Aspects on pathophysiological mechanisms in COPD

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1 Review doi: /j x Aspects on pathophysiological mechanisms in COPD Kjell Larsson From the unit of Lung and Allergy Research, National Institute of Environmental Medicine, Karolinska Institutet, Stockholm, Sweden Abstract. Larsson K (Karolinska Institutet, Stockholm, Sweden). Aspects on pathophysiological mechanisms in COPD (Review). J Intern Med 2007; 262: Chronic obstructive pulmonary disease (COPD) is a condition which is characterized by irreversible airway obstruction due to narrowing of small airways, bronchiolitis, and destruction of the lung parenchyma, emphysema. It is the fourth most common cause of mortality in the world and is expected to be the third most common cause of death by The main cause of COPD is smoking but other exposures may be of importance. Exposure leads to airway inflammation in which a variety of cells are involved. Besides neutrophil granulocytes, macrophages and lymphocytes, airway epithelial cells are also of particular importance in the inflammatory process and in the development of emphysema. Cell trafficking orchestrated by chemokines and other chamoattractants, the proteinase antiproteinase system, oxidative stress and airway remodelling are central processes associated with the development of COPD. Recently systemic effects of COPD have attracted attention and the importance of systemic inflammation has been recognized. This seems to have direct therapeutic implications as treatment with inhaled glucocorticosteroids has been shown to influence mortality. The increasing body of knowledge regarding the inflammatory mechanism in COPD will most likely have implications for future therapy and new drugs, specifically aimed at interaction with the inflammatory processes, are currently being developed. Keywords: chemokines, COPD, cytokines, emphysema inflammation, review. The designation chronic obstructive pulmonary disease (COPD), as we understand the condition today, was first used in the literature in 1964 [1]. During the 1970s and 1980s diagnosis such as chronic bronchitis, chronic obstructive bronchitis, emphysema and chronic bronchitis with emphysema were used and later on, international guidelines and recommendations were published on how to define the disease, today called COPD [2 5]. The causal relationship to tobacco smoking was established and the modern view on the disease is much based on a classic study by Fletcher et al. who prospectively followed 792 men for 8 years, between 1961 and 1969, in the United Kingdom [6]. In that study the authors distinguished two distinct components of chronic obstructive lung disease, the obstructive disorder and the hypersecretory disorder. The obstructive disorder originates from an intrinsic disease of the airways and emphysema whereas the hypersecretory disorder was characterized by chronic excessive bronchial mucus secretion. This view of two distinct conditions has been modified and chronic mucus hypersecretion is today regarded as a symptom associated with poor prognosis in patients with COPD [7]. In the study by Fletcher et al. [6] it was also shown that the rapid decline in lung function was influenced by smoking cessation. Those who stopped smoking did not regain lung function already lost but the subsequent loss of lung function was parallel to nonsmokers. The most common cause of COPD is tobacco smoking although the disease may also result from occupational exposure [8, 9]. Smoking does not lead to COPD in all smokers and in the study by Fletcher et al., airways obstruction was observed in 12% of the moderate smokers and 26% of the heavy smokers [6]. The prevalence of the disease increases as the individuals who suffer from it become older and in a ª 2007 Blackwell Publishing Ltd 311

2 recent study airflow obstruction was demonstrated in approximately 50% of the smokers who reached the age of 75 years [10]. The diagnosis of COPD is today based on lung function measurement and according to most recommendations a postbronchodilator ratio of forced expiratory volume in 1 s (FEV 1 ) vital capacity (VC) or forced vital capacity (FVC) below 0.7 is required for the diagnosis [11, 12]. In addition, staging of the disease is based on lung function assessed as FEV 1 in percentage of predicted value, although the definition of mild, moderate and severe disease varies somewhat between the different guidelines. The main characteristic of COPD is expiratory airflow limitation and the definition of the disease is based on spirometry (postbronchodilator FEV 1 FVC or VC ratio <0.7). As this definition will overestimate the prevalence of COPD in elderly subjects, we have, in Sweden, introduced the rule which implies that a postbronchodilator FEV 1 VC ratio <0.65 is required for the COPD diagnosis in individuals who are older than 65 years of age. The airflow limitation is caused by airway inflammation with mucosal oedema, increased airway secretion and airway remodelling leading to increased airway resistance and, as described above, by emphysema which alters mechanical properties of the lungs. All these factors co-operate and result in airway collapse during expiration and airway obstruction. It was early recognized that COPD is an inflammatory disease and that the mechanisms of airway obstruction differed from that observed in asthma. The pathological anatomical basis for COPD is altered structure and function of central airways (bronchitis), small airways (bronchiolitis), pulmonary vasculature (pulmonary hypertension) and lung parenchyma (emphysema). It could be assumed that the progress of the disease reflects the progress of emphysema but a number of studies have shown that smokers with no or only minor symptoms may have developed significant emphysema [13 15]. There is a correlation between lung function assessed by spirometry and emphysema score assessed by computed tomography (CT) or magnetic resonance but this relationship is not as clear as one may expect. A significant correlation between CT scores and spirometric tests has been demonstrated by some authors [14, 16, 17] whereas no such relationship was found by others [13]. Fain et al. found a relationship between spirometric lung function tests and emphysema assessed by diffusionweighted magnetic resonance imaging [18]. At the group level, smokers with severe emphysema have impaired lung function [reduced FEV 1, FVC and FEV 1 FVC, and increased functional residual capacity (FRC)] compared with smokers with mild emphysema [19]. Thus, not very surprisingly, there is a relationship, although not very strong, between the severity of emphysema and lung function assessed by spirometry. In general, there is a better correlation between the stage of emphysema and diffusion capacity for carbon monoxide (DLCO) [15 18]. There is a good correlation between elastic recoil pressure [measured at 90% of total lung capacity (TLC)] on the one hand and FEV 1, DLCO and lung density measured by CT on the other hand [20]. Airway obstruction in COPD is composed of reversible and irreversible components. The main contributing features of irreversible, fixed, airway obstruction are destructive processes such as emphysema and loss of alveolar-bronchial attachments and airway remodelling processes. The reversible component of airway obstruction in COPD is due to airway smooth muscle contraction, airway inflammation with plasma exudation and increase in inflammatory cells and mucus production and secretion. Previously it was recognized that the fast decline in lung function over time is an important feature of smokers who develop COPD. The annual decline in FEV 1 in healthy, middle-aged, nonsmokers is ml [6, 21] whereas the additional loss in smokers is, on average, ml more per year [22]. There is an inter-individual variation in this respect and there are smokers, the so-called rapid decliners, who may have an annual decline of FEV 1 >60 ml or even more than 100 ml. There is no consensus about the definition of rapid declining but 90 ml year has been suggested [6]. Low FEV 1 and rapid decline in COPD are associated with poor prognosis [23]. Rapid decline in lung function may also be related to exacerbations and there are data 312 ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262;

3 indicating that frequent exacerbation increases the decline in lung function over time [24]. Respiratory system: response to exercise Ventilation increases during exercise by increase in breathing rate and depth (tidal volume). In healthy subjects the increase in breathing rate and depth is adjusted in order to get the highest possible ventilation to the lowest possible energy costs. At low work load tidal volume is increased and at higher load both breathing rate and depth are increased up to approximately 75% of maximal work load after which only the frequency of breathing is increased [25]. Although there are inter-individual variations, this usually implies a breathing frequency of breaths per minute and a breathing depth, i.e. a tidal volume, of 50 60% of the VC at maximal work load [26]. If breathing rate is increased to a higher frequency the breathing would be shallow resulting in lower ventilation and vice versa, if the breathing depth (tidal volume) is too much enhanced during exercise the breathing frequency cannot be high enough to retain high ventilation. Thus increased ventilation during exercise is based on a harmonic increase in breathing rate and tidal volume. both forces of the chest wall (which at maximal inspiration are centripetally directed) and the recoil of the lungs. At maximal expiration (i.e. at residual volume, RV) the total forces of the respiratory system are centrifugally directed whereas the lung recoil forces are still, despite the compression of the lungs, centripetally directed (Fig. 1). The normal lung constitutes a three-dimensional network of tissue and air spaces which are composed of airways and alveoli. Alveolar walls are attached to the interstitial tissue and small, peripheral, airways creating a network which keeps the airways and alveoli open due to the opposing forces acting on the lungs. The patency of the small airways is thus dependent Forces influencing the lungs The lungs are influenced by centrifugal (directed outwards) and centripetal (directed inwards) forces which are regulated by activity of the breathing muscles and the elastic properties of the chest wall and the lungs. The lung volume at the end of a normal expiration represents the relaxation volume, functional residual capacity (FRC). At FRC centrifugal forces (represented by chest wall expansion) equal the centripetal forces (represented by elastic recoil forces of the lungs) and the respiratory system is at rest. During a deep inhalation the centrifugal forces of the chest wall are attenuated and the centripetal lung recoil forces are enhanced and the other way round, during expiration from FRC the