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Article |
National Heart and Lung Institute, Imperial College, London, United Kingdom
Abstract
Abstract I. Introduction II. Chronic Obstructive Pulmonary Disease as an Inflammatory Disease A. What is Chronic Obstructive Pulmonary Disease? B. Differences from Asthma C. Inflammatory Cells III. Lipid Mediators A. Prostanoids B. Leukotrienes C. Platelet-Activating Factor IV. Reactive Oxygen Species A. Formation B. Antioxidants C. Evidence for Increased Oxidative Stress D. Effects on Airway Function E. Effects of Antioxidants V. Nitric Oxide A. Formation B. Inhibition VI. Peptide Mediators A. Endothelins B. Bradykinin C. Tachykinins D. Complement Fragments VII. Chemokines A. Interleukin-8 B. Growth-Related Oncogene-{alpha} C. Epithelial Cell-Derived Neutrophil-Activating Peptide-78 D. CXC3 Chemokines E. Monocyte Chemoattractant Protein-1 F. Macrophage Inflammatory Protein G. Eosinophil-Selective Chemokines H. Lymphocyte-Selective Chemokines I. Dendritic Cell-Selective Chemokines J. CX3C Chemokines VIII. Cytokines A. Tumor Necrosis Factor-{alpha} B. Interleukin-1{beta} C. Interleukin-6 D. Interleukin-9 E. Granulocyte-Macrophage Colony Stimulating Factor F. Interleukin-10 G. Interleukin-12 H. Interleukin-13 I. Interleukin-17 J. Interferon-{gamma} IX. Growth Factors A. Transforming Growth Factors B. Epidermal Growth Factor C. Vascular-Endothelial Growth Factor D. Fibroblast Growth Factors X. Proteases A. Neutrophil Elastase B. Other Serine Proteases C. Cysteine Proteases D. Matrix Metalloproteinases E. Antiproteases XI. Conclusions
Chronic obstructive pulmonary disease (COPD) is a major and increasing global health problem that is now a leading cause of death. COPD is associated with a chronic inflammatory response, predominantly in small airways and lung parenchyma, which is characterized by increased numbers of macrophages, neutrophils, and T lymphocytes. The inflammatory mediators involved in COPD have not been clearly defined, in contrast to asthma, but it is now apparent that many lipid mediators, inflammatory peptides, reactive oxygen and nitrogen species, chemokines, cytokines, and growth factors are involved in orchestrating the complex inflammatory process that results in small airway fibrosis and alveolar destruction. Many proteases are also involved in the inflammatory process and are responsible for the destruction of elastin fibers in the lung parenchyma, which is the hallmark of emphysema. The identification of inflammatory mediators and understanding their interactions is important for the development of anti-inflammatory treatments for this important disease.
Chronic obstructive pulmonary disease (COPD1) is a major and increasing global health problem. It is predicted by the World Health Organization to become the third most common cause of death and the fifth most common cause of disability in the world by 2020 (Lopez and Murray, 1998
). Indeed, COPD is already the fourth most common cause of death and the only common cause of death in the United States that has increased over the last 30 years (Murray et al., 2001). Although there have been major advances in the understanding and management of asthma, COPD has been relatively neglected, and there are no current therapies that reduce the inevitable progression of this disease. However, because of the enormous burden of disease and escalating health care costs, which now exceed those of asthma by more than 3-fold, there is now renewed interest in the underlying cellular and molecular mechanisms (Barnes, 2000a) and a search for new therapies (Barnes, 2001c), resulting in a reevaluation of the disease (Barnes, 2002b, 2003).
Although many inflammatory mediators (now more than 100) have been identified in asthma (Barnes et al., 1998; Chung and Barnes, 1999), there is much less information about the production and role of mediators in COPD. COPD is a complex inflammatory disease that involves many different types of inflammatory and structural cells, all of which have the capacity to release multiple inflammatory mediators (Figs. 1 and 2). This suggests that mediator antagonists may have some potential as new therapies for COPD. However, because of the redundant effects of many inflammatory mediators, it unlikely that antagonism of a single mediator will provide major clinical benefit, as is the case in asthma. Although asthma and COPD both involve chronic inflammation in the respiratory tract, there are marked differences in the types of inflammatory cells involved and in the site of inflammation, making it likely that different patterns of mediators are involved. There is much less information available about the mediators of COPD than those of asthma. Unlike asthma, some patients with COPD also have systemic features of the disease, and these are also likely to be mediated via inflammatory mediators.
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II. Chronic Obstructive Pulmonary Disease as an Inflammatory Disease
A. What is Chronic Obstructive Pulmonary Disease?
COPD is characterized by slowly progressive development of airflow limitation that is poorly reversible, in sharp contrast to asthma where there is variable airflow obstruction that is usually reversible spontaneously or with treatment. A new definition of COPD has recently been adopted by the Global Initiative on Obstructive Lung Disease: "a disease state characterized by airflow limitation that is not fully reversible. The airflow limitation is usually progressive and associated with an abnormal inflammatory response of the lungs to noxious particles and gases" (www.goldcopd.com/workshop/index.html). For the first time, this definition encompasses the idea that COPD is a chronic inflammatory disease, and much of the recent research has focused on the nature of this inflammatory response.
COPD includes chronic obstructive bronchitis with fibrosis and obstruction of small airways, and emphysema with enlargement of airspaces and destruction of lung parenchyma, loss of lung elasticity, and closure of small airways (Fig. 3). The obstruction of peripheral airways due to inflammatory cell infiltration and fibrosis, together with inflammatory exudates in the lumen, correlate best with the severity of airflow obstruction, indicating the importance of chronic inflammation in COPD (Hogg et al., 2004). Chronic bronchitis, by contrast, is defined by a productive cough of more than 3 months' duration for more than two successive years; this reflects mucus hypersecretion and is not necessarily associated with airflow limitation. Most patients with COPD have all three pathological mechanisms (chronic obstructive bronchitis, emphysema, and mucus plugging) as all are induced by smoking, but they may differ in the proportion of emphysema and obstructive bronchitis. In developed countries, cigarette smoking is by far the most common cause of COPD, but there are several other risk factors, including air pollution (particularly indoor air pollution from burning fuels), poor diet, and occupational exposure. COPD is characterized by acceleration in the normal decline of lung function seen with age (Fig. 4). The slowly progressive airflow limitation leads to disability and premature death and is quite different from the variable airway obstruction and symptoms in asthma, which rarely progresses in severity.
