Chronic Obstructive Pulmonary Disease (COPD), Inflammatory Mechanisms of
Peter J. Barnes, National Heart and Lung Institute, Imperial College, London, UK
Airway obstruction in chronic obstructive pulmonary disease (COPD) is caused by narrowing of small airways as a result of inflammation and fibrosis and the disruption of their alveolar attachments as a result of emphysema. COPD is characterized by a complex inflammatory disease process that increases as the disease progresses, which leads to increasing airflow limitation. Many inflammatory cells and mediators have now been implicated in the pathogenesis of COPD. Increased numbers of macrophages, neutrophils, T-lymphocytes (particularly CD8+ cells) and B-lymphocytes have been observed, as well as the release of multiple inflammatory mediators (lipids, chemokines, cytokines, growth factors). Macrophages seem to play an important role in orchestrating the inflammatory process, which includes the recruitment of neutrophils and T-cells into small airways and lung parenchyma and the secretion of proteinases that lead to emphysema. A high level of oxidative and nitrative stress may amplify this inflammation through the reduction in histone deacetylase-2, which also results in corticosteroid resistance.
Chronic obstructive pulmonary disease (COPD) has now become a major global epidemic, and it is predicted to become the third leading cause of death and fifth leading cause of disability over the next decade (1). The increase in COPD is a particular problem in developing nations; in developed countries, is the only common cause of death that is increasing. COPD now has a world-wide prevalence of over 10% in men and is increasing toward this figure in women (2). Because of the enormous burden of disease and escalating health-care costs, there is now renewed interest in the underlying cellular and molecular mechanisms of COPD (3) and a search for new therapies (4). The definition of COPD adopted by the Global initiative on Obstructive Lung Disease encompasses the idea that COPD is a chronic inflammatory disease, and much recent research has focused on the nature of this inflammatory response (5). COPD is an obstructive disease of the lungs that slowly progresses over many decades leading to death from respiratory failure unless patients die of comorbidities such as heart disease and lung cancer before this stage. Although the most common cause of COPD is chronic cigarette smoking, some patients, particularly in developing countries, develop the disease from inhalation of wood smoke from biomass fuels or other inhaled irritants (2). However, only about 25% of smokers develop COPD, which suggests that genetic or host factors may predispose patients to its development, although these factors have not yet been identified. The disease is relentlessly progressive, and only smoking cessation reduces the rate of decline in lung function; as the disease becomes more severe, there is less effect of smoking cessation, and lung inflammation persists.
COPD as an Inflammatory Disease
The progressive airflow limitation in COPD is caused by two major pathological processes: remodeling and narrowing of small airways and destruction of the lung parenchyma with consequent destruction of the alveolar attachments of these airways as a result of emphysema (Fig. 1). This disease results in diminished lung recoil, higher resistance to flow and closure of small airways at higher lung volumes during expiration, which traps air in the lung. This trapped air leads to the characteristic hyperinflation of the lungs, which causes the sensation of dyspnea and limits exercise capacity. The major symptom of COPD is shortness of breath on exertion. Both the small airway remodeling and narrowing and the emphysema are caused by chronic inflammation in the lung periphery. Quantitative studies have shown that the inflammatory response in small airways and lung parenchyma increases as the disease progresses (6). A specific pattern of inflammation in COPD airways and lung parenchyma is observed with increased numbers of macrophages, T-lymphocytes, with predominance of CD8+ (cytotoxic) T-cells, and in more severe disease B-lymphocytes with increased numbers of neutrophils in the lumen (3). The inflammatory response in COPD involves both innate and adaptive immune responses. Multiple inflammatory mediators are increased in COPD and are derived from inflammatory cells and structural cells of the airways and lungs (7). A similar pattern of inflammation is observed in smokers without airflow limitation, but in COPD, this inflammation is amplified even more during acute exacerbations of the disease, which are usually precipitated by bacterial and viral infections (Fig. 2). The molecular basis of this amplification of inflammation is not yet understood but may be, at least in part, determined by genetic factors. Cigarette smoke and other irritants in the respiratory tract may activate surface macrophages and airway epithelial cells to release chemotactic factors that then attract circulating leukocytes into the lungs. Among chemotactic factors, chemokines predominate and therefore play a key role in orchestrating the chronic inflammation in COPD lungs and its amplification during acute exacerbations (8). These events might be the initial inflammatory events that occur in all smokers. However in smokers who develop COPD, this inflammation progresses into a more complicated inflammatory pattern of adaptive immunity and involves T- and B-lymphocytes and possibly dendritic cells along with a complicated interacting array of cytokines and other mediators (9).
