(Chest. 2004;125:1368-1371.)
© 2004
American College of Chest Physicians
Increased Responses to Inhaled Oxitropium Bromide in Asthmatic Patients With Active Hepatitis C Virus Infection*
Hiroshi Kanazawa, MD;
Kazuto Hirata, MD and
Junichi Yoshikawa, MD
* From the Department of Respiratory Medicine, Graduate School of Medicine, Osaka City University, Osaka, Japan.
Correspondence to: Hiroshi Kanazawa, MD, Department of Respiratory Medicine, Graduate School of Medicine, Osaka City University, 1-4-3, Asahi-machi, Abenoku, Osaka 545-8585, Japan; e-mail: kanazawa-h{at}med.osaka-cu.ac.jp
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Abstract
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Study objectives: The interaction between chronic hepatitis C virus (HCV) infection and bronchial asthma is of considerable interest. This study was designed to examine whether differences in airway responses to an inhaled anticholinergic agent exist between asthmatic patients with and without active HCV infection.
Design: Controlled cross-sectional analysis.
Setting: University hospital.
Patients: Sixteen HCV-negative asthmatic patients and 36 HCV-positive asthmatic patients.
Interventions: All HCV-positive patients received interferon (INF) therapy for 6 months (INF responders, 16 patients; INF nonresponders, 20 patients). No patient had received INF within 3 years of the start of the study.
Measurements and results: Airway hyperreactivity to methacholine (ie, the provocative concentration of methacholine causing a 20% fall in FEV1 [PC20]), maximal increase in FEV1, and forced expiratory flow between 25% and 75% of FVC (FEF25–75) after the administration of oxitropium bromide (200 µg) were examined. At the start of the study, the groups were well-matched with respect to age, body mass index, and baseline lung function, including methacholine PC20. The mean (SD) increase in FEV1 after oxitropium bromide administration was significantly greater in patients with active HCV (95 [7] mL) than in HCV-negative asthmatic patients (68 [12] mL) and asthmatic patients with inactive HCV infection (69 [6] mL; p < 0.001). The increase in FEF25–75 after oxitropium bromide administration was also significantly greater (250 [90] mL/s vs 170 [90] and 180 [80] mL/s, respectively; p < 0.029).
Conclusions: In patients with asthma, active HCV infection is associated with increased bronchodilator responses to inhaled oxitropium bromide. HCV infection may modulate acetylcholine-mediated airway tone.
Key Words: airway hyperreactivity • CD8+ T lymphocytes • cholinergic activity • muscarinic receptors
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Introduction
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Parasympathetic nerve stimulation releases acetylcholine onto M3 muscarinic receptors on airway smooth muscle, causing bronchoconstriction. The release of acetylcholine is limited by inhibitory M2 muscarinic receptors.1 Therefore, M2 receptor dysfunction leads to increased acetylcholine release and the potentiation of acetylcholine-induced bronchoconstriction. M2 receptor dysfunction has been described in some patients with asthma.2 Although the precise mechanisms of M2 receptor dysfunction are not fully understood, it has recently been reported3 that CD8+ T lymphocytes induced by viral infection are necessary for M2 receptor dysfunction, resulting in cholinergic activation in asthmatic airways.
Hepatitis C virus (HCV), an RNA virus that was first identified in 1989, is a major cause of posttransfusion and sporadic hepatitis. HCV can lead to persistent infection and progression to chronic liver disease. CD8+ T lymphocytes are typically observed within the hepatic parenchyma in patients with chronic HCV hepatitis.4 It has been suggested that HCV-specific CD8+ T lymphocytes may contribute to the development of disease.5 Currently, the interaction between chronic HCV infection and certain pulmonary disorders is of considerable interest.67 Moreover, a previous study8 reported that the numbers of lymphocytes in the lung are increased in patients with chronic HCV infection, suggesting that chronic HCV infection might be a trigger for the development of airway inflammation. Whether HCV infection affects lung function in asthma is unclear. This study was designed to examine whether differences in airway responses to inhaled anticholinergic agents exist between asthmatic patients with and without active HCV infection.