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Although COPD and asthma both involve inflammation in the respiratory tract, there are marked differences in the nature of the inflammatory process, with differences in inflammatory cells, mediators, response to inflammation, anatomical distribution, and response to anti-inflammatory therapy (Barnes, 2000b; Saetta et al., 2001) (Fig. 5). Some patients appear to share the characteristics of COPD and asthma, however. Rather than this representing a graded spectrum of disease, it is more likely that these patients have both of these common diseases at the same time.
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Histopathological studies show a predominant involvement of peripheral airways (bronchioles) and lung parenchyma, whereas asthma involves inflammation in all airways but without involvement of the lung parenchyma. There is obstruction of bronchioles, with fibrosis and infiltration with macrophages and T lymphocytes. There is destruction of lung parenchyma, as well as an increased number of macrophages and CD8+ (cytotoxic) T lymphocytes (Saetta et al., 1998). Bronchial biopsies show similar changes with an infiltration of macrophages and CD8+ cells and an increased number of neutrophils in patients with severe COPD (Di Stefano et al., 1998). Bronchoalveolar lavage (BAL) fluid and induced sputum demonstrate a marked increase in macrophages and neutrophils (Keatings et al., 1996; Pesci et al., 1998). In contrast to asthma, eosinophils are not prominent except during exacerbations or when patients have concomitant asthma (Fabbri et al., 1998, 2003).
COPD is a complex inflammatory disease that involves several types of inflammatory cells (Barnes et al., 2003). Although abnormal numbers of inflammatory cells have been documented in COPD, the relationship between these cell types and the sequence of their appearance and their persistence are largely unknown. Most studies have been cross-sectional based on selection of patients with different stages of the disease, and comparisons have been made between smokers without airflow limitation (normal smokers) and those with COPD who have smoked a similar amount. There are no serial studies, and selection biases (such as selecting tissue from patients suitable for lung volume reduction surgery) may give misleading results. Analysis of the cell profile in alveoli and small airways shows an increase in all of the cell types implicated in COPD, including macrophages, T lymphocytes, B lymphocytes, and neutrophils (Retamales et al., 2001).
1. Neutrophils. Increased numbers of activated neutrophils are found in sputum and BAL fluid of patients with COPD (Lacoste et al., 1993; Keatings et al., 1996), yet they are increased relatively little in the airways or lung parenchyma (Finkelstein et al., 1995). This may reflect their rapid transit through the airways and parenchyma. Neutrophils secrete serine proteases, including neutrophil elastase (NE), cathepsin G, and proteinase-3, as well as matrix metalloproteinase (MMP)-8 and MMP-9, which may contribute to alveolar destruction. These serine proteases are also potent mucus stimulants. Neutrophil recruitment to the airways and parenchyma involves adhesion to endothelial cells, and Eselectin is up-regulated on endothelial cells in the airways of COPD patients (Di Stefano et al., 1994). Adherent neutrophils then migrate into the respiratory tract under the direction of neutrophil chemotactic factors, which include interleukin (IL)-8 and leukotriene B4 (LTB4). Neutrophil survival in the respiratory tract may be increased by cytokines, such as granulocyte-macrophage colony stimulating factor (GM-CSF) and granulocyte colony stimulating factor.
The role of neutrophils in COPD is not yet clear. There is a correlation between the number of circulating neutrophils and fall in forced expiratory volume in 1 s (FEV1) (Sparrow et al., 1984). Neutrophil numbers in bronchial biopsies and induced sputum are correlated with COPD disease severity (Keatings et al., 1996; Di Stefano et al., 1998) and with the rate of decline in lung function (Stanescu et al., 1996). Smoking has a direct stimulatory effect on granulocyte production and release from the bone marrow, possibly mediated by GM-CSF and granulocyte colony stimulating factor released from lung macrophages (Terashima et al., 1997). Smoking may also increase neutrophil retention in the lung (MacNee et al., 1989). There is no doubt that the neutrophils recruited to the airways of COPD patients are activated, since there are increased concentrations of granule proteins, such as myeloperoxidase and human neutrophil lipocalin, in the sputum supernatant (Keatings and Barnes, 1997; Yamamoto et al., 1997; Peleman et al., 1999). These neutrophils also show an increase in the respiratory burst response, which correlates with the degree of airflow limitation (Richards et al., 1989).
Neutrophils have the capacity to induce tissue damage through the release of serine proteases and oxidants. Priming is a prerequisite for degranulation and superoxide anion generation in neutrophils (Condliffe et al., 1998). Neutrophils in the peripheral circulation show evidence of priming in COPD (Noguera et al., 2001), but this may result from, rather than contribute to, lung pathophysiology. There are several chemotactic signals that have the potential for neutrophil recruitment in COPD, including LTB4, IL-8, and related CXC chemokines, which are increased in COPD airways (Tanino et al., 2002; Traves et al., 2002). These mediators may be derived from alveolar macrophages and epithelial cells, but the neutrophil itself is a major source of IL-8 (Bazzoni et al., 1991).
Neutrophils from the circulation marginate in the pulmonary circulation and adhere to endothelial cells in the alveolar wall before passing into the alveolar space (Hogg and Walker, 1995). The precise route for neutrophil migration in large airways is less certain, but it is more likely that they reach the airway from the tracheobronchial circulation and migrate across postcapillary venules (Pettersen and Adler, 2002). The cellular mechanisms underlying neutrophil adhesion and transmigration differ between systemic and pulmonary circulations, and this might confer different properties on the neutrophils arriving from the alveolar or bronchial compartments. There may be significant differences in neutrophil transit times in different areas of the lung that may account for differential distribution of emphysema, for example, the upper lobe predominance in centrilobular emphysema. Little is known about survival and apoptosis of neutrophils in COPD airways. Theoretically, GM-CSF may prolong neutrophil survival, but it has proven difficult to culture neutrophils from sputum samples.