Figure 1. Small airways in COPD patients. The airway wall is thickened and infiltrated with inflammatory cells, predominately macrophages and CD8+ lymphocytes, with increased numbers of fibroblasts. In severe COPD, lymphoid follicles are observed, which consist of a central core of B-lymphocytes, surrounded by T-lymphocytes and are thought to indicate chronic exposure to antigens (bacterial, viral, or autoantigens). Similar changes are also reported in larger airways. The lumen is often filled with an inflammatory exudate and mucus. Peribronchial fibrosis occurs, and it results in progressive and irreversible narrowing of the airway. Airway smooth muscle may be increased slightly.
Figure 2. Amplification of lung inflammation in COPD. Normal smokers have a mild inflammatory response, which represents the normal (probably protective) reaction of the respiratory mucosa to chronic inhaled irritants. In COPD, this same inflammatory response is markedly amplified, and this amplification increases as the disease progresses. It is increased even more during exacerbations triggered by infective organisms. The molecular mechanisms of this amplification are currently unknown, but they may be determined by genetic factors or possibly latent viral infection. Oxidative stress is an important amplifying mechanism and may increase the expression of inflammatory genes through impairing the activity of histone deacetylase 2 (HDAC2), which is needed to switch off inflammatory genes.
Differences from asthma
Histopathological studies of COPD show a predominant involvement of peripheral airways (bronchioles) and lung parenchyma, whereas asthma involves inflammation in all airways (particularly proximal airways) but usually without involvement of the lung parenchyma (10). In COPD, the bronchioles become narrow, with fibrosis and infiltration with macrophages and T-lymphocytes, along with destruction of lung parenchyma and an increased number of macrophages and T-lymphocytes, with a greater increase in CD8+ (cytotoxic) than CD4+ (helper) cells (6) (Fig. 3). Bronchial biopsies show similar changes with an infiltration of macrophages and CD8+ cells and an increased number of neutrophils in patients with severe COPD. Bronchoalveolar lavage (BAL) fluid and induced sputum demonstrate a marked increase in macrophages and neutrophils. In contrast to asthma, eosinophils are not prominent except during exacerbations or when patients have concomitant asthma (10).
Figure 3. Inflammatory cells in COPD. Inhaled cigarette smoke and other irritants activate epithelial cells and macrophages to release several chemotactic factors that attract inflammatory cells to the lungs, including CC-chemokine ligand 2 (CCL2), which acts on CC-chemokine receptor 2 (CCR2) to attract monocytes, CXC-chemokine ligand 1 (CXCL1) and CXCL8, which act on CCR2 to attract neutrophils and monocytes (which differentiate into macrophages in the lungs) and CXCL9, CXCL10, and CXCL11, which act on CXCR3 to attract T-helper 1 (Th1) cells and type 1 cytotoxic T-cells (Tc1 cells). These inflammatory cells together with macrophages and epithelial cells release proteases, such as MMP-9, which cause elastin degradation and emphysema. Neutrophil elastase also causes mucus hypersecretion. Epithelial cells and macrophages also release TGF-β, which stimulates fibroblast proliferation and results in fibrosis in the small airways.
For many years it was believed that the inflammatory reaction in the lungs of smokers consisted of neutrophils and macrophages and that proteinases from these cells were responsible for the lung destruction in COPD. More recently it has been recognized that there is a prominent T-cell infiltration in the lungs of patients with COPD, with a predominance of CD8+ (cytotoxic) T-cells, although CD4+(helper) T-cells are also numerous. 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 not yet understood in detail (3). 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. No serial studies have been conducted, and selection biases (such as selecting tissue from patients suitable for lung volume reduction surgery) may give misleading results. Nonetheless, a progressive increase in the numbers of inflammatory cells in small airways and lung parenchyma are observed as COPD becomes more severe, even though the patients with most severe obstruction have stopped smoking for many years (6). This finding indicates the existence of some mechanisms that perpetuate the inflammatory reaction in COPD. This characteristic is in contrast to many other chronic inflammatory diseases, such as rheumatoid arthritis and interstitial lung diseases, in which the inflammation tends to diminish in severe disease. The inflammation of COPD lungs involves both innate immunity (neutrophils, macrophages, eosinophils, mast cells, NK cells, γδ-T-cells, and dendritic cells) and adaptive immunity (T and B cells).
