1)-anti-trypsin complex (NE-AAT), tumour necrosis factor receptor (p55) and interleukin-8 (IL-8) were measured in cell free supernatants. RESULTS: Three CF patients reported serious adverse events following BAL. NE was usually undetectable in both IS or BAL samples and NE-AAT concentrations did not differ consistently between the two sampling methods. IL-8 and p55 levels in the CF patients tended to be higher in IS samples compared with BAL samples (median 19,860 vs. 3,855 pg/ml and 2.55 vs. 0.29 ng/ml, respectively). There was a significant difference in mean p55 concentrations between CF, MA and HV in IS samples (P=0.003) but not in BAL samples (P=0.36). The difference in mean IL-8 concentrations in IS samples between subject groups was statistically different (P=0.023). CONCLUSIONS: IS samples can be safely obtained from CF patients. Analysis of IS samples can help to characterize the inflammatory process in the airways of CF patients. The serious adverse events following BAL in 3 CF patients highlight an inherent risk associated with this procedure.

Respiratory and laryngeal symptoms secondary to gastro-oesophageal reflux.

Gastro-oesophageal reflux may cause a range of laryngeal and respiratory symptoms. Mechanisms responsible include the proximal migration of gastric refluxate beyond the upper oesophageal sphincter causing direct irritation of the larynx and lower airway. Alternatively, refluxate entering the distal oesophagus alone may stimulate oesophageal sensory nerves and indirectly activate airway reflexes such as cough and bronchospasm. Recognising reflux as a cause for these extraoesophageal symptoms can be difficult as many patients do not have typical oesophageal symptoms (eg, heartburn) and clinical findings on laryngoscopy are not very specific. Acid suppression remains an effective treatment in the majority of patients but there is growing appreciation of the need to consider and treat non-acid and volume reflux. New opinions about the role of existing medical and surgical (laparoscopic techniques) treatment are emerging and a number of novel anti-reflux treatments are under development.

Common causes and current guidelines.

Chronic cough is a common and disabling symptom. Recent guidelines have attempted to provide direction in the clinical management of cough in both primary and secondary care. They have also provided a critical review of the available literature and identified gaps in current knowledge. Despite this they have been criticized for a reliance on a low quality evidence base. In this review, we summarize the current consensus on the clinical management of chronic cough and attempt to rationalize this based on recent evidence. We have also provided an overview of the likely pathophysiological mechanisms responsible for cough and highlighted areas, where knowledge deficits exist and suggest directions for future research. Such progress will be critical in the search for new and effective treatments for cough.

Calcitonin gene-related peptide relates to cough sensitivity in children with chronic cough.

Airway neuropeptides, in particular calcitonin gene-related peptide (CGRP), are likely to be important in the pathogenesis of chronic cough. The present authors evaluated the following: 1) the relationship between cough sensitivity and bronchoalveolar lavage (BAL) neuropeptides; and 2) the effect of reflux oesophagitis (RO) on cough, cough sensitivity and BAL neuropeptides in children not selected for cough. It was hypothesised that CGRP would be increased in children with chronic cough and would relate to cough sensitivity. Capsaicin cough sensitivity was performed in children undergoing gastro-duodenal endoscopy. CGRP, substance P and neurokinin A were measured in BAL obtained nonbronchoscopically. Children were defined as "coughers" if chronic cough was present. Coughers (n = 21) had significantly reduced cough sensitivity but were just as likely as noncoughers (n = 19) to have RO. The median CGRP was significantly higher in coughers with oesophagitis than in noncoughers with oesophagitis. CGRP significantly negatively correlated to cough sensitivity in coughers but not in noncoughers. Elevated calcitonin gene-related peptide, but not substance P or neurokinin A, is only associated with chronic cough in children when oesophagitis coexists. Calcitonin gene-related peptide in bronchoalveolar lavage relates to cough sensitivity and is likely to be important in the pathophysiology of chronic cough.

Idiopathic chronic cough: a real disease or a failure of diagnosis?

Despite extensive diagnostic evaluation and numerous treatment trials, a number of patients remain troubled by a chronic and uncontrollable cough. Eosinophilic bronchitis, atopic cough and non-acid reflux have been recently added to the diagnostic spectrum for chronic cough. In some cases, failure to consider these conditions may explain treatment failure. However, a subset of patients with persisting symptoms may be regarded as having an idiopathic cough. These individuals are most commonly female, of postmenopausal age and frequently report viral upper respiratory tract infections as an initiating event. This paper seeks to explore the validity of idiopathic cough as a distinct clinical entity.