However, although neutrophils have the capacity to cause elastolysis, this is not a prominent feature of other pulmonary diseases where chronic airway neutrophilia is even more prominent, including cystic fibrosis and bronchiectasis. This suggests that other factors are involved in the generation of emphysema. Indeed, there is a negative association between the number of neutrophils and the amount of alveolar destruction in COPD (Finkelstein et al., 1995), and neutrophils are not a prominent feature of parenchymal inflammation in COPD. However, it is likely that airway neutrophilia is linked to mucus hypersecretion in chronic bronchitis. Serine proteases from neutrophils, including neutrophil elastase, cathepsin G, and proteinase-3, are all potent stimulants of mucus secretion from submucosal glands and goblet cells in the epithelium (Sommerhoff et al., 1990; Witko-Sarsat et al., 1999).
2. Macrophages. Macrophages appear to play a pivotal role in the pathophysiology of COPD and can account for most of the known features of the disease (Shapiro, 1999; Barnes, 2004) (Fig. 6).
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There is a marked increase (5- to 10-fold) in the numbers of macrophages in airways, lung parenchyma, BAL fluid, and sputum in patients with COPD. A careful morphometric analysis of macrophage numbers in the parenchyma of patients with emphysema showed a 25- fold increase in the numbers of macrophages in the tissue and alveolar space compared with normal smokers (Retamales et al., 2001). Furthermore, macrophages are localized to sites of alveolar wall destruction in patients with emphysema (Finkelstein et al., 1995; Meshi et al., 2002). There is a correlation between macrophage numbers in the airways and the severity of COPD (Di Stefano et al., 1998).
Macrophages may be activated by cigarette smoke extract to release inflammatory mediators, providing a cellular mechanism that links smoking with inflammation in COPD. Alveolar macrophages also secrete elastolytic enzymes, including MMP-2, MMP-9, MMP-12, cathepsins K, L, and S, and neutrophil elastase taken up from neutrophils (Punturieri et al., 2000; Russell et al., 2002b). Alveolar macrophages from patients with COPD secrete more inflammatory proteins and have a greater elastolytic activity at baseline than those from normal smokers, and this is further increased by exposure to cigarette smoke (Lim et al., 2000b; Russell et al., 2002a, b). Macrophages demonstrate this difference even when maintained in culture for 3 days and therefore appear to be intrinsically different from the macrophages of normal smokers and nonsmoking normal control subjects (Russell et al., 2002b). The predominant elastolytic enzyme secreted by alveolar macrophages in COPD patients is MMP-9. Most of the inflammatory proteins that are up-regulated in COPD macrophages are regulated by the transcription factor nuclear factor-
B (NF-B), which is activated in alveolar macrophages of COPD patients, particularly during exacerbations (Di Stefano et al., 2002; Caramori et al., 2003).
The increased numbers of macrophages in smokers and COPD patients may be due to increased recruitment of monocytes from the circulation in response to monocyte-selective chemokines and T lymphocytes via the release of lymphocyte chemotactic factors. The increased numbers of macrophages in COPD may also be due to increased proliferation and prolonged survival in the lungs. Macrophages have a very low proliferation rate in the lungs, but we have demonstrated that there is some increase in cell proliferation measured by proliferative cell nuclear antigen (Tomita et al., 2002). Macrophages have a long survival time, so this is difficult to measure directly. However, in macrophages from smokers, there is markedly increased expression of the antiapoptotic protein Bcl-XL and increased expression of p21CIP/WAF1 in the cytoplasm (Tomita et al., 2002). This suggests that macrophages may have a prolonged survival in smokers and patients with COPD.
Corticosteroids are ineffective in suppressing inflammation, including cytokines, chemokines, and proteases, in patients with COPD (Keatings et al., 1997; Culpitt et al., 1999). In vitro the release of IL-8, TNF-
, and MMP-9 macrophages from normal subjects and normal smokers are inhibited by corticosteroids, whereas corticosteroids are ineffective in macrophages from patients with COPD (Culpitt et al., 2003). Curiously, this does not apply to GM-CSF, which does not appear to have increased secretion in COPD and is suppressed by corticosteroids, albeit to a lesser extent than in macrophages from normal smokers. The reasons for resistance to corticosteroids in COPD and, to a lesser extent, macrophages from smokers may be the marked reduction in activity of histone deacetylase (HDAC) (Ito et al., 2001a), which is recruited to activated inflammatory genes by glucocorticoid receptors to switch off inflammatory genes (Ito et al., 2000). The reduction in HDAC activity in macrophages is correlated with increased secretion of cytokines like TNF- and IL-8 and reduced response to corticosteroids. The reduction of HDAC activity on COPD patients may be mediated through oxidative stress and peroxynitrite formation.
Macrophages are phagocytic for bacteria and play an important role in host defense. The phagocytic potential of macrophages from COPD patients has not been explored, but it is possible that impaired phagocytosis may result in the increased bacterial load in the respiratory tract of patients with COPD. Macrophages recognize apoptotic cells via expression of phosphatidylserine, which interacts with specific receptors on the macrophage surface (Fadok et al., 2000). Ingestion of apoptotic granulocytes by macrophages induces the secretion of transforming growth factor (TGF)-
1 (Huynh et al., 2002). Neutrophil elastase cleaves the phosphatidylserine receptor and may thus impair the ability of macrophages to take up apoptotic neutrophils, resulting in increased numbers of apoptotic neutrophils in the airways (Vandivier et al., 2002).