Epithelial cells are activated by cigarette smoke to produce inflammatory mediators, which include tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and CXCL8 (IL-8). Epithelial cells in small airways may be an important source of transforming growth factor (TGF)-P, which then induces local fibrosis. Vascular endothelial growth factor (VEGF) seems to be necessary to maintain alveolar cell survival and blockade of VEGF receptors in rats induces apoptosis of alveolar cells and an emphysema-like pathology, which may be mediated via the sphingolipid ceramide (11). Airway epithelial cells are also important in defense of the airways, with mucus production from goblet cells, and secretion of antioxidants, antiproteases and defensins. It is possible that cigarette smoke and other noxious agents impair these innate and adaptive immune responses of the airway epithelium, which increases susceptibility to infection. The airway epithelium in chronic bronchitis and COPD often shows squamous metaplasia, which may result from increased proliferation of basal airway epithelial cells, but 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 (EGFR) show increased expression in airway epithelial cells of smokers and may contribute to basal cell proliferation, which results in squamous metaplasia and an increased risk of bronchial carcinoma (12).
Increased numbers of activated neutrophils are found in sputum and BAL fluid of patients with COPD (13), yet neutrophil levels are increased relatively little in the airways or lung parenchyma. This finding may reflect their rapid transit through the airways and parenchyma. The role of neutrophils in COPD is not yet clear; however, neutrophil numbers in induced sputum are correlated with COPD disease severity (13) and with the rate of decline in lung function. Smoking has a direct stimulatory effect on granulocyte production and release from the bone marrow and survival in the respiratory tract, which is possibly mediated by GM-CSF and G-CSF released from lung macrophages. Smoking may also increase neutrophil retention in the lung. Neutrophil recruitment to the airways and parenchyma involves adhesion to endothelial cells and E-selectin, which is upregulated on endothelial cells in the airways of COPD patients. Adherent neutrophils then migrate into the respiratory tract under the direction of neutrophil chemotactic factors. Several chemotactic signals have the potential for neutrophil recruitment in COPD, which include leukotriene (LT)B4, CXCL8, and related CXC chemokines, including CXCL1 (GRO-α) and CXCL5 (ENA-78), which are increased in COPD airways (14). These mediators may be derived form alveolar macrophages T-cells and epithelial cells, but the neutrophil itself may be a major source of CXCL8. Neutrophils from the circulation marginate in the pulmonary circulation and adhere to endothelial cells in the alveolar wall before passing into the alveolar space. The neutrophils recruited to the airways of COPD patients are activated because increased concentrations of granule proteins, such as myeloperoxidase and human neutrophil lipocalin, are found in the sputum supernatant (15). Neutrophils secrete serine proteases, which include neutrophil elastase, cathepsin G, and proteinase-3, as well as matrix metalloproteinase (MMP)-8 and MMP-9, which may contribute to alveolar destruction. Neutrophils have the capacity to induce tissue damage through the release of serine proteases and oxidants. However, whereas neutrophils have the capacity to cause elastolysis, this ability is not a prominent feature of other pulmonary diseases in which chronic airway neutrophilia is even more prominent, including cystic fibrosis and bronchiectasis. This comparison suggests that other factors are involved in the generation of emphysema. Indeed, neutrophils are not a prominent feature of parenchymal inflammation in COPD. It is likely that airway neutrophilia is more linked to mucus hypersecretion in chronic bronchitis. Serine proteases from neutrophils, which include neutrophil elastase, cathepsin G, and proteinase-3, are all potent stimulants of mucus secretion from submucosal glands and goblet cells in the epithelium. A marked increase in neutrophil numbers is observed in the airways in acute exacerbations of COPD, which accounts for the increased purulence of sputum. This finding may reflect increased production of neutrophil chemotactic factors, which include LTB4 and CXCL8 (16, 17)
Macrophages seem to play a pivotal role in the pathophysiology of COPD and can account for most of the known features of the disease (18) (Fig. 4). A marked increase (5-10-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 (19). Furthermore, macrophages are localized to sites of alveolar wall destruction in patients with emphysema, and a correlation is observed between macrophage numbers in the parenchyma and severity of emphysema (20). Macrophages may be activated by cigarette smoke extract to release inflammatory mediators, which includes TNF-α, CXCL8, and other CXC chemokines; CCL2 (MCP-1); LTB4; and reactive oxygen species. This release is a cellular mechanism that links smoking with inflammation in COPD. Alveolar macrophages also secrete elastolytic enzymes, which include MMP-2; MMP-9; MMP-12; cathepsins K, L, and S; and neutrophil elastase taken up from neutrophils (21). Alveolar macrophages from patients with COPD and with exposure to cigarette smoke secrete more inflammatory proteins and have a greater elastolytic activity at baseline than those from normal smokers (21). Macrophages demonstrate this difference even when maintained in culture for 3 days, and therefore they seem to be intrinsically different from the macrophages of normal smokers and nonsmoking normal control subjects (21). The predominant elastolytic enzyme secreted by alveolar macrophages in COPD patients is MMP-9. Most inflammatory proteins that are upregulated in COPD macrophages are regulated by the transcription factor nuclear factor-KB (NF-KB), which is activated in alveolar macrophages of COPD patients, particularly during exacerbations (22).