chest wall centrifugal forces are increased and the lung recoil force which are centripetally directed are diminished. At maximal inspiration the forces of the breathing muscles have to oppose Fig. 1 At functional residual capacity (FRC) the elastic forces of the lungs (blue curve), which are centripetally directed, equal the elastic forces of the chest wall (red curve), which are centrifugally directed (approximately 0.5 kpa) resulting in a situation where the respiratory system is at rest. Note that, at residual volume (RV), the elastic forces of the lung (blue curve) are still centripetally directed as are the elastic forces of the chest wall (red curve) at total lung capacity (TLC). At TLC a muscular force corresponding to the sum of chest wall and lung forces is needed to keep the respiratory system at maximal inspiration. ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262;

4 on the structure of this network and any possible structural damage alters the elastic properties and may influence ventilation. During expiration the transpulmonary pressure falls concurrently with the reduction in lung volumes. The point where the transluminal pressure during an expiration is zero, i.e. the point where the dilating (centrifugal) forces, constituted by the elastic recoil forces of the lung equal the compressing (centripetal) forces constituted by the pleural pressure, is called equal pressure point (EPP). Beyond EPP the airway will collapse during expiration. In healthy subjects EPP is located in the central airways during more than 60% of the first part of a forced expiration implying that the upstream segment includes peripheral and the major part of the central airways. During the last part of expiration EPP is moved towards peripheral airways and is located in the small airways (defined as airways with a diameter <2 mm) during the last quarter of expiration. Structural changes in the airways and lung parenchyma alter the mechanical properties of the tissue. Thus, in emphysema, EPP is moved more peripherally, and airway compression will occur at an earlier stage during expiration which will lead to hyperinflation. When air flows through the airways a pressure on the surrounding airway wall is produced. This pressure is inversely proportional to the flow rate, i.e. higher at low flow rates and lower at high flow rates, the socalled Bernoulli effect. At very high flow rates the pressure may even be negative and thus directed inwards resulting in airway narrowing or even airway collapse. Airways are compressed by high driving forces and by increased airway resistance. Thus, central airways may collapse in healthy subjects during cough and high expiratory flow may induce dynamic compression of the airways in asthmatic subjects with increased airway resistance. In COPD with emphysema the elastic properties of the airways are altered and the lungs become flabby leading to airway collapse at relatively low expiratory flow rates. When expiratory flow increases during exercise there is a compression and collapse of the airways during expiration which results in dynamic lung hyperinflation. As the disease progresses the airways will collapse during the expiratory phase of normal breathing which leads to increased FRC (hyperinflation) and breathing at a higher level of the pressure volume curve. This increasing of the operating lung volumes will improve the flow conditions at the expense of increased energy expenditure and increased dyspnoea. Dyspnoea is a subjective experience of breathing discomfort and constitutes a cardinal symptom in patients with COPD. Although the airway obstruction in COPD is characterized by expiratory airflow limitation, most patients with COPD experience inspiratory difficulties [27, 28]. This indicates that it is not the dynamic airway compression per se that explains the experience of breathing discomfort. Lung function tests such as FEV 1 and VC are poor predictors of dyspnoea and the correlation between these lung function indices and dyspnoea is poor [29 31]. Dyspnoea also predicts mortality, even better than annual decline in lung function [32]. There is a close relationship between dynamic lung hyperinflation and dyspnoea in COPD [27, 33] and it has also been shown that lung hyperinflation is a predictor of mortality [34]. In the study by Casanova et al. it was demonstrated that inspiratory capacity (IC, which is a reflection of e the operating lung volume, FRC, provided that TLC does not change) at the start of the study was related to 5 years survival; high IC was associated with prolonged survival [34]. In COPD end-expiratory volume (FRC) increases and IC decreases with increasing work load, and these parameters reflect dynamic lung hyperinflation during exercise [35]. Considering that hyperinflation is often present at rest, before exercise, the space for increasing tidal volume (i.e. the inspiratory reserve volume) is limited as the patient breathes close to TLC already at rest. The important implication of exercise-induced hyperinflation is that tidal volume cannot be sufficiently increased during exercise, i.e. the COPD patient is not capable of increasing the depth of breathing (i.e. the tidal volume) and is therefore reduced to increasing breathing rate to improve ventilation during exercise [27, 36]. This will lead to a situation in which increased breathing effort does not result in increased ventilation. O Donnell et al. showed that the ratio between transpulmonary pressure obtained during exercise maximal 314 ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262;