3. T Lymphocytes. There is an increase in the total numbers of T lymphocytes in lung parenchyma and peripheral and central airways of patients with COPD, with the greater increase in CD8+ than in CD4+ cells (Finkelstein et al., 1995; O'Shaughnessy et al., 1997; Saetta et al., 1999; Majo et al., 2001; Retamales et al., 2001). There is a correlation among the number of T cells, the amount of alveolar destruction, and the severity of airflow obstruction. There is also an increase in the absolute number of CD4+ T cells, but the ratio of CD4+/CD8+ cells is reversed in COPD. This is mainly found in smokers with COPD rather than smokers without evidence of airflow limitation (Majo et al., 2001). It is not known whether these cells are classified as Tc1 (interferon-
producing) or Tc2 (IL-4 producing) subtypes (Vukmanovic-Stejic et al., 2000), but there is evidence that the majority of T cells in COPD airways are of the Tc1 subtype (Saetta et al., 2002). CD8+ and CD4+ T cells show increased expression of activation markers compared with T cells in the circulation, although there is no clear difference between patients with COPD and normal controls (Leckie et al., 2003).
The mechanisms by which CD8+ and, to a lesser extent, CD4+ cells accumulate in the airways and lungs of patients with COPD are not yet understood. However, homing of T cells to the lung must depend on some initial activation followed by adhesion and selective chemotaxis. T cells in peripheral airways of COPD patients show increased expression of CXCR3, and there is increased secretion of CXCR3-activating chemokines in COPD airways (Saetta et al., 2002).
There is also an increase in the numbers of CD8+ cells in the circulation in COPD patients who do not smoke (de Jong et al., 1997; Kim et al., 2002) and an increase in Th1 type [interferon (IFN)- producing] CD4+ cells in COPD patients (Majori et al., 1999). This indicates that there may be chronic immune stimulation via antigens presented via the human leukocyte antigen class 1 pathway. Dendritic Cells may migrate from the airways to regional lymph nodes and stimulate proliferation of CD8+ and CD4+ T cells. CD8+ cells are typically increased in airway infections, and it is possible that the chronic colonization of the lower respiratory tract of COPD patients by bacterial and viral pathogens is responsible for this inflammatory response (Hill et al., 2000). It is also possible that protease-induced lung injury may uncover previously sequestered autoantigens or that cigarette smoke itself may damage airway epithelial cells and make them antigenic (Cosio et al., 2002).
The role of increased numbers of CD4+ cells in COPD, particularly in severe disease, is also unknown (Retamales et al., 2001); it is possible that they have immunological memory and play a role in perpetuating the inflammatory process in the absence of cigarette smoking. Natural killer (NK; CD56+) cells are the first line of defense against viral infections. Circulating NK cells are reduced in patients with COPD and have reduced phagocytic activity (Prieto et al., 2001), and similar findings are found in normal smokers (Zeidel et al., 2002), although no difference in NK cells was found in lung parenchyma of COPD patients (Majo et al., 2001). There is an increase in /
T cells in alveoli of smokers, whether they have airway obstruction or not (Majo et al., 2001).
The role of T cells in the pathophysiology of COPD is not yet certain. CD8+ cells have the capacity to cause cytolysis and apoptosis of alveolar epithelial cells through release of perforins, granzyme-B, and TNF- (Hashimoto et al., 2000). There is an association between CD8+ cells and apoptosis of alveolar cells in emphysema (Majo et al., 2001). In a mouse model of cigarette-induced emphysema, there is a predominance of T cells, which are directly related to the severity of emphysema (Takubo et al., 2002).
4. Eosinophils. The role of eosinophils in COPD is uncertain. There are some reports of increased numbers of inactive eosinophils in the airways and lavage of patients with stable COPD, whereas others have not found increased numbers in airway biopsies, BAL, or induced sputum (Turato et al., 2001). The presence of eosinophils in patients with COPD predicts a response to corticosteroids and may indicate coexisting asthma (Brightling et al., 2000; Papi et al., 2000). Increased numbers of eosinophils have been reported in bronchial biopsies and BAL fluid during acute exacerbations of chronic bronchitis (Saetta et al., 1994, 1996). Surprisingly, the levels of eosinophil basic proteins in induced sputum are as elevated in COPD as in asthma, despite the absence of eosinophils, suggesting that they may have degranulated and are no longer recognizable by microscopy (Keatings and Barnes, 1997). Perhaps this is due to the high levels of neutrophil elastase that have been shown to cause degranulation of eosinophils (Liu et al., 1999).
5. Dendritic Cells. Dendritic cells play a central role in the initiation of the innate and adaptive immune response (Banchereau et al., 2000). The airways and lungs contain a rich network of dendritic cells that are localized near the surface, so they are ideally located to signal the entry of foreign substances that are inhaled (Holt and Stumbles, 2000). Dendritic cells can activate a variety of other inflammatory and immune cells, including macrophages, neutrophils, and T and B lymphocytes (Huang et al., 2001). It therefore likely that the dendritic cell may play an important role in the pulmonary response to cigarette smoke and other inhaled noxious agents and may therefore be a key cellular element in COPD. The mechansism by which tobacco smoke activates the immune system is not yet understood, but a glycoprotein isolated from tobacco has powerful immunostimulatory actions (Francus et al., 1988). There is an increase in the number of dendritic cells in rat lungs exposed to cigarette smoke (Zeid and Muller, 1995) and in the airways and alveolar walls of smokers (Casolaro et al., 1988; Soler et al., 1989). Pulmonary histiocytosis is a disease caused by dendritic cell granulomata in the lung and is characterized by destruction of the lung parenchyma, which resembles emphysema (Tazi et al., 1999, 2000). The adult form of the disease occurs almost exclusively in smokers. In mice exposed to chronic cigarette smoke, there is an increase in dendritic cells in the airways and lung parenchyma (D'Hulst et al., 2002). The role of dendritic cells in recruiting other effector cells in COPD deserves further study.
6. Epithelial Cells. Airway and alveolar epithelial cells may be an important source of inflammatory mediators and proteases in COPD. Epithelial cells are activated by cigarette smoke to produce inflammatory mediators, including TNF-, IL-1, GM-CSF, and IL-8 (Mio et al., 1997; Hellermann et al., 2002; Floreani et al., 2003). Epithelial cells in small airways may be an important source of TGF-, which then induces local fibrosis (Takizawa et al., 2001). Vascular-endothelial growth factor (VEGF) appears to be necessary to maintain alveolar cell survival, and blockade of VEGF receptors (VEGFR2) in rats induces apoptosis of alveolar cells and an emphysema-like pathology (Kasahara et al., 2000).