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Materials and Methods
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Sixteen HCV-negative and 36 HCV-positive asthmatic patients were included in the study. All subjects were lifelong nonsmokers and met the American Thoracic Society guidelines for asthma.9 In short, they all had episodic cough, wheezing and dyspnea, and normal findings on chest radiographs. They also exhibited reduced FEV1 during asthma attacks and an increase of 
20% in FEV1 in response to ß2-adrenoceptor agonists. Their medications consisted of therapy with inhaled ß2-adrenoceptor agonists on demand and inhaled corticosteroids. Patients had received a stable course of medication for > 1 year. All patients were stable, had a history of mild asthma, and did not have other significant medical illnesses. These patients were selected from a region (Osaka City, Japan) with an extraordinarily high incidence of chronic HCV infection. Anti-HCV antibody levels were measured with a first-generation or second-generation enzyme-linked immunosorbent assay (Ortho Diagnostics; Tokyo, Japan). Serum HCV RNA was detected by reverse transcription and nested polymerase chain reaction with primers derived from the highly conserved 5'-untranslated region of the viral genome, as described previously.10 HCV-positive patients were defined as being anti–HCV-positive and HCV RNA-positive. All HCV-positive patients received interferon (INF) therapy for 6 months. We labeled cases inactive hepatitis if HCV RNA disappeared by the end of INF therapy and could not be detected at the time of this study. Sixteen of 36 asthmatic patients with chronic HCV infection were INF responders. No patient had taken INF within 3 years of the start of the study. All subjects gave their written informed consent for participation in the study, which was approved by the Ethics Committee of Osaka City University.
Methacholine inhalation challenge testing was performed in all subjects. All challenge tests were performed at 1:00 PM to eliminate the effects of diurnal variation. After baseline spirometry and inhalation of a diluent to establish the stability of FEV1, the subjects were instructed to take slow inspirations in each set of inhalations. On the following day, each subject was given oxitropium bromide (200 µg), and the changes in FEV1 and forced expiratory flow between 25% and 75% of FVC (FEF25–75) were examined. Spirometry was performed thereafter at 10-min intervals for 1 h, and increases in FEV1 and FEF25–75 was evaluated as the maximal increase after oxitropium bromide administration.
Statistical Analysis
All data are expressed as the mean (SD). Multiple comparisons among the groups were analyzed by one-way analysis of variance followed by Bonferroni correction. Statistical significance was defined as p < 0.05.
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Results
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The clinical characteristics of each group in this study are shown in Table 1
. The groups were well-matched with respect to age, body mass index, and baseline lung function. However, levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were significantly higher in patients with active HCV than in HCV-negative patients and patients with inactive HCV infection.
All subjects in this study exhibited airway hyperreactivity to methacholine. The provocative concentration of methacholine causing a 20% fall in FEV1 (PC20 methacholine) did not significantly differ among the three groups (HCV-negative patients, 2.68 [2.27] mg/mL; HCV-inactive patients, 3.16 [2.09] mg/mL; HCV-active patients, 3.27 [2.42] mg/mL) [Fig 1
]. However, the increase in FEV1 after oxitropium bromide administration was significantly greater in patients with active HCV (95 [7] mL) than in HCV-negative patients (68 [12] mL; p < 0.001) and patients with inactive HCV infection (69 [6] mL; p < 0.001) [Fig 2
]. The increase in FEF25–75 after oxitropium bromide administration was also significantly greater in patients with active HCV infection (250 [90] mL/s) than in HCV-negative patients (170 [90] mL/s; p = 0.009) and patients with inactive HCV infection (180 [80] mL/s; p = 0.029) [Fig 3
].
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Figure 2. Comparison of increase in FEV1 after oxitropium bromide administration in HCV-negative, HCV-inactive, and HCV-active asthmatic patients. All values are expressed as the mean ± SD. ** = p < 0.01, compared with the HCV-negative group; ## = p < 0.01, compared with the HCV-inactive group.
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Figure 3. Comparison of the increase in FEF25–75 after oxitropium bromide administration in HCV-negative, HCV-inactive, and HCV-active asthmatic patients. All values are expressed as the mean ± SD. ** = p < 0.01, compared with the HCV-negative group. # = p < 0.05, compared with HCV-inactive group.