Figure 4. Macrophages in COPD. Macrophages may play a pivotal role in COPD as they are activated by cigarette-smoke extract and secrete many inflammatory proteins that may orchestrate the inflammatory process in COPD. Neutrophils may be attracted by CXCL8, CXCL1, and LTB4, monocytes by CCL2, and CD8+ lymphocytes by CXCL10 and CXCL11. Release of elastolytic enzymes, such as MMP and cathepsins, cause elastolysis, and release of TGF-β1 and connective tissue growth factor (CTGF). Macrophages also generate ROS and NO, which together form peroxynitrite and may contribute to steroid resistance.
The increased numbers of macrophages in smokers and COPD patients may be caused by increased recruitment of monocytes from the circulation in response to the monocyte-selective chemokines CCL2 and CXCL1, which are increased in sputum and BAL of patients with COPD (14). Monocytes from patients with COPD show a greater chemotactic response to GRO-α than cells from normal smokers and nonsmokers, but this finding is not explained by an increase in CXCR2 (23). Interestingly, whereas all monocytes express CCR2, which is the receptor for CCL2, only ~30% of monocytes express CXCR2. It is possible that these CXCR2-expressing monocytes transform into macrophages that are more inflammatory. Macrophages also release the chemokines CXCL9, CXCL10, and CXCL11, which are chemotactic for CD8+ Tc1 and CD4+ Th1 cells, via interaction with the chemokine receptor CXCR3 expressed on these cells (24).
The increased numbers of macrophages in COPD are mainly caused by increased recruitment of monocytes, as macrophages have a very low proliferation rate in the lungs. Macrophages have a long survival time so the macrophage level is difficult to measure directly. However, in macrophages from smokers, a markedly increased expression of the antiapoptotic protein Bcl-XL and increased expression of p21CIP/WAF1 is observed in the cytoplasm (25). This finding suggests that macrophages may have a prolonged survival in smokers and patients with COPD. Once activated, macrophages will increase production of reactive oxygen species, nitric oxide, and lysosomal enzymes and will increase secretion of many cytokines, which include TNFα, IL-1β, IL-6, CXCL8, and IL-1β, among others. Activated macrophages are aimed at the more efficient killing of organisms and promote inflammation mainly by TNFα, IL-1β, and short-lived lipid mediators.
Corticosteroids are ineffective in suppressing inflammation, which include cytokines, chemokines, and proteases, in patients with COPD (26). In vitro, the release of CXCL8, TNF-α and MMP-9 macrophages from normal subjects and normal smokers are inhibited by corticosteroids, whereas corticosteroids are ineffective in macrophages from patients with COPD (27). 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-2 (HDAC2) (28), which is recruited to activated inflammatory genes by glucocorticoid receptors to switch off inflammatory genes. The reduction in HDAC activity in macrophages is correlated with increased secretion of cytokines like TNF-α and CXCL8 and reduced response to corticosteroids. The reduction of HDAC activity on COPD patients may be mediated through oxidative stress and peroxynitrite formation (29).