Airway epithelial cells are also important in defense of the airways. Mucus produced from goblet cells traps bacteria and inhaled particulates (Adler and Li, 2001). Epithelial cells secrete defensins and other cationic peptides with antimicrobial effects and are part of the innate defense system but are also involved in tissue repair processes (Aarbiou et al., 2002). They secrete antioxidants as well as antiproteases, such as secretory leukoprotease inhibitor (SLPI). Epithelial cells also transport IgA and are therefore involved in adaptive immunity (Pilette et al., 2001). It is possible that cigarette smoke and other noxious agents impair these innate and adaptive immune responses of the airway epithelium, thereby increasing susceptibility to infection.
The airway epithelium in chronic bronchitis and COPD often shows squamous metaplasia, which may result from increased proliferation of airway epithelial cells. Proliferation in basal airway epithelial cells, measured by proliferative cell nuclear antigen, is increased in some normal smokers but is markedly increased in patients with chronic bronchitis and correlates with the degree of squamous metaplasia (Demoly et al., 1994). The nature of the growth factors involved in epithelial cell proliferation, cell cycle, and differentiation in COPD are not yet known. Epithelial growth factor receptors show increased expression in airway epithelial cells of smokers and may contribute to basal cell proliferation, resulting in squamous metaplasia and an increased risk of bronchial carcinoma (Franklin et al., 2002).
As in asthma, lipid mediators derived from arachidonic acid may play an important role in the pathophysiology of COPD.
1. Prostaglandin E2. There is an increase in the concentration of prostaglandin (PG)E2 in exhaled breath of COPD patients (Montuschi et al., 2003) (Fig. 7). This is likely to be derived from cyclooxygenase-2 (COX-2), which is expressed in alveolar macrophages (Jiang et al., 2003). There is increased COX-2 expression in alveolar macrophages from patients with COPD compared with normal control subjects (Taha et al., 2000). This is presumably a result of induction by inflammatory cytokines such as TNF- and IL-1, which activate NF-B, the key regulator of COX-2 (Newton et al., 1997). Inflammatory cytokines may also activate sphingomyelinase in the cell membrane to generate ceramide, which may also upregulate COX-2 independently of NF-B (Newton et al., 2000).
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PGE2 is a bronchodilator of human airways (Pavord and Tattersfield, 1995) and inhibits the release of proinflammatory cytokines from monocytes (Meja et al., 1997) and acetylcholine release from airway cholinergic nerves (via prostaglandin E3 receptors) (Spicuzza et al., 1998), suggesting that it may have beneficial effects in COPD airways. Furthermore, PGE2 markedly enhances the anti-inflammatory actions of phosphodiesterase-4 inhibitors, which are in clinical development as anti-inflammatory therapy for COPD (Au et al., 1998). However, PGE2 also has potentially detrimental effects in stimulating mucus secretion and expression of mucin genes (MUC5AC, MUCB) (Borchers et al., 1999) and in sensitizing and activating airway sensory nerves to enhance coughing (Stone et al., 1992; Lee et al., 2002). Inhalation of the nonselective COX inhibitor indomethacin is reported to reduce mucus hypersecretion in patients with COPD (Tamaoki et al., 1992), but long-term trials of COX inhibitors (and in particular COX-2 inhibitors) have not yet been undertaken.
2. Prostaglandin F2. PGF2 is also increased in exhaled breath condensate of COPD patients (Montuschi et al., 2003). PGF2 is a bronchoconstrictor and also activates airway sensory nerves to produce cough (Nichol et al., 1990).
3. Thromboxane. Thromboxane (Tx) B2 concentrations are not increased in exhaled breath of patients with COPD (Montuschi et al., 2003). However, the concentration of the major metabolite of thromboxane 11-dehydro-TxB2 is increased in the urine of patients with COPD, and this is correlated with the degree of hypoxia and reversed by supplementary oxygen therapy (Davi et al., 1997). The elevated concentrations of 11-dehdroxy-TxB2 are almost normalized by low doses of aspirin, indicating that they are likely to be derived from platelets. Thromboxane is a potent pulmonary vasoconstrictor and may contribute to the pulmonary hypertension in hypoxic COPD patients. A thromboxane receptor antagonist seratrodast reduces urinary 11-dehydro-TxB2 in COPD patients and is reported to reduce symptoms of dyspnea, but it has no effect on airway function (Horiguchi et al., 2002).
1. Leukotriene B4. Human alveolar macrophages express cytosolic phospholipase A2 and release LTB4 and platelet-activating factor on activation (Shamsuddin et al., 1997). LTB4 is increased in exhaled breath condensate of patients with stable COPD (Montuschi et al., 2003) (Fig. 7) and is further increased during exacerbations (Biernacki et al., 2003). LTB4 is also increased in the sputum of patients with COPD, particularly during exacerbations (Hill et al., 1999; Woolhouse et al., 2002; Beeh et al., 2003c). Plasma concentrations of LTB4 are also reported to be increased in COPD patients (Seggev et al., 1991). The cellular source of LTB4 in COPD is likely to be from alveolar macrophages and neutrophils.
LTB4 is a potent chemoattractant of neutrophils through the activation of BLT1 receptors that are expressed predominantly on neutrophils. BLT2 receptors are expressed on T lymphocytes (Yokomizo et al., 2000). BLT1 antagonists, such as LY29311 (2-[2-propyl-3-[3-[2-ethyl-4-(4-fluorophenyl)-5-hydroxphenoxy]propoxy]phenoxy]benzoic acid), have now been developed for the treatment of neutrophilic inflammation (Silbaugh et al., 2000). BLT1-receptor antagonists inhibits the neutrophil chemotactic activity of sputum from COPD patients, indicating the potential clinical value of such drugs (Crooks et al., 2000; Beeh et al., 2003c), but they only give about 25% inhibition, indicating that other neutrophil chemotactic factors are also involved. LTB4 antagonists have also been shown to reverse lipopolysaccharide-induced survival of neutrophils from COPD patients (Lee et al., 2000).
