The Proceedings of the American Thoracic Society 2:150-156 (2005)
© 2005 The American Thoracic Society
Overview of Virus-induced Airway Disease
Sebastian L. Johnston
Department of Respiratory Medicine, National Heart and Lung Institute and Wright Fleming Institute of Infection and Immunity, Imperial College London, London, United Kingdom
Correspondence and requests for reprints should be addressed to Sebastian L. Johnston, M.D., Ph.D., Professor of Respiratory Medicine, Department of Respiratory Medicine, National Heart & Lung Institute and Wright Fleming Institute of Infection & Immunity, Imperial College London, Norfolk Place, London W2 1PG, UK. E-mail: s.johnston{at}imperial.ac.uk
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ABSTRACT
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Acute exacerbations of asthma and chronic obstructive pulmonary disease (COPD) are the major cause of morbidity, mortality, and health costs of both diseases. Currently available treatments are poorly effective in both acute treatment of and prevention of acute exacerbations. New treatments for intervention and prophylaxis are therefore required; to facilitate their development, we must understand the causes and mechanisms of exacerbations. Respiratory viral infections (2/3 rhinoviruses) precipitate 80% or more of asthma exacerbations in children, and the majority of exacerbations of asthma and COPD in adults, but mechanisms of virus-induced lower airway inflammation and of host resistance against respiratory viruses are poorly understood. Development of in vitro experimental models of virus infection has identified interferon-ß and nitric oxide as possible therapeutic targets to augment antiviral immunity, and nuclear factor-
B as a target for development of anti-inflammatory therapies. In vivo models could also serve to identify and validate targets and as an experimental system to test candidate molecules as they emerge into clinical studies. Studies in asthma have paved the way for development of an asthma model; a similar experimental model in COPD would accelerate development of new therapies for these common diseases with enormous burdens of illness.
Key Words: acute exacerbations • asthma • chronic obstructive pulmonary disease • virus infections
Asthma now affects up to 10 to 33% of children and 5 to 10% of adults in Westernized communities (1). Although the underlying disease of chronic asthma has major health impacts, acute exacerbations of asthma are the major cause of morbidity and mortality. The health costs associated with acute exacerbations, in terms of time off school and work, primary care consultations, and hospital admissions, are enormous. Asthma mortality remains at unacceptably high levels despite increased use of prophylactic medication in recent years (2), indicating that new therapies are needed to reduce the frequency and impact of acute exacerbations of this disease.
Chronic obstructive pulmonary disease (COPD) is the second most prevalent chronic respiratory disease, affecting mostly adults and the elderly. Its prevalence is also increasing, and it is predicted to become the third leading cause of death worldwide by 2020 (3). It is currently the fourth leading cause of death in the United States, and the only common cause of death that is increasing. As with asthma, although the clinical and health care impact of chronic COPD is a major concern, it is acute exacerbations of this disease that are associated with the greatest morbidity and mortality. Acute exacerbations of COPD are an enormous problem to primary care and respiratory physicians each winter and account for most of the healthcare spending of COPD (4). Each winter the increase in acute admissions and bed occupancy consequent upon acute exacerbations of COPD causes major logistic problems to hospital administrators and healthcare workers.
This article will therefore assess the importance of virus infections as precipitants of acute exacerbations of both asthma and COPD. It will then discuss mechanisms of susceptibility to infection and mechanisms of virus induction of inflammation, in order to point toward possible candidates for the development of new therapies for virus-induced exacerbations of airway disease.