Patients with gastro-oesophageal reflux disease and cough have impaired laryngopharyngeal mechanosensitivity.

BACKGROUND: Laryngopharyngeal sensitivity (LPS) is important in preventing pulmonary aspiration and may be impaired by anaesthesia and stroke. It has been suggested that gastro-oesophageal reflux disease (GORD) may also impair LPS, although the underlying mechanism is unclear. The aim of this study was to compare LPS in patients with chronic cough and GORD with healthy subjects and to determine the effect of laryngopharyngeal infusions of both acid and normal saline on LPS. METHODS: Fifteen patients with chronic cough and GORD and 10 healthy subjects without GORD underwent LPS testing using the fibreoptic endoscopic evaluation of swallowing with sensory testing (FEESST) technique. LPS, as measured by the lowest air pressure required to elicit the laryngeal adductor reflex (LAR), was determined both before and after laryngopharyngeal infusions of normal saline and 0.1 N hydrochloric acid performed on separate days. RESULTS: The mean baseline LAR threshold of the patient group was significantly higher (9.5 mm Hg, range 6.0-10.0) than in normal subjects (3.68 mm Hg, range 2.5-5.0; p<0.01). Retest thresholds were not significantly different. In normal subjects LAR thresholds were significantly raised after acid but not after saline infusion (p = 0.005). There were no complications associated with the procedure. CONCLUSIONS: Patients with cough and GORD have significantly reduced LPS to air stimuli compared with healthy subjects which could potentially result in an increased risk of aspiration. Exposure to small amounts of acid significantly impaired the sensory integrity of the laryngopharynx.

Cough . 6: Which investigations are most useful in the diagnosis of chronic cough?

There is no consensus as to the best diagnostic strategy for chronic cough. Many protocols combine empirical trials of treatment with laboratory investigations. More precise diagnostic tools and improved therapeutic options are required. Until then, the management of chronic cough will remain a clinical challenge.

Acute and chronic cough.

Individuals are generally content to self-medicate for acute cough. It is only when the cough becomes persistent that they seek medical assistance. It is not known why patients cough in association with an acute upper respiratory tract infection (URTI), although interest has focused on how viruses may influence airway sensory nerve function and contribute to heightened cough reflex sensitivity. Why some patients develop a persistent cough following a viral URTI is also unclear. Much more is known about the causes and aggravants of chronic cough although there is no broad agreement as to the best way to manage such patients. Asthma, upper gastrointestinal dysfunction and rhinitis are frequently associated with chronic cough, although the impact of cough in suppurative lung disease and interstitial lung fibrosis is rarely considered. The development of effective treatments for cough remains a challenge and will require co-operation between clinicians, scientists and the pharmaceutical industry.

Idiopathic cough, prevalence and underlying mechanisms.

A number of authors have reported a significant percentage of patients whose cause of cough remains undetermined despite a systematic evaluation as recommended in a number of International Guidelines. This subset of patients, which may be regarded as having an idiopathic cough, are often female and of peri or postmenopausal age. Sex hormones may influence the cough reflex in disease or contribute to the chronic lymphocytic airway inflammation seen in some cases and to the association with organ specific autoimmune disease reported. This paper seeks to investigate some of the possible causes of idiopathic cough.

A retrospective survey of diagnosis and management of patients presenting with chronic cough to a general chest clinic.

Respiratory physicians often encounter patients with chronic cough. The evaluation and outcome of such patients by centres with established diagnostic protocols has been well described. By contrast, little is known about patients referred to general respiratory clinics where no such protocol exists. We describe the findings of a retrospective survey of all new patient referrals with chronic cough to a general respiratory clinic over a 12-month period. A diagnosis of asthma or chronic airflow obstruction was made in 43% of patients. Gastro-oesophageal reflux and postnasal drip syndrome, together accounting for over 60% of diagnoses in specialist clinics, were infrequently identified in our study (4% and 2% respectively). At follow-up, 43% of patients reported persistent symptoms, contrasting the excellent treatment response reported by specialist clinics. In general respiratory clinics where a specific diagnostic protocol is not in place, these differences in diagnosis and outcome may be due to inadequate investigation or may reflect a different patient population.