2. Cysteinyl-leukotrienes. Cysteinyl-leukotrienes are increased in asthma and largely derived from mast cells, but there is no evidence that they are increased in COPD. Thus, exhaled breath condensate shows an increase in concentration of cysteinyl-LTs in adults and children with asthma, but not in patients with COPD (Csoma et al., 2002; Montuschi and Barnes, 2002b; Montuschi et al., 2003). There is no scientific rationale for the use of cysteinyl-leukotriene receptor antagonists, such as montelukast, in the treatment of COPD. However, it has recently been reported that montelukast improves some of the symptoms of COPD, although there is no improvement in objective lung function measurements (Rubinstein et al., 2004). This might indicate an effect on some other aspects of COPD, such as mucus secretion.
Platelet-activating factor (PAF) is a potent chemoattractant and activator of neutrophils. PAF is also produced by and activates alveolar macrophages (Shindo et al., 1998). There are no reports of PAF production in COPD patients, and there are no studies of PAF antagonists, so the role of PAF in COPD remains unknown.
Oxidative stress is an important feature of COPD and there is increasing evidence that it is involved in its pathophysiology (Fig. 8). Oxidative stress occurs when reactive oxygen species (ROS) are produced in excess of the antioxidant defense mechanisms and result in harmful effects, including damage to lipids, proteins, and DNA. There is increasing evidence that oxidative stress is an important feature in COPD (Repine et al., 1997; Henricks and Nijkamp, 2001; MacNee, 2001).
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Inflammatory and structural cells that are activated in the airways of patients with COPD produce ROS, including neutrophils, eosinophils, macrophages, and epithelial cells (MacNee, 2001). Superoxide anions (O2·-) are generated by NADPH oxidase, and this is converted to hydrogen peroxide (H2O2) by superoxide dismutases. H2O2 is then dismuted to water by catalase. O2·- and H2O2 may interact in the presence of free iron to form the highly reactive hydroxyl radical (·OH). O2·- may also combine with NO to form peroxynitrite, which also generates ·OH (Beckman and Koppenol, 1996). Oxidative stress leads to the oxidation of arachidonic acid and the formation of a new series of prostanoid mediators called isoprostanes, which may exert significant functional effects (Morrow, 2000), including bronchoconstriction and plasma exudation (Kawikova et al., 1996; Okazawa et al., 1997; Janssen, 2001).
Granulocyte peroxidases, such as myeloperoxidase in neutrophils, play an important role in the generation of oxidative stress. In neutrophils, H2O2 generated from O2·- is metabolized by myeloperoxidase in the presence of chloride ions to hypochlorous acid, which is a strong oxidant. Myeloperoxidase is also able to nitrate tyrosine residues, as can peroxynitrite (Eiserich et al., 1998; van der Vliet et al., 1999a; Gaut et al., 2002).
The lung is also exposed to exogenous oxidants, which presumably summate with endogenous ROS production to further enhance oxidative stress in the lungs. Cigarette smoke itself is a potent source of oxidants, and the gas phase has been estimated to contain over 1015 free radicals (Pryor and Stone, 1993). Some forms of air pollution, including ozone, nitrogen dioxide, and diesel particulates, are also an oxidative stress and are associated with increased prevalence and increased numbers of exacerbations of COPD (Sunyer, 2001).
The normal production of oxidants is counteracted by several endogenous antioxidant mechanisms in the human respiratory tract (Cantin et al., 1990). Antioxidants may be enzymatic or nonenzymatic. The major enzymatic antioxidants in the airways are catalase, superoxide dismutase (SOD), glutathione peroxidase, glutathione S-transferase, xanthine oxidae, and thioredoxin. The nonenzymatic category of antioxidant defenses includes low molecular weight compounds such as glutathione, ascorbate, urate, -tocopherol, bilirubin, and lipoic acid. Concentrations of these antioxidants vary, depending on both subcellular and anatomic location. For example, glutathione is 100-fold more concentrated in the airway epithelial lining fluid compared with plasma (van der Vliet et al., 1999b). Oxidant stress activates the inducible enzyme heme oxygenase-1, converting heme and hemin to biliverdin with the formation of carbon monoxide (Choi and Alam, 1996). Biliverdin is converted via bilirubin reductase to bilirubin, which is a potential antioxidant. Heme oxygenase-1 is widely expressed in human airways (Lim et al., 2000a), and carbon monoxide production is increased in COPD (Montuschi et al., 2001). In the lung, intracellular antioxidants are expressed at relatively low levels and are not induced by oxidative stress, whereas the major antioxidants are extracellular (Comhair and Erzurum, 2002). Extracellular antioxidants, particularly glutathione peroxidase, are markedly up-regulated in response to cigarette smoke and oxidative stress. The glutathione system is the major antioxidant mechanism in the airways. There is a high concentration of reduced glutathione in lung epithelial lining fluid (Cantin et al., 1990), and concentrations are higher in cigarette smokers. Extracellular glutathione peroxidase is an important antioxidant in the lungs and may be secreted by epithelial cells and macrophages, particularly in response to cigarette smoke or oxidative stress (Avissar et al., 1996). Extracellular glutathione peroxidase inactivates H2O2 and O2·- but also reactive nitrogen species (Comhair and Erzurum, 2002). Extracellular antioxidants also include the dietary antioxidants vitamin C (ascorbic acid) and vitamin E (-tocopherol), uric acid, lactoferrin, and extracellular SOD3. SOD3 is highly expressed in human lung, but its role in COPD is not yet clear (Bowler and Crapo, 2002).
C. Evidence for Increased Oxidative Stress
There is considerable evidence for increased oxidative stress in COPD (Repine et al., 1997; MacNee, 2001). As discussed above, oxidant stress is derived from cigarette smoke and from inflammatory cells, such as activated macrophages and neutrophils. Epidemiological evidence indicates that reduced dietary intake of antioxidants may be a determinant of COPD, and population surveys have linked a low dietary intake of the antioxidant ascorbic acid with declining lung function (Britton et al., 1995; Schunemann et al., 2001).