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ASSOCIATION OF VIRUS INFECTIONS WITH EXACERBATIONS OF ASTHMA
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Recent studies indicate that respiratory virus infection precipitates the majority of acute exacerbations of asthma in all age groups (5, 6). Respiratory viral infection has now been shown in a number of separate studies to be associated with approximately 80 to 85% of acute asthma exacerbations in children (5, 7–9). There are fewer published studies in adults, but a time series analysis has strongly associated the seasonal pattern of viral respiratory infections with acute admissions for asthma in adults (6). Studies that have directly investigated the association vary in their results according to study design and virus detection methods, with studies that employ polymerase chain reaction (PCR) virus detection being much more successful than those relying on standard virology. For example, one published study employing serology and culture on nasopharyngeal aspirates had a virus detection rate of 29% (10), while another study that sampled the lower airway and employed PCR for all common respiratory viruses had a detection rate of 76% (11). Further studies are needed, but the latter study suggests that respiratory viruses are also associated with the majority of acute asthma exacerbations in adults.
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EVIDENCE OF A CAUSAL RELATIONSHIP BETWEEN VIRUS INFECTION AND ASTHMA EXACERBATION
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All the studies published to date demonstrate an association in time between virus infection and asthma exacerbation. However, studies have not investigated whether the relationship is causal. Evidence of a causal relationship comes from a study in adults admitted to the emergency room with asthma exacerbations (11). Viruses were detected in sputum in 76% of these admissions, and sputum samples were further assessed for inflammatory markers, including inflammatory cell numbers, and for markers of cellular activation, including eosinophil cationic protein and myeloperoxidase (MPO). Sputum samples were also assessed for levels of lactate dehydrogenase (LDH) as a marker of virus-induced lower airway cell damage. Logistic regression analysis indicated that among all clinical and inflammatory parameters assessed, LDH levels were the single strongest predictor of severity of the exacerbations measured in terms of length of hospital stay, indicating that the degree of virus induced lower airway cell damage was the major driver of severity of the exacerbation (11). This study strongly suggests that virus infection is the major factor causing exacerbations of asthma and also that severity of infection is the major determinant of the severity of the exacerbation.
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AVAILABLE TREATMENT FOR ACUTE EXACERBATIONS OF ASTHMA
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Options for therapy of acute asthma exacerbations are currently of limited efficacy. Intervention by doubling the dose of inhaled steroids has been shown to be ineffective in two separate studies (12–14). Treatments for exacerbations of asthma therefore include inhaled bronchodilators and systemic/oral steroids. These treatments are partially effective, but have significant side effects and do not address the cause of the exacerbation, nor specific mechanisms involved in its pathogenesis.
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PREVENTION OF ACUTE EXACERBATIONS OF ASTHMA
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Inhaled steroids are the mainstay of treatment for asthma and are remarkably effective at treating some aspects of the disease. However, in adults with persistent asthma even optimal therapy is only able to reduce the frequency of exacerbations by around 40% (15). In school-age children, moderate doses in inhaled steroids (400 µg beclomethasone daily) are completely ineffective at reducing exacerbation frequency, duration, or severity (16).
In preschool children with virus-induced wheeze, a 5-day course of oral steroids at the onset of the next exacerbation has recently been shown to be ineffective in reducing the duration or severity of the exacerbation. Remarkably, this treatment was ineffective even in a subgroup of children with primed systemic eosinophils (17).
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NEED FOR NEW THERAPIES FOR ASTHMA EXACERBATIONS
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These studies indicate that a major component of the disease asthma—acute exacerbations—is largely unresponsive to therapy that is remarkably effective at treating other components of the disease. This strongly suggests that the pathogenesis of acute asthma exacerbations is different from that of the underlying disease, and therefore that different therapies are likely to be required to effectively treat or prevent asthma exacerbations.
New therapies for both the prevention and treatment of asthma exacerbations are therefore urgently required. In order to identify targets for the development of new treatments of asthma exacerbations, it is necessary first to seek a detailed understanding of the mechanisms of asthma exacerbations.
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VIRUS TYPES PRECIPITATING ACUTE EXACERBATIONS OF ASTHMA
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In all the above studies on etiology of asthma exacerbations in children, rhinoviruses were numerically the most important virus type, accounting for around 66% of infections detected (5, 7–9). In the studies in asthma in children, they were associated with 50% of total exacerbations. Rhinovirus infections are therefore the major cause of asthma exacerbations and the most appropriate cause with which to investigate the pathogenesis of acute asthma exacerbations.