Although eosinophils are the predominant leukocyte in asthma, their role in COPD is much less certain. Increased numbers of eosinophils have been described in the airways and BAL of patients with stable COPD, whereas others have not found increased numbers in airway biopsies, BAL, or induced sputum. The presence of eosinophils in patients with COPD predicts a response to corticosteroids and may indicate coexisting asthma (30). Increased numbers of eosinophils have been reported in bronchial biopsies and BAL fluid during acute exacerbations of chronic bronchitis (31). Surprisingly, the levels of eosinophil basic proteins in induced sputum are as elevated in COPD, as in asthma, despite the absence of eosinophils, which suggests that they may have degranulated and are no longer recognizable by microscopy (15). Perhaps this finding is caused by the high levels of neutrophil elastase that have been shown to cause degranulation of eosinophils.
Dendritic cells play a central role in the initiation of the innate and adaptive immune response, and it is believed that they provide a link between them (32). The airways and lungs contain a rich network of dendritic cells that are localized near the surface, so that they are located ideally to signal the entry of foreign substances that are inhaled. Dendritic cells can activate a variety of other inflammatory and immune cells, which include macrophages and neutrophils, as well as T- and B-lymphocytes, so dendritic cells may play an important role in the pulmonary response to cigarette smoke and other inhaled noxious agents. However, an increase in dendritic cells is not observed in the airways of COPD patients in contrast to asthma patients (33)
An increase in the total numbers of T-lymphocytes is observed in lung parenchyma as well as in peripheral and central airways of patients with COPD; a greater increase is observed in CD8+ than CD4+ cells (6, 24). A correlation is observed between the numbers of T-cells and the amount of alveolar destruction and the severity of airflow obstruction. Furthermore, the only significant difference in the inflammatory cell infiltrate in asymptomatic smokers and smokers with COPD is an increase in T-cells, mainly CD8+, in patients with COPD. An increase in the absolute number of CD4+ T-cells, albeit in smaller numbers, is evidenced in the airways of smokers with COPD, and these cells express activated STAT-4, which is a transcription factor that is essential for activation and commitment of the Thi lineage, and IFN-γ.
The ratio of CD4+:CD8+ cells are reversed in COPD. Most T-cells in the lung in COPD are of the Tci and Thi subtypes (24). A marked increase is observed in T-cells in the walls of small airways in patients with severe COPD, and the T-cells are formed into lymphoid follicles, which surround B-lymphocytes (6).
The mechanisms by which CD8+, and to a lesser extent CD4+ cells, accumulate in the airways and parenchyma of patients with COPD is not yet understood (34). However, homing of T-cells to the lung must depend on some initial activation (only activated T-cells can home to the organ source of antigenic products), then adhesion and selective chemotaxis. CD4+ and CD8+ T-cells in the lung of COPD patients show increased expression of CXCR3, which is a receptor activated by the chemokines CXCL9, CXCL10, and CXCL11, all of which are increased in COPD (35). Increased expression of CXCL10 by bronchiolar epithelial cells is observed, and it could contribute to the accumulation of CD4+ and CD8+ T-cells, which preferentially express CXCR3 (36) (Fig. 5). 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. It is possible that cigarette-induced lung injury may uncover previously sequestered autoantigens, or cigarette smoke itself may damage lung interstitial and structural cells and make them antigenic (37). The role of increased numbers of CD4+ cells in COPD, particularly in severe diseas,e is also unknown (19); however, it is now clear that T-cell help is required for the priming of cytotoxic T-cell responses, for maintaining CD8+ T-cell memory, and for ensuring CD8+ T-cell survival. It is also possible that CD4+ T-cells have immunological memory and play a role in perpetuating the inflammatory process in the absence of cigarette smoking. In a mouse model of cigarette-induced emphysema, there is a predominance of T-cells that are directly related to the severity of emphysema (38).
The role of T-cells in the pathophysiology of COPD is not yet certain, although they have the potential to produce extensive damage in the lung. CD8+ cells have the capacity to cause cytolysis and apoptosis of alveolar epithelial cells through release of perforins, granzyme-B, and TNF-α (39, 40). An association between CD8+ cells and apoptosis of alveolar cells is observed in emphysema (41). Apoptotic cells are powerful sources of antigenic material that could reach the DC and perpetrate the T-cell response. In addition, CD8+ T-cells also produce several cytokines of the Tc1 phenotype, which include TNF-α, lymphotoxin, and IFN-γ, and evidence suggests that CD8+ in the lungs of COPD patients expresses IFN-γ (42). All these cytokines would enhance the inflammatory reaction in the lung besides the direct killing by CD8+ cells. COPD has been considered an autoimmune disease triggered by smoking, as previously suggested (37), and the presence of highly activated oligoclonal T-cells in emphysema patients supports this conclusion (43). Evidence suggests that anti-elastin antibodies exist in experimental models of COPD and in COPD patients (44). In addition to activated Th1 cells, some evidence indicates an increase in Th2 cells that express IL-4 in BAL fluid of COPD patients in COPD patients (45).
