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Published ahead of print on January 10, 2008, doi:10.1164/rccm.200703-457OC
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American Journal of Respiratory and Critical Care Medicine Vol 177. pp. 585-592, (2008)
© 2008 American Thoracic Society
doi: 10.1164/rccm.200703-457OC

Original Article

Effect of Pressure Stress Applied to the Airway on Cough-Reflex Sensitivity in Guinea Pigs

Johsuke Hara1,2, Masaki Fujimura1, Akihito Ueda1, Shigeharu Myou1, Yoshitaka Oribe1,3, Noriyuki Ohkura1, Toshiyuki Kita1,4, Masahide Yasui1 and Kazuo Kasahara1

1 Respiratory Medicine, Cellular Transplantation Biology, Kanazawa University Graduate School of Medicine, Kanazawa, Ishikawa, Japan; 2 Department of Respiratory Medicine, Ishikawa Prefectural Central Hospital, Kanazawa, Ishikawa, Japan; 3 Department of Internal Medicine, Keiju Medical Center, Nanao, Ishikawa, Japan; and 4 Department of Respiratory Medicine, National Hospital Organization Kanazawa Medical Center, Kanazawa, Ishikawa, Japan

Correspondence and requests for reprints should be addressed to Johsuke Hara, M.D., Respiratory Medicine, Cellular Transplantation Biology, Kanazawa University Graduate School of Medicine, 13-1, Takara-machi, Kanazawa City, Ishikawa, 920-8641, Japan. E-mail: hara{at}ipch.jp


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: We hypothesized that cough stress of the airway wall results in a self-perpetuating cough-reflex cycle in which antigen-induced increase in cough-reflex sensitivity results in pathologic cough, and the cough in turn further amplifies cough-reflex sensitivity.

Objectives: To examine cough-reflex sensitivity in an experimental animal model.

Methods: We developed an experimental guinea pig model in which airway collapse similar to that in cough was induced by rapid negative pressure applied to the airway of artificially ventilated animals. We examined the influence of this stimulus on cough-reflex sensitivity to inhaled capsaicin and bronchoalveolar lavage (BAL) cell components. After the termination of artificial ventilation, the number of coughs due to capsaicin was measured, and BAL was performed.

Measurements and Main Results: Capsaicin cough-reflex sensitivity and the number of BAL neutrophils were increased 6 hours after stimulus application, decreasing to control levels by 24 hours. Cough-reflex sensitivity or BAL cell components were not changed in the absence of stimulus application. The number of BAL neutrophils correlated significantly with the number of coughs. Hydroxyurea inhibited the stimulus-induced increase in the number of coughs and airway neutrophil accumulation.

Conclusions: Our findings suggest that cough itself is a traumatic mechanical stress to the airway wall that induces neutrophilic airway inflammation and cough-reflex hypersensitivity. Cough stress to the airway wall results in a self-perpetuating cough-reflex cycle.

Key Words: cough • cough-reflex sensitivity • hydroxyurea • negative mechanical pressure stress • neutrophilic airway inflammation



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
Patients with chronic nonproductive cough usually have enhanced cough-reflex sensitivity to inhaled tussive agents such as capsaicin or citric acid. Little is known about the underlying mechanisms of the increased cough response.

What This Study Adds to the Field
The cough stress to airway wall may induce a self-perpetuating cough-reflex cycle.

 
Cough is one of the most common symptoms of respiratory disease. Patients with chronic nonproductive cough usually have an enhanced cough-reflex sensitivity to inhaled tussive agents such as capsaicin or citric acid (1, 2). Little is known about the underlying mechanisms of the increased cough response. On the basis of our clinical experience, we hypothesized that cough stress to the airway wall creates a self-perpetuating cough-reflex cycle in which an antigen-induced increase in cough-reflex sensitivity results in pathologic cough that in turn further amplifies the cough-reflex sensitivity. We developed an experimental guinea pig model, in which airway collapse simulating cough was induced in response to rapid negative pressure applied to the airway of artificially ventilated animals, and we examined the influence of this stimulus on cough-reflex sensitivity to inhaled capsaicin, bronchoalveolar lavage (BAL) cell components, and histology of the airway wall.