5 Ventilation inspiratory pressure (P transpulm during exercise P maximal inspiratory pressure), i.e. what efforts have been devoted to breathing in the numerator and tidal volume predicted VC (V T VC % predicted value ), i.e. what is the ventilation, i.e. the benefit, that comes out of it, in the denominator was highly correlated with dyspnoea as assessed with the Borg scale [27]. The condition in which increased work of breathing only results in a minor increase in ventilation has been designated as neuro-mechanical dissociation [27]. The improvement of dyspnoea during exercise observed after inhalation of a bronchodilator is well correlated with a reduction in FRC and increased tidal volume during exercise [37, 38] (Fig. 2). Inflammation COPD prebronchodilatation V T COPD postbronchodilatation Normal Time (exercise) Fig. 2 In chronic obstructive pulmonary disease (COPD) breathing is performed at higher operational lung volumes [i.e. at higher functional residual capacity (FRC)] than in healthy subjects. Inhalation of a bronchodilator reduces FRC enabling increased tidal volume during exercise. The physiological lung function impairment described above is a result of a chronic inflammatory process in the lungs, although the link between inflammatory mechanisms and physiological dysfunction is not fully understood. COPD was early recognized as an inflammatory disease and airway inflammation starts at an early stage, many years prior to the onset of clinical symptoms [39]. Autopsy studies have shown that bronchiolitis starts early in young healthy smokers V T V T [39]. Thus, the small airway disease in smokers may progress for many years without giving any symptoms or indications of lung function impairment as assessed by spirometry [40]. Although airway inflammation is located in both large and small airways, central airway inflammation does not seem to contribute much to airway obstruction [39, 41] (Fig. 3). Bronchiolitis with considerable small airway obstruction due to the inflammatory reaction [42 44] and by a more peripheral distribution of goblet cells (goblet cell metaplasia) [44], peribronchiolar fibrosis [42] and thickening of the bronchial smooth muscle layer [43] thus precede, by many years, the time when clinical symptoms of COPD become present. As the disease progresses, airway obstruction is related to inflammatory changes in small airways and structural changes in the airway wall as well as peribronchiolar fibrosis [40, 45, 46]. There is also a progress of the emphysema which is an important cause of respiratory failure in COPD. As described above there is no clear relationship between emphysema and lung function, at least not in mild and moderate disease. Many cells contribute to the inflammatory reaction in COPD and there seems to be a clear relationship between the severity of emphysema and the content of inflammatory cells in lung tissue and airways alveoli. Irritating stimuli in the airways activate a variety of cells to produce and release chemotactic factors which leads to attraction of other inflammatory cells. Chemokines constitute an important group of chemotactic factors which induce attraction of inflammatory cells into the airways and lungs. More than 50 chemokines have been identified which all bind to surface receptors of the seven-transmembrane (7TM) type. The chemokine receptors are divided into four subfamilies CXC, CC, C and CX 3 C where C indicates the position of cysteine residues separated by a single amino acid (CXC) or adjacently located (CC) in the two most important subfamilies. Each chemokine may bind on one or more receptors and each receptor may bind to one or more chemokines (Fig. 4). Chemokines may act as agonists when binding to one receptor and as antagonists when binding to another receptor [47 50]. ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262;

6 Fig. 3 Exposure, target cells, mediators, tissue effects and outcomes in the inflammatory processes in chronic obstructive pulmonary disease. Fig. 4 CXC- and CC-chemokines and their receptors. Green letters and arrows indicate stimulation, red letters and arrows indicate inhibition and blue letters indicate ligands with both activating and inhibitory properties. Neutrophils It was previously demonstrated that the number of neutrophil granulocytes was increased in the airways in COPD [51] and that these cells are of importance in the inflammatory reaction (Fig. 5). It has also been shown that sputum from COPD patients with a 1 -antitrypsin deficiency has enhanced neutrophil chemotactic activity compared with COPD patients with normal serum levels of a 1 -antitrypsin [52]. Although tumour necrosis factor (TNF) and interleukin-1b (IL-1b) do not exert direct chemotactic activity for 316 ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262;