1. Pulmonary Oxidative Stress. There is abundant evidence for increased oxidative stress in the lungs of patients with COPD. Using electromagnetic spin trapping, a marked increase in xanthine/xanthine oxidase activity has been detected in BAL fluid of COPD patients (Pinamonti et al., 1998). A specific marker lipid peroxidation, 4-hydoxy-2-nonenal, which forms adducts with basic amino acid residues in proteins, can be detected by immunocytochemistry and has been detected in lungs of patients with COPD (Rahman et al., 2002). This signature of oxidative stress is localized to airway and alveolar epithelial cells, endothelial cells, and neutrophils.
2. Exhaled Markers of Oxidative Stress. There are several markers of oxidative stress that may be detected in the breath, and several studies have demonstrated increased production of oxidants in exhaled air or breath condensates (Kharitonov and Barnes, 2001; Montuschi and Barnes, 2002a; Paredi et al., 2002). Ethane, a volatile hydrocarbon formed through lipid peroxidation, is increased in the breath of COPD patients, and the concentration correlates with disease severity (Paredi et al., 2000). There is an increased concentration of H2O2 in exhaled breath condensate of patients with COPD, particularly during exacerbations (Dekhuijzen et al., 1996; Nowak et al., 1999). There is also an increase in the concentration of 8-iso prostaglandin F2 (8-isoprostane) in exhaled breath condensate, which is found even in patients who are ex-smokers (Montuschi et al., 2000) and is increased further during acute exacerbations (Biernacki et al., 2003). 8-Isoprostane is also increased in the breath of normal smokers, but to a lesser extent than in COPD, suggesting that there is an exaggeration of oxidative stress in COPD. Malondialdehyde and thiobarbituric acid reactive substances, which are markers of lipid peroxidation, are also increased in exhaled breath condensate of patients with COPD (Nowak et al., 1999; Corradi et al., 2003).
3. Systemic Markers of Oxidative Stress. There is also evidence for increased systemic markers of oxidative stress in patients with COPD as measured by biochemical markers of lipid peroxidation (Rahman et al., 1996). Increased plasma concentrations of malondialdehyde have been reported in COPD patients (Calikoglu et al., 2002). 8-Isoprostane is increased in the urine of patients with COPD and further increased during exacerbations (Pratico et al., 1998). The interaction of O2·- and NO forms peroxynitrite, which forms stable 3-nitrotyrosine adducts, as a footprint of oxidative stress, as discussed below.
ROS have several effects on the airways, which would have the effect of increasing the inflammatory and destructive response in COPD. These effects may be mediated by direct actions of ROS on target cells in the airways but may also be mediated indirectly via activation of signal transduction pathways and transcription factors and via the formation of oxidized mediators, such as isoprostanes and hydroxyl-nonenal.
1. Effects on Transcription Factors. ROS activate NF-B, which switches on multiple inflammatory genes resulting in amplification of the inflammatory response (Barnes and Karin, 1997). The molecular pathways by which oxidative stress activates NF-B have not been fully elucidated, but there are several redox-sensitive steps in the activation pathway (Janssen-Heininger et al., 2000). Many of the stimuli that activate NF-B appear to do so via the formation of ROS, particularly H2O2. ROS activate NF-B in an epithelial cell line (Adcock et al., 1994) and increase the release of proinflammatory cytokines from cultured human airway epithelial cells (Rusznak et al., 1996). Oxidative stress results in activation of histone acetyltransferase activity, which opens up the chromatin structure and is associated with increased transcription of multiple inflammatory genes (Rahman, 2003; Tomita et al., 2003).
Another transcription factor that activates inflammatory genes is activator protein-1, a heterodimer of Fos and Jun proteins. As with NF-B, there are several redox-sensitive steps in the activation pathway (Xanthoudakis and Curran, 1996).
2. Effects on Signal Transduction Pathways. Oxidants also activate mitogen-activated protein (MAP) kinase pathways. H2O2 is a potent activator of extracellular regulated kinases (ERK) and p38 MAP kinase pathways that regulate the expression of many inflammatory genes and survival in certain cells and spreading of macrophages (Ogura and Kitamura, 1998). Indeed, many aspects of macrophage function are regulated by oxidants through the activation of multiple kinase pathways (Forman and Torres, 2002).
3. Effects on Target Cells. H2O2 directly constricts airway smooth muscle in vitro (Rhoden and Barnes, 1989), and hydroxyl radicals (OH-) potently induce plasma exudation in airways (Lei et al., 1996). 8-Isoprostane (or 8-epi-prostaglandin F2), the predominant isoprostane formed by the nonenzymatic oxidation of arachidonic acid in humans, is a potent constrictor of animal and human airways in vitro, an effect that is largely mediated via thromboxane receptors (Kawikova et al., 1996). In rat airways, oxidant stress increases cholinergic nerve-induced bronchoconstriction, an effect that may be due to oxidant damage of acetylcholinesterase (Ohrui et al., 1991). 8-Isoprostane also has direct effects on airway nerves (Spicuzza et al., 2001).
Little is known about the effects of ROS on the vasculature. ·OH potently induces plasma exudation in rodent airways (Lei et al., 1996), and 8-isoprostane is a potent inducer of plasma exudation in airways (Okazawa et al., 1997), but their effects on pulmonary vessels are not known.
In rats, oxidative stress increases airway mucus secretion, an effect that is blocked by cyclo-oxygenase inhibitors (Adler et al., 1990). The effect of oxidative stress may be mediated via the activation of epidermal growth factor receptors (EGFR) on submucosal glands (Takeyama et al., 2000). Neutrophil elastase is a potent stimulant of mucus secretion and increases the expression of mucin genes (MUC5AC); its effects are inhibited by dimethylthiourea, a purported scavenger of ·OH (Fischer and Voynow, 2000). Oxidative stress may induce proliferation of airway epithelial cells, and this effect also appears to be mediated via activation of EGFR (Tamaoki et al., 2004).
4. Effects on Inflammatory Response. The increased oxidative stress in the airways of COPD patients may play an important pathophysiological role in the disease by amplifying the inflammatory response in COPD. This may reflect the activation of NF-B and activator protein-1, which then induce a neutrophilic inflammation via increased expression of IL-8 and other CXC chemokines, TNF- and MMP-9. Oxidative stress may therefore serve to amplify the ongoing chronic inflammatory response in COPD and may be an important mechanism leading to increased inflammation during acute exacerbations.