   METHODS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Male albino Hartley-strain guinea pigs (body weight, 550–600 g), obtained from Sankyou Laboratory Service (Toyama, Japan), were used. They were quarantined at the Animal Research Center of Kanazawa University. All animal procedures in this study conformed to the standards set out in the Guidelines for the Care and Use of Laboratory Animals at the Takara-machi campus of Kanazawa University.

Study Design
Experimental protocol 1.
Naive guinea pigs were assigned to one of four groups: normal control (NA), negative control (NE), or positive control (PO10 or PO20) (n = 8–9 per group). Guinea pigs were anesthetized with an intraperitoneal injection of 30 mg/kg sodium pentobarbital and placed in the supine position. Animals in the NA group were tracheotomized only. After the trachea was cannulated with a polyethylene tube (outside diameter, 2.5 mm; inside diameter, 2.1 mm), animals in the other groups were artificially ventilated with a small-animal respirator (model 1680; Harvard Apparatus Co., Inc., South Natick, MA) adjusted to a tidal volume of 10 ml/kg at a rate of 60 strokes/minute for 1 minute. Animals in the PO groups were exposed to the negative mechanical pressure stimulus; animals in the PO10 group were exposed to a negative pressure of 10 cm H2O and animals in the PO20 group were exposed to a negative pressure of 20 cm H2O. Animals in the NE group were ventilated artificially without exposure to negative pressure. After stimulus presentation, the trachea and skin were sutured, and the animals were allowed to awaken from anesthesia. Cough-reflex sensitivity to inhaled capsaicin was measured at 6, 12, and 24 hours after stimulus presentation, after which the animals were anesthetized and subjected to BAL. Increases in cough-reflex sensitivity to inhaled capsaicin and neutrophil count in the BAL fluid (BALF) were significant in the PO20 group but not in the PO10 group. Therefore, we examined the time course of cough-reflex sensitivity and examined BALF in the PO20 group compared with the NE group.

Three additional groups of guinea pigs (NA, NE, and PO20) were prepared for histologic examination (n = 5–6 per group).

Experimental protocol 2.
Naive guinea pigs were pretreated with hydroxyurea (HU) or saline (Sal) and assigned to one of six groups: (1) normal control with HU (NA-HU group), (2) normal control with Sal (NA-Sal group), (3) negative control with HU (NE-HU group), (4) negative control with Sal (NE-Sal group), (5) positive control with HU (PO20-HU group), and (6) positive control with Sal (PO20-Sal group) (n = 8–9 per group). Guinea pigs pretreated with Sal received 1.0 ml/kg of 0.15 mM NaCl daily for 5 days via intraperitoneal injection, and those pretreated with HU received 1.0 ml/kg of 800 mg/ml HU solution daily for 5 days via intraperitoneal injection. Cough-reflex sensitivity to inhaled capsaicin was measured 6 hours after stimulus presentation, after which the animals were anesthetized and subjected to BAL.

Experimental Apparatus for the Application of Negative Mechanical Pressure Stress
The apparatus used to apply negative mechanical pressure stress to the airway of artificially ventilated animals is shown in Figure 1. An electromagnetic valve (model USG3-6-1-H; CKD Corporation, Aichi, Japan) was linked with ventilation and this valve opened 20 times during the 60-second artificial ventilation period. The valve was kept open for 0.2 seconds, during which negative pressure induced by a large aspirator (model MERA MS-005; Izumi Koika Kogyo, Tokyo, Japan) was applied to the animals in the PO groups. Changes in resistance to lung inflation, the lateral pressure of the tracheal tube (pressure at the airway opening [Pao], cm H2O), were measured with a pressure transducer (model TP-603T; Nihon Koden Kogyo, Tokyo, Japan). Examples of Pao measurements in the NE and PO groups are shown in Figures 2A and 2B.


Figure 1
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  		Figure 1. Experimental apparatus for presentation of negative mechanical pressure to the airway wall of artificially ventilated guinea pigs. An electromagnetic valve was linked with ventilation and this valve opened 20 times during the 60-second artificial ventilation period. The valve was kept open for 0.2 seconds during which negative pressure induced by a large aspirator was presented to the airway in the positive control (PO) groups. Changes in resistance to lung inflation, via lateral pressure on the tracheal tube (pressure at the airway opening [Pao], cm H2O), were measured with a pressure transducer.