Sensory neuropeptides induce histamine release from bronchoalveolar lavage cells in both nonasthmatic coughers and cough variant asthmatics.

BACKGROUND: Sensory neuropeptides have been suggested to play a role in the pathogenesis of a number of respiratory diseases including asthma and chronic non-productive cough. OBJECTIVES: To investigate the action of sensory neuropeptides on airway mast cells obtained by bronchoalveolar lavage (BAL). METHODS: BAL was performed on 23 nonasthmatic patients with cough (NAC), 11 patients with cough variant asthma (CVA) and 10 nonatopic controls. Washed lavage cells were stimulated (20 min, 37 degrees C) with calcitonin gene-related peptide (CGRP), neurokinin A (NKA) and substance P (25 and 50 micromol/L). RESULTS: The neuropeptides tested induced histamine release in all groups studied. Only CGRP (50 micromol/L) induced significantly more histamine release from both NAC and CVA patients compared with control subjects (P = 0.038 and 0.045, respectively). CONCLUSION: Regardless of aetiology, mast cells from patients with chronic cough appear to have an increased responsiveness to CGRP compared with controls. The results of the present study suggest that the role of CGRP in chronic cough should be further investigated.

Bronchoalveolar lavage findings in patients with chronic nonproductive cough.

Mast cells and eosinophils may play a role in the pathophysiology of chronic cough in nonasthmatics. It is unknown, however, whether degranulation of these cells occurs in the airways of such patients. Thirty-five nonsmoking patients referred with a chronic nonproductive cough (mean cough duration 76.2 months) were evaluated using a comprehensive diagnostic protocol. Bronchoalveolar lavage (BAL) cell differentials and BAL histamine, tryptase and eosinophilic cationic protein (ECP) concentrations were determined. Ten nonsmoking healthy volunteers served as controls. Diagnostic subgroups were identified: eight postnasal drip syndrome (PNDS), seven cough variant asthma (CVA), seven gastro-esophageal reflux (GOR), seven dual aetiology and six idiopathic. Nonasthmatic coughers (NAC) were characterized as those patients without bronchial hyperresponsiveness on histamine challenge and whose cough had either responded to therapy for PNDS or GOR or failed to improve with antiasthma therapy. There was a significant increase in both eosinophil and mast cell numbers (p<0.05) and in histamine levels (p = 0.027) when NAC patients were compared with controls. Tryptase and ECP levels were elevated in 7 of 23 and 6 of 23 NAC patients, respectively. In conclusion, airway inflammatory cell numbers are not only increased but also activated, suggesting an important role for airways inflammation in the pathophysiology of chronic nonproductive cough.

Adenosine induces histamine release from human bronchoalveolar lavage mast cells.

Previous studies have shown that in vitro adenosine enhances histamine release from activated human lung mast cells obtained by enzymic dispersion of lung parenchyma. However, adenosine alone has no effect on histamine release from these cells. Given the evidence for direct activation of mast cells after endobronchial challenge with adenosine and previous studies indicating that mast cells obtained at bronchoalveolar lavage are a better model for asthma studies than those obtained by enzymic dispersion of lung tissue, the histamine-releasing effect of adenosine was examined on lavage mast cells. Bronchoalveolar lavage fluid was obtained from patients attending hospital for routine bronchoscopy (n=54). Lavage cells were challenged with adenosine or adenosine receptor agonists (20 min, 37 degrees C) and histamine release determined using an automated fluorometric assay. Endogenous adenosine levels were also measured in lavage fluid (n=9) via an HPLC method. Adenosine alone caused histamine release from lavage mast cells in 37 of 54 patients with a maximal histamine release of 20.56+/-2.52% (range 5.2-61%). The adenosine receptor agonists (R)-N6-(2-phenylisopropyl)adenosine, 5'-N-ethylcarboxamidoadenosine and CGS21680 also induced histamine release from lavage mast cells. Preincubation of lavage mast cells with the adenosine receptor antagonist xanthine amine congener caused significant inhibition of the response to adenosine (P=0.007). There was an inverse correlation between endogenous adenosine levels in the lavage fluid and the maximal response to in vitro adenosine challenge of the lavage cells. The findings of the present study indicate a means by which adenosine challenge of the airways can induce bronchoconstriction and support a role for adenosine in the pathophysiology of asthma. The results also suggest that cells obtained from bronchoalveolar lavage fluid may provide the ideal model for the testing of novel, adenosine receptor, targeted therapies for asthma.