5. Effect on Proteases. Oxidative stress may also impair the function of antiproteases such as 1-antitrypsin and SLPI and thereby accelerates the breakdown of elastin in lung parenchyma (Taggart et al., 2000).
6. Effect on Steroid Responsiveness. Corticosteroids are much less effective in COPD than in asthma and do not reduce the progression of the disease (Pauwels et al., 1999; Vestbo et al., 1999; Burge et al., 2000; Lung Health Study Research Group, 2000). In contrast to patients with asthma, those with COPD do not show any significant anti-inflammatory response to corticosteroids (Keatings et al., 1997; Culpitt et al., 1999). Alveolar macrophages from patients with COPD show a marked reduction in responsiveness to the anti-inflammatory effects of corticosteroids, compared with cells from normal smokers and nonsmokers (Culpitt et al., 2003). Recent studies suggest that there may be a link between oxidative stress and the poor response to corticosteroids in COPD. Oxidative stress impairs binding of glucocorticoid receptors to DNA and the translocation of these receptors from the cytoplasm to the nucleus (Hutchison et al., 1991; Okamoto et al., 1999). Corticosteroids switch off inflammatory genes by recruiting HDAC2 to the active transcription site, and by deacetylating the hyperacetylated histones of the actively transcribing inflammatory gene, they are able to switch off its transcription and thus suppress inflammation (Ito et al., 2000; Barnes et al., 2003). In cigarette smokers and patients with COPD, there is a marked reduction in activity of HDAC and reduced expression of HDAC2 in alveolar macrophages (Ito et al., 2001a) and an even greater reduction in HDAC2 expression in peripheral lung tissue (Ito et al., 2001b). This reduction in HDAC activity is correlated with reduced expression of inflammatory cytokines and a reduced response to corticosteroids. This may result directly or indirectly from oxidative stress and is mimicked by the effects of H2O2 in cell lines (Ito et al., 2001b).
7. Effects on Apoptosis. Oxidative stress may also induce apoptosis in endothelial and epithelial cells (Haddad, 2004). Apoptosis of type 1 pneumocytes may be contributory to the development of emphysema, and this might be induced by cytotoxic T lymphocytes or by inhibition of vascular-endothelial growth factor receptors (Kasahara et al., 2000; Majo et al., 2001). ROS may induce apoptosis by activating the NF-B pathway, by direct DNA damage via activation of poly-ADP-ribose, and via the generation of 4-hydroxy-nonenal. Apoptosis signal-regulating kinase-1 is held in an inactive conformation by thioredoxin, and when oxidized by ROS, this triggers apoptotic pathways (Gotoh and Cooper, 1998).
8. Systemic Effects. The systemic oxidative stress in COPD may contribute to the systemic effects seen in severe disease. For example, impaired redox balance in skeletal muscle cells may be contributory to the muscle weakness, fatigability, and wasting seen in some patients (Langen et al., 2003).
In view of the persuasive evidence presented above that oxidative stress is important in the pathophysiology of COPD, antioxidants are a logical approach to therapy (MacNee, 2000; Barnes, 2001c).
Several antioxidants have also been administered to patients with COPD to explore their effects on lung function. N-Acetyl cysteine was developed as a mucolytic agent but also acts as an antioxidant by increasing the formation of glutathione. Although small-scale trials failed to demonstrate any clear clinical benefit, more recent meta-analyses have shown a small but significant clinical benefit in COPD, particularly in reducing exacerbations (Grandjean et al., 2000; Poole and Black, 2001). This benefit is not shared by other mucolytics and is therefore likely to be due to the antioxidant effect of N-acetyl cysteine. These results should encourage the development of more effective antioxidants in the future. Currently available antioxidants are rather weak, but more potent drugs, including spin-trap antioxidants (nitrones) and stable glutathione analogs, are currently in clinical development (Cuzzocrea et al., 2001).
1. Nitric Oxide. NO is generated in COPD from the enzyme inducible NO synthase (iNOS), which is expressed in macrophages and lung parenchyma of patients with COPD, particularly in patients with severe disease (Ichinose et al., 2000; Maestrelli et al., 2003). NO is markedly increased in exhaled breath of patients with mild asthma, reflecting the inflammatory process in the airways, but in patients with COPD exhaled NO levels are little raised above normal (Maziak et al., 1998; Corradi et al., 1999; Rutgers et al., 1999) but are more clearly increased during exacerbations (Maziak et al., 1998; Agusti et al., 1999).
2. Peroxynitrite. The reason why exhaled NO may be elevated in COPD as much as in asthma may be because exhaled NO levels are depressed by cigarette smoking and oxidative stress, since NO combines avidly with superoxide anions to form peroxynitrite. This is supported by the fact that nitrate concentrations, formed by metabolism of peroxynitrite, are increased in breath condensate and sputum of cigarette smokers and patients with COPD (Corradi et al., 2001; Kanazawa et al., 2003c). Generation of superoxide anions from neutrophils also decreases the amount of NO formed by an epithelial cell line in vitro, as NO is consumed to form peroxynitrite (Jones et al., 1998). There is also a reduction in the undefined "peroxynitrite inhibitory activity" in sputum of COPD patients (Kanazawa et al., 2003c). Peroxynitrite reacts with tyrosine residues in certain proteins to form 3-nitrotyrosine, which may be detected immunologically. There is increased 3-nitrotyrosine immunoreactivity in sputum macrophages from patients with COPD (Ichinose et al., 2000). There is also an increase in the tyrosine nitration of proteins in sputum of COPD patients compared with normal controls, and this is correlated with disease severity (Sugiura et al., 2004).
Oxidative stress and peroxynitrite may also reduce HDAC2 levels, thereby inducing resistance to the antiinflammatory actions of corticosteroids (Barnes et al., 2004). This may be the result of nitration of critical tyrosine residues in the structure of HDAC2, which impairs its enzymatic activity (Ito et al., 2004