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EXPERIMENTAL MODELS OF VIRUS-INDUCED ASTHMA EXACERBATIONS
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There is no small animal model of rhinovirus infection in which to investigate disease pathogenesis; the work that has been carried out to date has therefore been carried out in in vitro models of rhinovirus infection of respiratory epithelial cells, or in human experimental infection studies. In vitro models have proven extremely useful in investigating the molecular regulation of rhinovirus-induced proinflammatory mediators (18–24) and have identified potentially antiinflammatory responses that could be harnessed in future treatment strategies (18). Most of the studies to date, however, have concentrated on mechanisms of induction of acute inflammation in response to virus infection. These studies have investigated a number of proinflammatory mediators including interleukin (IL)-6, IL-8, intercellular adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule (VCAM). For all mediators that have been investigated to date, the transcription factor nuclear factor-B (NF-B) is required for full induction to occur (19–24). These studies indicate that treatment strategies targeting mechanisms of induction of NF-B by virus infection could have broad antiinflammatory activity and be of potential benefit in the context of virus-induced exacerbations of airway disease. Some candidates are currently in early clinical development, and the results of these studies are awaited with great interest.
In vitro models can also be used to study responses of primary bronchial epithelial cells ex vivo. This approach has recently been used to demonstrate differences in innate immune responses between subjects with asthma and those without asthma (see below for more detail) (25).
One disadvantage of in vitro models is that they involve only a single cell type and do not reflect the complexity of in vivo infections where complex interactions between cells occur. Furthermore, it is clearly desirable that observations made in in vitro systems are tested and confirmed in the in vivo situation.
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IN VIVO EXPERIMENTAL INFECTION MODELS OF VIRUS-INDUCED ASTHMA EXACERBATIONS
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Experimental infection studies have generated important findings that have considerably advanced our understanding of the pathogenesis of virus-induced asthma exacerbations: rhinovirus infections have been shown to lead to long-lasting excessive airway narrowing in volunteer subjects with asthma (26); to induce sputum markers of eosinophil activation, IL-8, and neutrophilia (27, 28); to induce bronchial infiltration with eosinophils, CD4, and CD8 T lymphocytes (29); and to induce activation of prostaglandin and leukotriene synthetic pathways (30). Rhinovirus induction of nitric oxide has also been identified as a potential protective response to infection, identifying another target for development of new therapies (31).
Experimental infection with rhinovirus in subjects with mild asthma thus provides a unique in vivo human model of virus-induced asthma exacerbations suitable for investigating the mechanisms of exacerbations. This model also offers a number of very important advantages over studies of natural virus-induced asthma exacerbations, including the ability to study subjects at baseline before virus infection; the selection of subjects for study according to specific criteria such as atopy, asthma severity, and use of medication; knowledge of the virus type and infective dose; accurate timing of investigations relative to the timing of infection; and the ability to carry out invasive investigations before, during, and after infection, including bronchoscopy for sampling of the lower airway.
These advantages are crucial in making the human experimental infection model the best model in which the mechanisms of virus-induced inflammation can practically be investigated. However, none of the above studies has been successful in demonstrating differences between normal subjects and subjects with asthma in their inflammatory responses to rhinovirus infection. Further studies investigating differences between volunteer subjects with asthma and normal volunteer subjects are therefore urgently required.
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INCREASED SUSCEPTIBILITY TO RHINOVIRUS INFECTION IN INDIVIDUALS WITH ASTHMA
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Recent work indicates that individuals with asthma are more susceptible to naturally occurring rhinovirus infection than normal individuals in that lower respiratory tract symptoms and changes in peak expiratory flow were both more severe and of longer duration in the subjects with asthma than in the normal subjects (32). However, the reasons for this increased susceptibility in individuals with asthma have to date been largely unknown.