 

Figure 2
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  		Figure 2. Examples of changes in airway opening pressure (Pao) in guinea pigs not exposed to negative mechanical pressure (A) or those exposed to negative mechanical pressure (PO) (B).

 
Measurement of Cough-Reflex Sensitivity
Cough-reflex sensitivity was measured at 6, 12, and 24 hours after the 1-minute artificial ventilation period with or without the negative pressure stress. Each conscious guinea pig was placed in a custom-built, airtight, transparent plastic box consisting of a head chamber (1,600-ml volume) isolated from a body chamber, and pressure in the body chamber was recorded. Coughs were detected as a change in pressure, with rapid inspiration followed by rapid expiration. Movements of the guinea pigs were monitored visually to control for motion- and sneezing-related changes in pressure. Coughs were counted by a trained observer and recognized by the characteristic animal posture and the pressure transducer recordings. Increasing concentrations of capsaicin solution (10–6 M, 10–4 M) were inhaled for 2 minutes from a nebulizer (model 646; DeVilbiss Co., Somerset, PA) operated by compressed air at 1.6 L/minute (Air Pump AP-115AN; Iwaki Co., Ltd., Tokyo, Japan). The nebulizer output was 37 µl/minute. The number of coughs was counted during the 2-minute inhalation of each capsaicin solution and for an additional minute. The total number of coughs during each 3-minute period was recorded for each concentration of capsaicin. Different animals were used for each 3-minute measurement period to avoid the possibility of tachyphylaxis to repeated inhalation of capsaicin.

BAL
After the measurement of cough-reflex sensitivity to capsaicin, animals were anesthetized with an intraperitoneal injection of 75 mg/kg of sodium pentobarbital and placed in the supine position. The trachea was cannulated with a polyethylene tube (outside diameter, 2.5 mm; inside diameter, 2.1 mm), and BAL was performed twice with 10 ml of 0.15 mM NaCl. Cells in the BALF were stained with Turk solution and counted in duplicate with a hemocytometer (in a Burker chamber). Differential cell counts were performed on a smear prepared by cytocentrifugation and Wright-Giemsa staining.

Effect of Negative Pressure Stress on Time Course of Cough-Reflex Sensitivity, BAL Cell Components, and Concentration of Substance P in BALF
Analysis of BALF was performed at 1, 3, 6, 12, and 24 hours after the 1-minute artificial ventilation in animals in the PO20 and NE groups (n = 8–9 per group). Animals at 1 and 3 hours were not fully awakened from the anesthesia; therefore, the inhalation of increasing concentrations of capsaicin solution was performed, but the number of coughs was not measured at these time points. After the examination of cough-reflex sensitivity to capsaicin, BAL was performed. Different animals were used for each measurement to avoid the possibility of tachyphylaxis to repeated inhalation of capsaicin.

The concentration of substance P in BALF was measured with a commercial enzyme immunoassay kit (Cayman Chemical Co., Ann Arbor, MI). This kit is a competitive assay that provides accurate measurements of substance P with a working range of 3.9–500 pg/ml.

Histologic Examination
Animals, under deep anesthesia, were exsanguinated from the abdominal aorta 6 hours after negative mechanical pressure stress. The airway and lungs of each animal were excised en bloc by opening the chest. The trachea was then cannulated and inflated with 10% formalin via the tracheal cannula. The airway and lungs were fixed for at least 48 hours and embedded in paraffin. The trachea and main bronchus up to 5 mm past the main carina and the lower lobes of the left lung were sectioned and stained with hematoxylin and eosin. The number of neutrophils in the epithelium of the left main bronchus was counted by light microscopy at a magnification of x400 and expressed as the number of cells/mm of airway basement membrane, which was measured with an objective micrometer.

We also counted the number of neutrophils in the airway epithelium in the cartilaginous and noncartilaginous airways of the lower lobe of the left lung with Win ROOF, image analysis software (Mitani Co., Ltd., Fukui, Japan) as the number of cells/mm2 of airway mucosa.

Drugs
The following chemicals were used: sodium pentobarbital (Abbott Laboratories, North Chicago, IL), 0.15 mM NaCl (Otsuka Pharmaceutical Co., Ltd., Osaka, Japan), capsaicin (Sigma-Aldrich, St. Louis, MO), and HU (Wako Pure Chemical Industries, Ltd., Osaka, Japan).