7 Fig. 5 Crucial effects of neutrophil granulocytes in chronic obstructive pulmonary disease. Double arrows indicate autocrine effects. neutrophils, these cytokines are capable of up-regulating adhesion molecules on endothelial cells and neutrophils, thereby contributing to the accumulation of neutrophils in the airways [53]. The number of sub-epithelial neutrophilic granulocytes is higher in severe COPD than in mild disease and higher in mild disease than in smokers without COPD [54]. There is a relationship between lung function and emphysema assessed by CT on the one hand and the number of sputum neutrophils on the other hand [55]. Furthermore, the decline in lung function over time seems to be related to the amount of neutrophil granulocytes present in the sputum [56]. It has also been demonstrated that the number of glandular neutrophils and macrophages is increased in patients with COPD [57]. Moreover, the number of neutrophils in peripheral blood is increased in COPD compared with healthy nonsmokers [58], and a relationship between lung function decline and the number of circulating neutrophils has been shown [59]. Neutrophils are recruited to the airways by a number of different signals. High levels of neutrophilic chemoattractants such as leukotriene B 4 (LTB 4 ), [60], CXCL8 (IL-8) [60, 61], CXCL1 (growth-related oncogene-a, GRO-a) [62] and CXCL5 (epithelial neutrophil activating protein 78, ENA-78) [61] have been demonstrated in the airways of patients with COPD. The level of CXCL8 in sputum is positively correlated with the number of neutrophils [60, 63 65] and relates negatively to lung function in patients with COPD [65]. An association between CXCL8 level in sputum and irreversible airway obstruction has been demonstrated in COPD [64]. Chemotaxis of neutrophils is induced by activation of CXCR2, a high affinity receptor to which CXCL1, CXCL2, CXCL3, CXCL5, CXCL6 and CXCL8 bind [49]. Sputum levels of LTB 4 are elevated in COPD and the increase seems to be related to airway bacterial load [66]. LTB 4 has been shown to be responsible for 30% [67] or almost half and CXCL8 for approximately one-third of the neutrophil chemotactic activity of sputum from patients with COPD [60]. LTB 4 is further elevated in association with acute exacerbations [66 68]. LTB 4 is also involved in the prolonged cell survival of neutrophils induced by a number of stimuli and an LTB 4 antagonist has been shown to totally ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262;

8 inhibit prolonged neutrophil survival induced by lipopolysaccharides (LPS) [69]. In addition, inhibition of 5-lipoxygenase (5-LO) or 5-LO-activating protein (FLAP) abolish prolonged neutrophil survival induced by granulocyte-macrophage colony stimulating factor (GM-CSF) and dexamethasone respectively [70]. Interleukin-8 (CXCL8), which binds to CXCR1 and CXCR2 [71, 72], is one of approximately 50 described chemokines and constitutes a strong stimulus for neutrophil chemotactic activity. The chemokine receptors CXCR1 and CXCR2 are up-regulated in the bronchial mucosa from patients with COPD during acute exacerbation whereas the expression is not different from healthy controls during the stable phase of the disease [73]. The sources of CXCL8 in COPD are primarily airway epithelial cells, macrophages and neutrophils [74, 75]. During acute exacerbations CXCL8 in sputum has been found to increase further [67], and the mrna expression of CXCL5 and CXCL8 in bronchial mucosa is up-regulated [73]. There are however results which do not confirm the increase of CXCL8 in sputum during acute exacerbations [68]. Furthermore, recent findings indicate that the chemotactic responsiveness in neutrophils is not increased in COPD [76]. In the study by Yoshikawa et al., the neutrophil response to chemotactic stimuli (CXCL8, fmlp) was even diminished in patients with severe COPD as was the response to sputum supernatant in smokers, with and without COPD [76]. By binding to the low affinity receptor CXCR1, CXCL8 is also involved in adhesion of neutrophils to endothelial cells by enhancing shedding and binding of leucocyte selectin, an adhesion protein that is expressed on neutrophils [77]. Furthermore, macrophage antigen 1 (Mac 1, CD11b CD18) is expressed on neutrophils and plays an important role for neutrophil migration. Noguera et al. showed that circulating neutrophils from patients with COPD had an enhanced surface expression of CD11b CD18 compared with neutrophils from nonsmokers, both at baseline and after activation with TNF [78], a finding that has also been confirmed by others [79]. There are also data suggesting that the levels of the soluble CD11b CD18-ligand, intercellular adhesion molecule- 1 (ICAM-1) which is up-regulated by TNF and IL-1b [80, 81], is increased in COPD compared with nonsmokers [82]. These results were, however, contradicted by the findings of low levels of soluble ICAM-1 in a study by Noguera et al. [83]. In patients with COPD the number of neutrophils is increased in the airway lumen (assessed by airway lavage and sputum analyses) whereas no such increase is observed in the airway mucosa, indicating a rapid migration of the cells across the epithelium into the airways [56, 84, 85]. This assumption is supported by the finding of up-regulation of E-selectin and ICAM-1 on mucosal vessels and bronchial epithelial cells in COPD [86]. There are also other indications of neutrophil activation in COPD and sputum markers of neutrophil activity, such as myeloperoxidase (MPO) and neutrophil elastase (NE), are elevated in patients with COPD [87]. There was a correlation between MPO and NE activity and also between these two activity markers and the chemoattractants CXCL8 and LTB 4 [87]. When neutrophils migrate from the blood into the tissue they have to be deformed. Cigarette smoke decreases the deformability of neutrophil granulocytes [88] which may explain the slow wash out rate of neutrophils from the lungs in smokers [89]. This may implicate that smoking increases lung inflammation also by decreased transit time of neutrophils in the lungs. Eosinophils The occurrence of eosinophil granulocytes may vary amongst smokers with COPD. The number of eosinophils in the bronchial mucosa has been shown to be increased in COPD [90] and so have markers of eosinophil activity [91]. It has been suggested that those with increased numbers of eosinophils and mast cells in the airway may constitute a specific COPD phenotype [92]. The number of mononuclear cells expressing CCL11 (eotaxin) mrna in the bronchial mucosa was higher in smokers with chronic bronchitis (of whom a few also had bronchial obstruction) than in 318 ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262;