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ROLE OF TYPE 1 INTERFERONS AND APOPTOSIS IN INNATE ANTIVIRAL IMMUNITY
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Using primary bronchial epithelial cells from volunteers with asthma and normal volunteers, we have recently described a novel mechanism for increased susceptibility to rhinovirus infection in the volunteers with asthma. Primary bronchial epithelial cells from subjects with asthma were cultured ex vivo and infected with rhinovirus 16. Asthmatic bronchial epithelial cells were found to have a defect in innate immune responses to rhinovirus infection, with profoundly impaired production of IFN-ß and apoptosis in response to virus infection, resulting in increased virus replication (25). These studies have identified IFN-ß as a novel therapeutic approach to augment antiviral immunity in the treatment/prevention of rhinovirus-induced asthma exacerbations.
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ARE TYPE 1 ACQUIRED IMMUNE RESPONSES IMPAIRED IN VIVO IN ASTHMA?
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In addition to innate immune responses, acquired immune responses are also important in contributing to antiviral immunity. An important component of the acquired antiviral immune response is the production of cytokines such as IFN-
by Th1 and/or Tc1 T cells. IFN- has a number of potentially antiviral actions, including the activation of alveolar macrophages, and it interacts with other antiviral cytokines such as tumor necrosis factor-
to bring about airway epithelial cell apoptosis favoring efficient elimination of rhinovirus infection (33).
There is some evidence for a deficiency in Th1 cytokine production in response to rhinovirus in asthma. We have compared the responses of peripheral blood mononuclear cells (PBMCs) from atopic subjects with asthma and nonatopic normal subjects to exposure to rhinovirus in vitro. Exposure of PBMCs in vitro to rhinovirus resulted in the induction of IFN-, IL-10, IL-12, and IL-13 assessed by protein levels in supernatants in both subject groups. IL-4 was induced only in subjects with asthma, and PBMCs from subjects with asthma produced significantly lower levels of IFN- and IL-12 and higher levels of IL-10 than PBMCs from normal healthy volunteers (34).
The relevance of PBMC responses to rhinovirus in vitro to antiviral responses during acute infection in the respiratory tract is not known. However, a further study carried out solely in subjects with asthma suggested that there was indeed clinical and biologic relevance, as there was an inverse correlation between induction of nonspecific airway hyperresponsiveness by rhinovirus infection and levels of rhinovirus induced IFN- in PBMC cultures (35). This indirect evidence of impaired type 1 acquired immune responses to rhinovirus in asthma certainly justifies further studies investigating whether this is true in the airway during acute infection.
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STUDIES OF AIRWAY TYPE 1 AND TYPE 2 RESPONSES TO RHINOVIRUS IN SUBJECTS WITH ASTHMA AND IN NORMAL SUBJECTS
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We have previously reported increased numbers of both CD4 and CD8 T cells in the bronchial mucosa in response to rhinovirus infection in both subjects with asthma and in normal subjects (29). However, there were no differences between subject groups in the numbers of lymphocytes recruited to the bronchial mucosa during the infection.
There is as yet no published study comparing type 1 and type 2 cytokine responses to rhinovirus infection in subjects with asthma and normal subjects. Gern and coworkers have suggested that such a study is necessary by reporting evidence solely in subjects with asthma that the balance of Th1/Th2 cytokine levels in the airway has an influence on both symptoms and virus clearance in vivo. In patients with asthma, sputum IFN-/IL-5 mRNA ratios correlated inversely with cold symptoms, whereas the ratio was higher by approximately one log in those that had cleared virus by Day 14, compared with those that still had virus detectable 14 days after rhinovirus inoculation (36). Further studies investigating the role of both innate and acquired immune responses in asthma exacerbations are clearly required to identify other candidates for the development of new therapies.