Statistical Analysis
All data are shown as mean ± SEM. Statistical differences were determined by analysis of variance followed by Fisher's protected least significant difference test (Statview; SAS Institute, Cary, NC), excluding the concentration of substance P in the BALF. Differences in the concentration of substance P in BALF were analyzed by Mann-Whitney U test. The relation between the number of BAL neutrophils and the number of coughs induced by capsaicin was analyzed by simple regression analysis. A P value of less than 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Cough-Reflex Sensitivity 6 Hours after the Presentation of Negative Pressure Stress
The number of coughs induced by inhaled capsaicin is shown in Figure 3. Animals in the PO10 group were exposed to a negative pressure of 10 cm H2O and animals in the PO20 group were exposed to a negative pressure of 20 cm H2O. Animals in the NE group were artificially ventilated in the absence of negative pressure application. The number of coughs elicited by inhaled capsaicin was significantly increased in a negative pressure intensity–dependent fashion (Figure 3). No significant difference in cough-reflex sensitivity was observed between the NA and NE groups (Figure 3).


Figure 3
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  		Figure 3. Cough-reflex sensitivity to inhaled capsaicin 6 hours after negative pressure stress in guinea pigs. Open bars, normal control group (tracheotomy alone); dotted bars, negative control group (tracheotomy and ventilation); hatched bars, PO10 group (exposed to –10 cm H2O negative mechanical pressure); solid bars, PO20 group (exposed to –20 cm H2O negative mechanical pressure). **P < 0.01 versus negative control group. n = 8–9 animals per group.

 
Pretreatment with HU completely inhibited the negative pressure–induced increase in the number of capsaicin-induced coughs (Figure 4).


Figure 4
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  		Figure 4. Cough-reflex sensitivity to inhaled capsaicin 6 hours after negative pressure stress in guinea pigs pretreated with 0.15 mM NaCl (saline [Sal]) or hydroxyurea (HU). Open bars, normal control group (tracheotomy alone); dotted bars, negative control group (tracheotomy and ventilation); solid bars, PO20 group (exposed to –20 cm H2O negative mechanical pressure). **P < 0.01 versus PO20 group pretreated with 0.15 mM NaCl. n = 8–9 animals per group.

 
BALF Analysis 6 Hours after the Presentation of Negative Pressure Stress
The total number of cells, the number of neutrophils, and the percentage of neutrophils in BALF were significantly increased in a negative pressure intensity–dependent manner (Figure 5). There was no significant difference in the percentage of neutrophils or the total number of cells or neutrophils between the NE and NA groups (Figure 5). There was also a significant increase in the number of macrophages in a negative pressure intensity–dependent fashion (data not shown). No significant difference in the percentage of macrophages, lymphocytes, or eosinophils, or in the number of lymphocytes or eosinophils, was observed between groups (data not shown).


Figure 5
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  		Figure 5. Bronchoalveolar lavage cell findings 6 hours after negative pressure stress. Open bars, normal control group (tracheotomy alone); dotted bars, negative control group (tracheotomy and ventilation); hatched bars, PO10 group (exposed to –10 cm H2O negative mechanical pressure); solid bars, PO20 group (exposed to –20 cm H2O negative mechanical pressure). **P < 0.01 versus negative control group. n = 8–9 per group.

 
Effects of HU on BALF cell counts and cell differentiation are shown in Figure 6. HU inhibited the negative pressure stress–induced increase in the number and percentage of neutrophils. HU also decreased the total number of cells and macrophages in the NA, NE, and PO20 groups (data not shown). No significant influence of HU was observed on the number of BAL lymphocytes or eosinophils or on the percentage of macrophages, lymphocytes, or eosinophils (data not shown).


Figure 6
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  		Figure 6. Bronchoalveolar lavage cell findings 6 hours after negative pressure in guinea pigs pretreated with 0.15 mM NaCl (saline [Sal]) or hydroxyurea (HU). Open bars, normal control group (tracheotomy alone); dotted bars, negative control group (tracheotomy and ventilation); solid bars, PO20 group (exposed to –20 cm H2O negative mechanical pressure). **P < 0.01 versus PO20 group pretreated with 0.15 mM NaCl. n = 8–9 animals per group.