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Discussion
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In this study, we compared bronchodilator responses to a muscarinic receptor antagonist in asthmatic patients with and without active HCV. We found that airway responses to methacholine administration did not differ. However, increases in FEV1 and FEF25–75 after oxitropium bromide administration were significantly greater in patients with active HCV than in HCV-negative patients and patients with inactive HCV infection. These findings suggest that differences in airway responses to inhaled oxitropium bromide administration exist between asthmatic patients with and without active HCV infection.
The mechanisms predisposing asthmatic patients with chronic HCV infection to increased cholinergic activity are unclear. Viral infections characteristically elicit strong CD8+ T lymphocytosis.11 Although CD8+ T lymphocytes are important effectors of cell-mediated immunity, their precise role in the pathogenesis of asthma is unclear. Previous studies have determined that acute severe asthma is associated with the infiltration of large numbers of CD8+ T lymphocytes into both proximal and distal lung tissues,12 and that an aberrant CD8+ T lymphocyte population arising in response to viral infection was observed in the bronchi of subjects experiencing severe asthma attacks.13 Interestingly, it has been reported3 that an increase in CD8+ T lymphocytes in response to viral infection contributes to M2 receptor dysfunction in a guinea pig model of airway hyperreactivity, and that CD8+ T lymphocyte depletion prevents M2 receptor dysfunction and airway hyperreactivity. Thus, CD8+ T lymphocytes play a role in the M2 receptor dysfunction of sensitized virus-infected guinea pigs. Several reports1415 also have suggested that chronic viral infection induces increases in the numbers of CD8+ T lymphocytes in both the airways and lung parenchyma, which is consistent with the expected virus-specific CD8+ T lymphocyte response. It therefore seems likely that CD8+ T lymphocytes activated by chronic HCV infection contribute to immune responses in the airways of asthmatic patients. Therefore, we hypothesized that CD8+ T lymphocytes activated by chronic HCV infection may induce cholinergic activation in asthma patients, at least in part through M2 receptor dysfunction.
It was not surprising that methacholine responsiveness did not differ among the three groups. We speculated that asthmatic patients with active HCV infection are not more hyperresponsive to methacholine than the other two groups, since airway responses to the exogenous administration of muscarinic receptor agonist could be similar in these patients with active HCV infection and M2 receptor dysfunction. These hypotheses would be strengthened if HCV-specific CD8+ T lymphocytes could be found in the lungs of asthmatic patients with chronic HCV infection. However, we do not know whether there is an increased incidence of asthma in patients with chronic HCV infection. Another possible explanation for our results is that abnormal liver function in asthmatic patients with active HCV infection might have important effects on the metabolism of and responses to anticholinergic drugs, since significantly higher levels of AST and ALT were observed in patients with active HCV infection. Since all subjects in this study received inhaled therapy, rather than systemic therapy, this possibility appears to be less likely. Indeed, we found no relationship between the degree of abnormal liver function and bronchodilator responses to oxitropium bromide in our study subjects.
There are many methodological limitations to the present study. Although we have discussed the role of CD8+ T lymphocytes on cholinergic activity as a possible explanation for our results, we have no data on lung CD8+ T lymphocyte populations in the setting of HCV and asthma. A comparison of inflammatory markers and immunologic patterns between asthmatic patients with and without active HCV infection should be performed. In conclusion, our findings suggest that active HCV infection is associated with increased bronchodilator responses to inhaled oxitropium bromide in asthmatic patients. HCV infection may modulate acetylcholine-mediated airway tone.
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Acknowledgements
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We thank Yukari Matsuyama for her help in the preparation and editing of the manuscript.
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Footnotes
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Abbreviations: ALT = alanine aminotransferase; AST = aspartate aminotransferase; FEF25–75 = forced expiratory flow between 25% and 75% of FVC; HCV = hepatitis C virus; INF = interferon; PC20 methacholine = concentration of methacholine causing a 20% fall in FEV1
This work was supported by a Grant-in-Aid for Scientific Research (15590820) from the Japan Society for the Promotion of Science. All authors declare that there is no conflict of interest in this study.
Received for publication May 5, 2003.
Accepted for publication October 30, 2003.
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Abstract
Introduction