Figure 5. T-lymphocytes in COPD. Epithelial cells and macrophages are stimulated by interferon-γ (IFNγ) to release the chemokines CXCL9, CXCL10, and CXCL11, which together act on CXC-chemokine receptor 3 (CXCR3) expressed on Th1 cells and Tc1 cells to attract them into the lungs. Tc1 cells, through the release of perforin and granzyme B, induce apoptosis of type 1 pneumocytes, which thereby contributes to emphysema. IFNy released by Th1 and Tc1 cells then stimulates release of CXCR3 ligands, which results in a persistent inflammatory activation.
Many inflammatory mediators have now been implicated in COPD, which include lipids, free radicals, cytokines, chemo- kines, and growth factors (7). These mediators are derived from inflammatory and structural cells in the lung and interact with each other in a complex manner.
The profile of lipid mediators in exhaled breath condensates of patients with COPD shows an increase in prostaglandins and leukotrienes (46). A significant increase in PGE2 and F2a and an increase in LTB4 but not cysteinyl-leukotrienes is observed. This increase is a different pattern to that observed in asthma, in which increases in thromboxane and cysteinyl-leukotrienes have been shown. The increased production of prostanoids in COPD is likely to be secondary to the induction of cyclo-oxygenase-2 (COX2) by inflammatory cytokines, and increased expression of COX2 is found in alveolar macrophages of COPD patients. LTB4 concentrations are also increased in induced sputum, and concentrations of LTB4 are increased even more in sputum and exhaled breath condensate during acute exacerbations (16). LTB4 is a potent chemoattractant of neutrophils, which acts through high-affinity BLT1-receptors. A BLT1-receptor antagonist reduces the neutrophil chemotactic activity of sputum by approximately 25% (47). Recently BLT1-receptors have been identified on T-lymphocytes, and evidence indicates that LTB4 is involved in recruitment of T-cells.
Oxidative stress occurs when reactive oxygen species (ROS) are produced in excess of the antioxidant defense mechanisms and result in harmful effects, which include damage to lipids, proteins, and DNA. Increasing evidence suggests that oxidative stress is an important feature in COPD (48). Inflammatory and structural cells that are activated in the airways of patients with COPD produce ROS, such as neutrophils, eosinophils, macrophages, and epithelial cells. Superoxide anions (∙O2-) are generated by NADPH oxidase and 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. 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, such as bronchoconstriction and plasma exudation (49) (Fig. 6).
Figure 6. Oxidative stress in COPD. Oxidative stress plays a key role in the pathophysiology of COPD and amplifies the inflammatory and destructive process. ROS from cigarette smoke or from inflammatory cells (particularly macrophages and neutrophils) result in several damaging effects in COPD, which include decreased antiprotease defenses, such as α1 -antitrypsin (AT) and secretory leukoprotease inhibitor (SLPI), activation of NF-KB resulting in increased secretion of the cytokines CXCL8 and TNF-αs, increased production of isoprostanes, and direct effects on airway function. In addition, recent evidence suggests that oxidative stress induces steroid resistance.
The normal production of oxidants is counteracted by several antioxidant mechanisms in the human respiratory tract (48). The major intracellular antioxidants in the airways are catalase, SOD, and glutathione, which is formed by the enzyme y-glutamyl cysteine synthetase, and glutathione synthetase. In the lung, intracellular antioxidants are expressed at relatively low levels and are not induced by oxidative stress, whereas the major antioxidants are extracellular. Extracellular antioxidants, particularly glutathione peroxidase, are markedly upregulated in response to cigarette smoke and oxidative stress. Extracellular antioxidants also include the dietary antioxidants vitamin C (ascorbic acid) and vitamin E (a-tocopherol), uric acid, lactoferrin, and extracellular superoxide dismutase, which is highly expressed in human lung, but its role in COPD is not yet clear.