 
Time Course of Effect of Negative Pressure Stress on Cough-Reflex Sensitivity
The time course of the effect of negative pressure stress on cough-reflex sensitivity to inhaled capsaicin in the PO20 and NE groups is shown in Figure 7. Although the number of 10–4 M capsaicin–induced coughs was significantly increased in the PO20 group versus the NE group 6 and 12 hours after the application of negative pressure stress, the number of coughs was much greater at 6 hours than after 12 hours. The increase in capsaicin-induced coughs decreased nearly to the level of the NE group by 24 hours. There was no change in cough-reflex sensitivity in the NE group over 24 hours.


Figure 7
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  		Figure 7. Time course of the effect of negative pressure stress on cough-reflex sensitivity to inhaled capsaicin. Dotted bars, negative control group (tracheotomy and ventilation); solid bars, PO20 group (exposed to –20 cm H2O negative mechanical pressure). **P < 0.01 versus negative control group at each time. n = 8–9 per group. NS = not significant. 10–6 and 10–4 refer to concentrations of capsaicin (in M) for left and right panels, respectively.

 
Time Course of Effect of Negative Pressure Stress on BAL Cell Components
Time courses of the effects of negative pressure stress on the total number of cells and neutrophils and on the percentage of neutrophils in BALF in the PO20 and NE groups are shown in Figure 8. The total number of cells and neutrophils and the percentage of neutrophils in BALF were increased in the PO20 group 6 hours after negative pressure stress, followed by a decrease to the levels of the NE group by 24 hours. Neither the number nor the percentage of neutrophils in the NE group changed during the 24-hour period.


Figure 8
Figure 8
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  		Figure 8. Time course of the effect of negative pressure stress on the total number of cells (A) and neutrophils (B) and the percentage of neutrophils (C) in bronchoalveolar lavage fluid. Dotted circles, negative control group (tracheotomy and ventilation); solid circles, PO20 group (exposed to –20 cm H2O negative mechanical pressure). **P < 0.01 versus negative control group at each time. n = 8–9 per group.

 
A significant increase in the number of macrophages was observed in the PO20 group versus the NE group 6 hours after negative pressure stress (data not shown). There was no significant difference in the number of lymphocytes or eosinophils between the two groups during the 24-hour period. The percentage of macrophages, lymphocytes, or eosinophils was also not significantly different between the two groups during this period (data not shown).

Time Course of the Effect of Negative Pressure Stress on the Concentration of Substance P in BALF
The time course of the effect of negative pressure stress on the concentration of substance P in BALF in the PO20, NE, and NA groups is shown in Figure 9. A significant increase in substance P concentration was observed 6 hours after negative pressure stress in the PO20 group. There was no significant difference in the substance P concentration between the NE and NO groups during 24-hour period after the surgical procedure.


Figure 9
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  		Figure 9. Time course of the effect of negative pressure stress on the concentration of substance P in bronchoalveolar lavage fluid. Open circles, normal control; dotted circles, negative control group (tracheotomy and ventilation); solid circles, PO20 group (exposed to –20 cm H2O negative mechanical pressure). Horizontal bars indicate the median values of each group. **P < 0.01 versus negative control group at each time. n = 5–8 per group.

 
Relation between the Number of Coughs Induced by Inhaled Capsaicin and Number of BALF Neutrophils
The number of BALF neutrophils was significantly correlated with the number of coughs induced by low and high concentrations of capsaicin (Figure 10).


Figure 10
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  		Figure 10. Relation between number of bronchoalveolar lavage neutrophils and number of coughs elicited by inhaled capsaicin. r = 0.421, P = 0.0007 (left); r = 0.413, P = 0.0009 (right). 10–6 and 10–4 refer to concentrations of capsaicin (in M) for left and right panels, respectively.

 
Histology
The number of neutrophils in the epithelium is shown in Table 1. The number of neutrophils in the PO20 group was significantly greater than the numbers in the NA and NE groups.


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  		TABLE 1. NEUTROPHIL COUNT IN THE BRONCHIAL MUCOSA*

 

   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we investigated whether negative mechanical pressure stress to the airway influences cough-reflex sensitivity to capsaicin or induces airway inflammation. Results showed that mechanical stress enhanced the cough-reflex sensitivity to capsaicin, and this was accompanied by neutrophil accumulation in the airway. In addition, the number of BALF neutrophils correlated significantly with the number of coughs induced by inhaled capsaicin. Pretreatment with HU completely inhibited the mechanical stress–induced increase in cough-reflex sensitivity and airway neutrophil accumulation. We failed to investigate the relation between the neutrophil count in bronchial tissue and cough-reflex sensitivity to inhaled capsaicin in the present study.