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ROLE OF VIRAL INFECTIONS AS PRECIPITANTS OF EXACERBATIONS OF COPD
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There has been a great deal of recent interest in the mechanisms of virus induced asthma exacerbations. This interest has been fuelled by the earlier epidemiologic studies demonstrating the importance of virus infection as precipitants of exacerbations. Acute exacerbations of COPD are also an enormous cause of morbidity and mortality, thus the etiology of COPD exacerbations is also of major interest.
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ROLE OF BACTERIAL INFECTIONS AS PRECIPITANTS OF EXACERBATIONS OF COPD
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A number of different studies have shown that bacterial infections are detected in potentially pathogenic quantities in around 50% of COPD exacerbations (37). However, bacteria can also be detected in potentially pathogenic quantities in around 30% of patients with COPD when stable (38, 39). These data indicate that the importance of the association of bacterial infection with exacerbations of COPD is still uncertain. Nonetheless, several studies have demonstrated clinical improvement with antibacterial therapy (40, 41). These data confirm a causal role for bacterial infection in a percentage of exacerbations, particularly those with purulent sputum (42). However, although antibiotics have a specific role in some acute exacerbations, the percentage in which they are useful is not likely to be more than 50% and their therapeutic impact is still disappointing (40, 42). Treatment of COPD exacerbations is therefore a major unmet medical need, and alternative etiologies other than/as well as bacterial infection need to be investigated.
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VIRUS INFECTIONS AS PRECIPITANTS OF EXACERBATIONS OF COPD
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Evidence of Association
As with adult asthma, there are relatively few studies on the association of respiratory viral infection with acute exacerbations of COPD. Studies employing standard virologic techniques have reported only low detection rates of up to 20% (43). However, a recent study employing PCR in nasal aspirates has implicated respiratory virus infections in 39% of acute exacerbations of COPD (44). This study also reported that virus-induced exacerbations were more severe and took longer to recover from than those in which no virus was detected, suggesting that viruses may be an important trigger for COPD exacerbations and that their role has previously been underestimated.
This interpretation is supported by a study by the same authors that employed PCR for rhinoviruses in both nasal aspirates and in sputum samples from the same patients. This study demonstrated that rhinoviruses were detected more often in sputum than in nasal samples (45), suggesting that the 39% detection rate obtained in nasal samples (44) was likely to be a considerable underestimate of the true contribution. This interpretation has been confirmed by a recent study involving PCR detection of viruses in both nasal and sputum samples, in which viruses were detected in 56% of acute exacerbations of COPD requiring hospitalization (46).
Evidence of Underestimation of the Role of Viruses in Association Studies
Epidemiologic assessment of the relationship between virus infection and COPD exacerbations depends on sampling for viruses when patients report lower respiratory tract symptoms (43–46). Sampling the upper respiratory tract for viruses when patients present with lower respiratory tract symptoms may give a falsely low detection rate, as sampling may occur well after the peak of virus load has passed in the upper respiratory tract. Further evidence to support this interpretation comes from the East London COPD study in which prospective diary card recordings indicated that symptoms of a common cold were associated with 65% of exacerbations, whereas virus detection was only positive in 39% (44). These data strongly suggest the true association of respiratory virus infection with COPD exacerbations is likely to be higher than reported and is likely in the region of two thirds. Further investigation into the mechanisms of respiratory virus induction of COPD exacerbations could lead to the development of new therapies for the substantial majority of COPD exacerbations.
Evidence of Causation
These studies provide evidence of an association between viral infection and acute exacerbations of COPD, and indicative evidence of a causal relationship, in that there was an association with exacerbation severity. However, a causal role for viral infection in inducing acute exacerbations of COPD has not yet been established.
Evidence of a causal role would be strengthened by the demonstration that experimental infection of patients with stable COPD with a respiratory virus infection induces an exacerbation of COPD. Studies of this kind have been carried out for many years in investigating the etiology and pathogenesis of exacerbations of asthma. However, studies of experimental virus infection in patients with COPD have never previously been carried out.