9 healthy nonsmokers [93]. Acute exacerbations of COPD is accompanied by an increased number of eosinophils and eosinophil activity markers in the airways [90, 93 95]. In patients with chronic bronchitis the expression of CCL5 (RANTES) was also increased in the bronchial mucosa following an exacerbation [93]. Both CCL5 and CCL11 are chemoattractants for eosinophils [96, 97]. In a study by Vachier et al. the number of eosinophil granulocytes in bronchial and nasal mucosa was higher in smokers without COPD than in smokers who suffered from the disease [98]. There are also data suggesting that the presence of eosinophils in the airways of patients with COPD is positively associated with lung function variability over time [64]. This is interesting considering that the occurrence of eosinophils in the airway of smokers with COPD predicts a favourable outcome of glucocorticosteroid therapy [99 101]. Macrophages monocytes Macrophages constitute an early line of the airway defence against invading agents and, due to their multiple functions, macrophages are involved in most pathological conditions of the airways and play an important role in COPD (Fig. 6). There are different types of macrophages and in the human lung both alveolar and interstitial macrophages can be distinguished [102, 103]. Alveolar macrophages have been shown to have higher phagocytic capacity than interstitial macrophages [102] which are more similar to blood monocytes than are alveolar macrophages. Fig. 6 Crucial effects of macrophages in chronic obstructive pulmonary disease. ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262;

10 There are many mechanisms which lead to the recruitment of macrophages in the lungs and airways in COPD. The number of macrophages in the airways and pulmonary tissue is increased in COPD [54, 104] and there are indications that macrophages are of direct importance for the development of emphysema [105]. The monocytic chemotactic activity in bronchoalveolar lavage (BAL) fluid is enhanced in smokers compared with nonsmokers, and was related to the number of BAL fluid macrophages and to the decline in lung function [106]. Monocyte chemotactic protein-1 (MCP-1, CCL2) produced by monocytes, T lymphocytes, fibroblasts and other cells [74], macrophage inflammatory protein-1a (MIP-1a, CCL3) most likely produced by T lymphocytes [107] and CXCL1 (GRO-a) produced by a variety of cells such as bronchial epithelial cells and alveolar macrophages [108] have the capability of attracting cells of different types, e.g. monocytes. In COPD the level of CCL2 and CXCL1 is increased in sputum (mainly representing central airways) whereas no such increase has been found in BAL fluid (representing peripheral airways and alveoli) [62]. However, Capelli et al. found increased level of CCL2 in BAL fluid in smokers and COPD patients compared with healthy nonsmokers [109]. CCL3 immunoreactivity was increased in airway epithelial cells from patients with severe COPD compared with patients with mild disease who, in turn, expressed more CCL3 than did healthy controls [54]. In that study a negative correlation between CCL3 expression and lung function assessed as FEV 1 was found [54]. The levels of CCL2 and CCL3 did not differ between asymptomatic smokers with and without emphysema [61]. Monocytes are activated by CXCL8 [110, 111] and the chemotactic response of peripheral blood mononuclear cells to CXCL1 (GRO-a) and CXCL7 (NAP-2) is increased in patients with COPD compared with controls [112]. This may possibly indicate a mechanism for increased recruitment of monocytes macrophages to the airways in COPD. Alveolar macrophages and neutrophil granulocytes produce and release CXCL8, an important chemoattractant for neutrophil granulocytes, when activated by a variety of stimuli, such as LPS and IL-1b. Alveolar macrophages from patients with COPD have an enhanced propensity to release CXCL8, both at baseline and upon stimulation, compared with alveolar macrophages from smokers without airway obstruction [113]. These findings indicate that recruitment of neutrophils by secretion of CXCL8 from macrophages most likely is an important mechanism for the development of airway obstruction in COPD. Stimulation of alveolar macrophages induces production of interleukin-10 (IL-10), which is a cytokine with anti-inflammatory properties. It has been demonstrated that IL-10 in sputum is lower in smokers (with and without COPD) than in healthy nonsmokers [114] which may imply that IL-10 is of importance for counterregulation of the airway inflammation in chronic bronchitis and COPD. On the other hand, IL-10 may be increased in alveolar macrophages from smokers, and IL-10 has been shown to increase the release of tissue inhibitors of metalloproteinases (TIMP-1) from human alveolar