Many investigators have devised a system to induce cough. In animal studies, inhalation of citric acid or capsaicin is used to evaluate cough-reflex sensitivity. Cough and bronchoconstriction are distinct but interrelated reflexes. It is speculated that inhalation of concentrations of capsaicin greater than 10–4 M, when delivered at a high flow rate, induces bronchoconstriction as well as coughing (3, 4). The number and intensity of coughs elicited by aerosolized 10–4 M capsaicin in naive guinea pigs are considered to be less than those of cough attacks in patients with chronic cough. Therefore, we developed the present guinea pig model, in which a coughlike reaction is induced by negative mechanical pressure stress. In animal studies, the pattern of cough varies depending on which part of the respiratory tract is stimulated (5). It is considered that cough consists of forced rapid expiration after deep inspiration (6, 7). The bronchi and intrathoracic trachea are compressed and narrowed during the forced rapid expiration of coughing. This narrowing results from a transmural pressure gradient between extraluminal and intraluminal pressures (7). Immediately before the start of the expiration phase of coughing, when the larynx is closed, both extraluminal and intraluminal pressures of the trachea and bronchi become positive. In the expiration phase of coughing, when the larynx is opened, intraluminal pressure is suddenly decreased to ambient pressure, resulting in the huge transmural pressure that compresses the tracheal and bronchial mucosa. In our experimental model, sudden negative pressure was presented to the airway to simulate the expiratory phase of coughing after the development of positive airway pressure. This simulation method has been used by several research groups (8–10).

It has been reported that patients with nonasthmatic dry cough, idiopathic cough (11), or chronic cough (12, 13) have a significantly greater proportion of neutrophils in induced sputum than do normal control subjects. These patients also have cough hypersensitivity to capsaicin compared with normal control subjects (14). However, Niimi and colleagues reported no correlation between submucosal neutrophils and cough-reflex sensitivity in patients with nonasthmatic cough (15). Although it is unknown about the interaction of cough-reflex sensitivity and neutrophilic inflammation, we speculate that mechanical stress—that is, repeated cough itself—can induce neutrophilic airway inflammation. It has been reported that cyclic stretch of the airway induces expression of several cytokines and chemokines, including the following: macrophage inflammatory protein (MIP)-2 in rat BALF (16), MIP-2, IL-6, and tumor necrosis factor (TNF)-{alpha} in isolated perfused mouse lungs (17); TNF-{alpha}, IL-1β, IL-6, MIP-2, and IFN-{gamma} in isolated rat lung (18); and IL-8 in human bronchial epithelial cells (19, 20). It has also been shown that positive end-expiratory pressure causes neutrophil adhesion and recruitment mediated by P-selectin, endothelin, and intercellular adhesion molecule-1 in the tracheal vasculature of rats and mice (21, 22). TNF-{alpha} and IL-8 are significantly increased in induced sputum of nonasthmatic patients with chronic dry cough (11). Nightingale and coworkers reported that the percentage of neutrophils in induced sputum from healthy subjects increases at 8 and 24 hours compared with the baseline value (23). Coughing, hypertonic saline, and/or sputum itself may cause the increase in neutrophils in induced sputum. On the basis of their findings that neutrophilia in induced sputum was found in all causes of cough, Jatakanon and colleagues speculated that the act of coughing itself may contribute to the release of proinflammatory cytokines such as TNF-{alpha} and IL-8 (11). It is unknown whether inflammatory cytokines, such as TNF-{alpha} and IL-8, influence cough-reflex sensitivity. It is likely that other mediators, such as prostaglandins or bradykinin, which are produced directly via cytokine activation, enhance cough-reflex sensitivity. In the present study, cough-reflex sensitivity remained increased even after BALF neutrophils had returned to control levels by 12 and 24 hours after negative pressure stress. The discrepancy in the time courses of increased cough-reflex sensitivity and BALF neutrophilia suggests that cytokines, chemokines, and chemical mediators produced by neutrophils, but not accumulated neutrophils per se, increase cough-reflex sensitivity.