ROS have several effects on the airways and parenchyma and increase the inflammatory response. ROS activate NF-KB, which switches on multiple inflammatory genes resulting in amplification of the inflammatory response. The molecular pathways by which oxidative stress activates NF-KB have not been fully elucidated, but several redox-sensitive steps must be followed in the activation pathway. 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 (50). Exogenous oxidants may also be important in worsening airway disease. Considerable evidence suggests increased oxidative stress in COPD (48). Cigarette smoke itself contains a high concentration of ROS. Inflammatory cells, such as activated macrophages and neutrophils, also generate ROS, as discussed above. Several markers of oxidative stress may be detected in the breath, and several studies have demonstrated increased production of oxidants, such as H2O2, 8-isoprostane, and ethane, in exhaled air or breath condensates, particularly during exacerbations (16).
The increased oxidative stress in the lung epithelium of a COPD patient may play an important pathophysiological role in the disease by amplifying the inflammatory response in COPD. This increase may reflect the activation of NF-KB and AP-1, which then induce a neutrophilic inflammation via increased expression of CXC chemokines, TNF-a, and MMP-9. Oxidative stress may also impair the function of antiproteases such as a ^antitrypsin and SLPI, and thereby accelerates the breakdown of elastin in lung parenchyma. Corticosteroids are much less effective in COPD than in asthma and do not reduce the progression or mortality of the disease. 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 (27). In patients with COPD, a marked reduction in activity of HDAC and reduced expression of HDAC2 is observed in alveolar macrophages and peripheral lung tissue (28), which is correlated with increased expression of inflammatory cytokines and a reduced response to corticosteroids. This finding may result directly or indirectly from oxidative stress and is mimicked by the effects of H2O2 in cell lines (51).
The increase in exhaled NO is less marked in COPD than in asthma, partly because cigarette smoking reduces exhaled NO. Recently exhaled NO has been partitioned into central and peripheral portions and this shows reduced NO in the bronchial fraction but increased NO in the peripheral fraction, which includes lung parenchyma and small airways (52). The increased peripheral NO in COPD patients may reflect increased expression of inducible NO synthase in epithelial cells and macrophages of patients with COPD (53). NO and superoxide anions combine to from peroxynitrite, which nitrates certain tyrosine residues in protein, and increased expression of 3-nitrotyrosine is observed in peripheral lung and macrophages of COPD patients (53). Tyrosine nitration of HDAC2 may lead it impaired activity and degradation of this enzyme, which results in steroid resistance (51).
Cytokines are the mediators of chronic inflammation, and several have been implicated in COPD (7). An increase in concentration of TNF-α is observed in induced sputum in stable COPD with an additional increase during exacerbations (13, 17). TNF-α production from peripheral blood monocytes is also increased in COPD patients and has been implicated in the cachexia and skeletal muscle apoptosis found in some patients with severe disease. TNF-α is a potent activator of NF-KB, which may amplify the inflammatory response. Unfortunately anti-TNF therapies have not proved to be effective in COPD patients. IL-1β and IL-6 are other proinflammatory cytokines that may amplify the inflammation in COPD and may be important for systemic circulation.
Chemokines are small chemotactic cytokines that play a key role in the recruitment and activation if inflammatory cells through specific chemokine receptors. Several chemokines have now been implicated in COPD and have been of particular interest since chemokine receptors are G-protein coupled receptors, for which small molecule antagonists have now been developed (8). CXCL8 concentrations are increased in induced sputum of COPD patients and increase even more during exacerbations (13, 17). CXCL8 is secreted from macrophages, T-cells, epithelial cells, and neutrophils. CXCL8 activates neutrophils via low-affinity specific receptors CXCR1, and is chemotactic for neutrophils via high-affinity receptors CXCR2, which are also activated by related CXC chemokines, such as CXCL1. CXCL1 concentrations are markedly elevated in sputum and in BAL fluid of COPD patients, and this chemokine may be more important as a chemoattractant than CXCL8, acting via CXCR2 that are expressed on neutrophils and monocytes (14). CXCL1 induces significantly more chemotaxis of monocytes of COPD patient compared with those of normal smokers, and it may reflect increased turnover and recovery of CXCR2 in monocytes of COPD patients (23). CXCL5 shows a marked increase in expression in airway epithelial cells during exacerbations of COPD; this increase is accompanied by a marked up regulation of epithelial CXCR2.