macrophages [115]. This effect of IL-10 in TIMP production may be directly mediated through interaction at the transcriptional level and through inhibition of TNF production in alveolar macrophages [115]. Lymphocytes A good correlation was found between emphysema assessed by CT and tissue CD8-positive cells and CD4-positive cells in the air spaces [104]. In COPD the involvement of T-lymphocytes is dominated by CD8 + cells which are found in increased number in the bronchial wall, around pulmonary vessels and lymph nodes leading to a low CD 4 + CD8 + ratio [ ]. In COPD there seems to be a relationship between the proportion of circulating CD8 + cells and impairment of diffusion capacity; patients with COPD, and normal transfer factor had an increased proportion of circulating CD8 + cells whereas those with impaired DLCO had a low proportion of circulating CD8 + [119]. The finding of an inverse correlation between CD8 + cells and lung function, measured as FEV 1 [84, 120], may lead to the conclusion of a direct pathophysiological importance of these cells for lung function decline in COPD. Such a causal 320 ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262;

11 relationship has, however, not been proved, although it was recently shown that the interaction between CXCL10 and CXCR3 induced secretion of matrix metalloproteinase-12 (MMP-12) from alveolar macrophages suggesting a role of the T cells in the destructive processes leading to emphysema [121]. In that study it was also demonstrated that COPD is associated with a high proportion of T lymphocytes, both CD4 + and CD8 +, expressing chemokine receptors characteristic of T-helper 1 cells (CXCR3 and CCR5) [121]. The c-interferon (IFN-c)-induced chemokines CXCL9 (monokine induced by IFN-c, Mig), CXCL10 (IFN-cinducible protein 10, IP-10) and CXCL11 (IFN-inducible T-cell a-chemoattractant) are produced by neutrophils and macrophages and bind to the CXC receptor 3 (CXCR3) on T cells, epithelial cells and macrophages. The CXCR3 is expressed on activated Th1 [122] and to a very small extent on Th2 cells [123], and chemokines that activate CXCR3 attract T lymphocytes (Th1, Tc1) which, in turn, release IFN-c which induces enhanced production and release of CXCR3 activating chemokines. Thus CXCL9 and CXCL10 are active chemoattractants for stimulated, but not resting T cells [124]. In COPD enhanced expression of CXCL10 and CXCR3 has been demonstrated in peripheral airway mucosal cells co-expressing CD8 and IFN-c [125]. In COPD the expression of CXCL10 is elevated in airway smooth muscle cells and CXCL10 secretion is stimulated by a combination of TNF and IFN-c [126]. Airway T-lymphocytes from patients with COPD show an enhanced response to CXCL10 [125]. The chemokines CXCL9, CXCL10 and CXCL11 also antagonize the CCR3, a receptor for CCL11 (eotaxin) and other CCLs on eosinophils, basophils and Th2-cells [127, 128] resulting in diminished migration of these cells. Epithelial cells The airway epithelium is multifunctional and consists of a number of different cell types such as ciliated columnar epithelial cells, goblet cells, Clara cells, basal cells and alveolar epithelial cells (Fig. 7). In chronic bronchitis there is an increase in submucosal glands and the ciliated epithelial cells are to a great extent replaced by goblet cells (goblet cell metaplasia) [129] and squamous epithelial cells. These structural changes lead to mucus hypersecretion which, perse, seems to be related to increased lung function deterioration and impaired prognosis in COPD [7]. One important function of the epithelium is to form the first line of defence and to act as a barrier towards inhaled particulates and micro-organisms. Other roles of the epithelium are to participate in the immunological responses, wound repair and airway remodelling. Airway epithelial cells actively participate in the inflammatory response and produce and secrete a number of inflammatory mediators and cytokines. When stimulated with pro-inflammatory agents such as cigarette smoke [130], organic dust [ ], TNF [134] or Th2 cytokines (IL-4 IL-13) [135], the airway epithelium releases high amounts of CXCL8, which is a potent neutrophil chemoattractant. Interleukin-17 from T-lymphocytes [136, 137], preferentially from the Th1 subtype, induces release of the chemoattractants CXCL1 (GRO-a), CXCL5 (ENA-78), CXCL6 (GCP-2) and CXCL8 (IL-8) from bronchial epithelial cells [138, 139]. Bronchial epithelial cells also produce CCL5 (RANTES) [140] which is a chemoattractant for eosinophils. When inflammatory cells migrate into the airways adhesion proteins at the cell surface are of importance for cell migration. Bronchial epithelial cells and endothelial cells express ICAM-1 [141] which binds to integrins on leucocytes. There is a variety of integrins which are all composed of an a-unit (CD11) and a b-unit (CD18). Macrophage antigen (Mac-1, CD11b CD18) and lymphocyte function-associated antigen-1 (LFA-1, CD11a CD18) are located on neutrophils [142] and mediate neutrophil binding to ICAM-1 on epithelial and endothelial cells. Cigarette smoke induces increased expression of ICAM-1 on human bronchial epithelial cells which is associated with increased capacity of the epithelial cells to bind mononuclear cells [143]. Bronchial epithelial cells also produce transforming growth factor-b (TGF-b) which has been shown in ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262;