HU is a member of a group of compounds that inhibit the enzyme ribonucleoside diphosphate reductase and is specific for the S phase of the cell cycle. HU has been used in numerous studies of circulating leukocyte depletion in animals (24–26). Thompson and colleagues reported that HU does not decrease neutrophils in the tracheal lamina propria (24). O'Byrne and coworkers reported that HU decreases neutrophils in the airway epithelium but not in the BALF (25). Our current findings in BALF of animals treated with HU are consistent with these findings reported previously. HU inhibited both the mechanical stress–induced increase in cough-reflex sensitivity and airway neutrophil accumulation. In addition, the BAL neutrophil count correlated significantly with the number of coughs induced by inhaled capsaicin. As the direct influence of HU on cough-reflex sensitivity is unexplored, based on our results in the control groups, there may be the possibility that neutrophilic airway inflammation increases cough-reflex sensitivity.

Previous studies have reported that inactivation of neutral endopeptidase (NEP) is caused by airway epithelium damage (27–29). In the airway, NEP is the major degrading enzyme of substance P (30). In this respect, the inactivation of NEP activity may cause cough-reflex hypersensitivity to capsaicin in our guinea pig model. Substance P has been shown to induce chemotaxis in human neutrophils (31) and to release IL-8 from human polymorphonuclear leukocytes and TNF-{alpha} from human monocytes (32). Furthermore, nonasthmatic patients with sputum neutrophilia have high levels of substance P (33). Therefore, in our guinea pig model, substance P may play an important role in the release of cytokines and accumulation of neutrophils into the airway.

The receptor for capsaicin, termed vanilloid receptor (VR)-1, is expressed in guinea pigs. VR-1 mediates cough induced by capsaicin (34). Increased expression of VR-1 has also been reported in humans with chronic cough (35). Therefore, in guinea pig airway exposed to negative mechanical pressure stress, epithelial damage may induce an increase in VR-1 expression, thereby contributing to enhanced cough-reflex sensitivity. This possibility should be examined in future studies.

Vagal afferent nerves are involved in the initiation of the cough reflex. Afferent neuronal subtypes, C fibers, rapidly adapting receptors (RARs), and slowly adapting stretch receptors are identified on the basis of their physicochemical sensitivity, adaptation to sustained lung inflation, neurochemistry, origin, myelination, conductive velocity, and sites of termination in the airways (36). RARs are activated by the dynamic mechanical forces that accompany lung inflation and deflation and become more active as the rate and volume of lung inflation increases (37–39). RAR activity is believed to be increased in guinea pigs, rats, and newborns of all species (36).

Results of the present study showed that negative mechanical pressure stress applied to the airway induced an increase in cough-reflex sensitivity 6 hours after application. It is unknown whether natural or induced cough causes cough-reflex hypersensitivity with a similar time course. Induced cough is considered to be weaker and less frequent than that induced by mechanical stress. Natural cough is considered to be a strong stress to the airway. Generally, natural cough is accompanied by airway inflammation. There is no known method for suppressing cough without suppressing inflammation. Therefore, investigation of changes in cough-reflex sensitivity between subjects with airway inflammation and natural cough and those without natural cough is not currently possible. Investigation of continuous changes in cough-reflex sensitivity in healthy subjects with extremely strong induced cough, causing muscle pain, urinary incontinence, or rib fracture, is also not possible.

Both natural cough and induced cough continue for some period and then resolve. The mechanism underlying this resolution is not well known compared with the sneeze reflex. We showed that mechanical stress to the airway induced an increase in neutrophilic airway inflammation and cough-reflex sensitivity. It would be interesting to determine whether neutrophilic airway inflammation and cough-reflex sensitivity are reinforced or weakened upon repeated mechanical stimulation.

In conclusion, the present study clearly showed that negative mechanical pressure stress applied to the airway to simulate cough attack induced neutrophilic airway inflammation resulting in increased cough-reflex sensitivity in guinea pigs. Mechanical stress applied to the airway thus results in a self-perpetuating cough-reflex cycle. The influence of repeated mechanical pressure stress on the airway will be examined in future studies.


   FOOTNOTES
 
Supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture (no. 17607003) of the Japanese government.

Originally Published in Press as DOI: 10.1164/rccm.200703-457OC on January 10, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form March 20, 2007; accepted in final form January 4, 2008


   REFERENCES
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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