CCL2 is increased in concentration in COPD sputum and BAL fluid (14) and plays a role in monocyte chemotaxis via activation of CCR2. CCL2 seems to cooperate with CXCL1 in recruiting monocytes into the lungs. The chemokine CCL5 (RANTES) is also expressed in airways of COPD patients during exacerbations and activates CCR5 on T cells and CCR3 on eosinophils, which may account for the increased eosinophils and T-cells in the wall of large airways that have been reported during exacerbations of chronic bronchitis. As discussed above, CXCR3 are upregulated on Tc1 and Th1 cells of COPD patients with increased expression of their ligands CXCL9, CXCL10, and CXCL11.
Several growth factors have been implicated in COPD and mediate the structural changes that are found in the airways. TGF-β1 is expressed in alveolar macrophages and airway epithelial cells of COPD patients, and it is released from epithelial cells of small airways. TGF-β is released in a latent from and activated by various factors, which include MMP-9. TGF-β may play an important role in the characteristic peribronchiolar fibrosis of small airways, either directly or through the release of connective tissue growth factor (Fig. 7). TGF-β downregulates β2-adrenergic receptors by inhibiting gene transcription in human cell lines, and it may reduce the bronchodilator response to β-agonists in airway smooth muscle. Alveolar macrophages produce TGF-α in greater amounts than TGF-β, which may be a major endogenous activator of EGFR that plays a key role in regulating mucus secretion in response to many stimuli, which include cigarette smoke. Cigarette smoke activates TNF-α-converting enzyme on airway epithelial cells, which results in the shedding of TGF-α and the activation of EGFR, resulting in increased mucus secretion (54) (Fig. 8).
VEGF is a major regulator of vascular growth and is likely to be involved in the pulmonary vascular remodeling that occurs as a result of hypoxic pulmonary vasoconstriction in severe COPD. Increased expression of VEGF is observed in pulmonary vascular smooth muscle of patients with mild and moderate COPD, but paradoxically a reduction in is indicated expression in severe COPD with emphysema. Inhibition of VEGF receptors using a selective inhibitor induces apoptosis of alveolar endothelial cells in rats, which results in emphysema; this finding seems to be driven by oxidative stress. In addition, VEGF is also an important proinflammatory cytokine produced by epithelial and endothelial cells, macrophages, and activated T-cells, which acts by increasing endothelial cell permeability, by inducing expression of endothelial adhesion molecules and via its ability to act as a monocyte chemoattractant. VEGF also stimulates the expression of CXCL10 and its receptor CXCR3. Thus VEGF is likely an intermediary between cell-mediated immune inflammation and the associated angiogenesis reaction.
Figure 7. TGF-β in COPD. TGF-β is released in a latent form that may be activated by MMP-9. It may then cause fibrosis directly through effects on fibroblasts or indirectly via the release of CTGF. TGF-β may also downregulate β2-adrenoceptors on cells such as airway smooth muscle to diminish the bronchodilator response to p-agonists.
Figure 8. Epidermal growth factor receptors (EGFR) in COPD. EGFR play a key role in the regulation of mucus hypersecretion, with increased expression of mucin genes (MUC5AC, MUCB) and differentiation of goblet cells and hyperplasia of mucus-secreting cells. These effects are mediated via the activation of mitogen-activated protein (MAP) kinases. EGFR are activated by TGF-α, which is in turn activated by tumor necrosis factor-a converting enzyme (TACE), activated via release of oxidants from cigarette smoke and neutrophils. EGFR may also be activated by EGF.
In summary, cigarette smoke exposure induces a florid inflammatory response in the lung that involves structural and inflammatory cells and a large array of inflammatory mediators. The interaction of these complex steps eventually leads to airway remodeling and obstruction and emphysema, albeit in only about 25% of chronic smokers. Of interest, the main difference between smokers who develop COPD and the ones who do not seems to be the presence of an adaptive immune response with CD8+, CD4+, and B-cells, which express obvious signs of being activated effector cells. It is likely that genetic and epigenetic factors (such as histone acetylation) are involved in determining the progression of the inflammatory cascade, as it is supported by animal models, where different strains seem to have different sensitivities to cigarette smoke. COPD is a complex inflammatory disease, and the interactions between different inflammatory cells and mediators are still uncertain. More research into these mechanisms is needed to identify novel targets that may lead to the discovery of more effective therapies that can prevent disease progression and reduce the high mortality of this common disease.
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