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VIRUS TYPES PRECIPITATING ACUTE EXACERBATIONS OF COPD
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Although all respiratory viruses have been detected in association with exacerbations of COPD, in all the above studies, rhinoviruses were numerically the most important virus type, accounting for around 66% of infections detected (44–46). The studies of antiviral immunity to, and proinflammatory mechanisms of, rhinovirus infection in asthma set a precedent suggesting that similar models of COPD exacerbation could be invaluable in investigating the molecular mechanisms of COPD exacerbations. Further, identification of such mechanisms could lead to development of novel therapeutic approaches.
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LACK OF EFFICACY OF AVAILABLE TREATMENT FOR ACUTE EXACERBATIONS OF COPD
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Treatments for acute exacerbations of COPD include inhaled bronchodilators and systemic/oral steroids. These treatments are partially effective but have significant side effects and do not address the cause of the exacerbation, nor specific mechanisms involved in its pathogenesis. Furthermore, the therapeutic effect of steroids in COPD exacerbations is disappointing—reducing the absolute treatment failure rate by only about 10%, increasing FEV1 by only about 100 ml, and shortening the hospital stay by 1 to 2 days (47).
Antibiotics have a specific role in some acute exacerbations of COPD, especially in those with more severely impaired lung function and in which purulent sputum is a feature; however, the overall treatment effect of antibiotic therapy during COPD exacerbations is disappointing (40, 42).
As with asthma, current therapy is also disappointing in the prevention of COPD exacerbations. Even optimal therapy with combined long-acting ß-agonists and inhaled corticosteroids, or with the long-acting anticholinergic tiotropium, are only able to reduce exacerbation frequency by around 20% (48, 49). Although this is an important therapeutic effect, it leaves the vast majority of exacerbations still occurring despite best available treatment.
Thus also similar to the situation with asthma, the major cause of morbidity and mortality in COPD is largely unresponsive to currently available therapies, and a new approach to therapy is required. Again, before such alternative therapies can be developed, we must seek a detailed understanding of the mechanisms of COPD exacerbations. Specifically, targeting of molecular and cellular pathways involved in virus induction of lower airway inflammation may identify new targets for therapy.
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AIRWAY INFLAMMATION IN COPD
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There is a specific pattern of inflammation in the airways and lung parenchyma of patients with COPD characterized by increased numbers of neutrophils, macrophages, and lymphocytes. This inflammation predominantly affects small airways and lung parenchyma and increases in intensity with the severity of the disease (50, 51). This inflammatory response may underlie the accelerated loss of lung function that is characteristic of COPD and is increased by exacerbations (52). The inflammation found in COPD appears to be an amplification of the normal inflammatory response to inhaled noxious agents (cigarette smoke or other irritants) with increased secretion of neutrophil products such as IL-8, Gro, and matrix metalloproteinase-9 (51). The molecular mechanisms of this exaggerated inflammatory response are not yet fully understood, nor is it understood how this exaggerated inflammation is further augmented during exacerbations. Understanding the molecular mechanisms of increased airway inflammation at acute exacerbation will likely provide novel targets for future therapy.
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AIRWAY INFLAMMATION IN COPD EXACERBATIONS
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Studying airway inflammation during acute exacerbations of COPD is difficult, as such studies require either bronchoscopy, or induced sputum induction at a time when patients are acutely ill. Although it is accepted that COPD exacerbations are associated with increased airway inflammation, there is conflicting data available on the nature of inflammatory changes occurring during acute exacerbations. Some studies report increased airway eosinophilia (53, 54), whereas others document increased markers of neutrophilic inflammation—the granulocyte activation marker MPO and the neutrophil chemoattractant IL-8—at exacerbation compared 
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ABSTRACT
ASSOCIATION OF VIRUS INFECTIONS...