12 Fig. 7 Crucial effects of airway epithelial cells in chronic obstructive pulmonary disease. both animal models [ ] and in human epithelial cells [147]. Apart from being active in the airway fibrosis processes, TGF-b has the capability of downregulating b 2 -adrenoceptors in guinea-pig, human tracheal smooth muscle cells and fibroblasts through reduction in the rate of b 2 -adrenoceptor gene transcription [148, 149]. The production and secretion of mucin (MUAC5AC) from airway cells are stimulated by cigarette smoke [150, 151], bacterial components [152, 153], oxidative stress [154], and pro-inflammatory cytokines such as TNF and IL-1b [155, 156]. Takeyama et al. showed that activation of neutrophils by CXCL8 or TNF increased epidermal growth factor receptor (EGFR) tyrosine phosphorylation and up-regulated mrna and protein expression of MUC5AC in a human pulmonary mucoepidermoid cell line [154]. In that study EGFR tyrosine phosphorylation and MUC5AC synthesis were not inhibited by antibodies to EGFR ligands such as epidermal growth factor (EGF) and TGF-a, and no increase in TGF-a was found in the neutrophil supernatant indicating that EGFR ligands were not responsible for the increased mucin synthesis [154]. Baginski et al. showed that TGF-a induced MUC5AC mucin in human airway epithelial cells [157]. The authors also demonstrated that TNF induced gene expression and release of TGF-a in airway epithelial cells [157]. It seems likely that mucin production induced by cigarette smoke is induced by EGFR ligands, such as TGF-a, through activation of TNF converting enzyme (TACE) [150]. Human bronchial epithelial cells express both CXCR1 and CXCR2 but these receptors do not seem to be up-regulated by pro-inflammatory cytokines such as 322 ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262;

13 TNF and IFN-c in concentrations that induce a multifold increase in CXCL8 release [158]. Oxidative stress Inflammatory cells, such as macrophages, granulocytes and airway epithelial cells, generate reactive oxygen species when activated. Together with inhaled reactive oxygen and nitrogen species, which are abundant in cigarette smoke, these endogenous oxidants may constitute a major oxidative burden to the lungs. In healthy subjects there is a balance between oxidants and antioxidants keeping the extracellular environment in a reduced state. Oxidative stress arises if this balance is disturbed, i.e. if oxidative influences increase or if antioxidative influences decrease. Superoxide anions, which are formed when one electron is added to oxygen, are rapidly converted to hydrogen peroxide catalysed by extracellular superoxide dismutase [159] which is expressed in human airways [160, 161]. Reactive hydroxyl radicals, which are formed when one electron is added to hydrogen peroxide, are nonenzymatically converted to water. There are multiple consequences of oxidative stress in the human airways and lungs. Early data suggested that antiproteinases may be inactivated by exogenously and endogenously derived oxidants [ ]. It has also been shown that proteinases induce formation of reactive oxygen species in bronchial epithelial cells and fibroblasts in vitro [165]. Macrophages from smokers produce more superoxide anions following stimulation with platelet-activating factor than did macrophages from nonsmokers indicating more sensitive macrophages in smokers [166]. Oxidative injury to the airway epithelium may also be of importance for the pathogenesis of COPD [167]. Reactive oxygen species stimulate mucus secretion [168] and have the capability of inhibiting ciliary activity in bronchial epithelial cells [169]. Interestingly, it has been shown that a superoxide dismutase mimetic (M40419) not only blocks the oxidative stress induced by the inhibition of vascular endothelial growth factor (VEGF) but also emphysema induced by VEGF inhibition [170]. Indications of oxidative processes in the airway can be obtained by measurements of oxidative products in exhaled breath condensates. The level of H 2 O 2 in exhaled breath condensate is increased in patients with COPD in a stable phase and even more increased during an acute exacerbation [171]. Isoprostane is a lipid peroxidation product which is produced by peroxidation of arachidonic acid and catalysed by free radicals. It has been demonstrated that 8-isoprostane in exhaled breath condensate is increased in patients with COPD and that this increase is observed both in smokers and ex-smokers [172]. Proteinases The main task of the proteinases is to exhibit proteolytic activity and by that render invading harmful agents, e.g. of innocuous microbial origin. The two groups of proteinases that have been suggested as important in COPD are classified according to the active moiety of the enzyme. Thus serine proteinases contain serine and metalloproteinases contain Zn 2+ at the active site. Proteinases are produced and released by circulating and resident cells such as granulocytes, monocytes, lymphocytes, endothelial cells, fibroblasts and other cells. The most extensively studied proteinase is neutrophil elastase (NE), a serine proteinase originating from neutrophil granulocytes and, to some extent, monocytes but not from macrophages. NE is released following cell activation and cellular death, and most likely plays an important role in the destruction of alveolar elastin [173], and has, in animal experiments, been shown to cause emphysema [174, 175]. Degradation of elastin, a major component in elastic fibres, is a characteristic feature of a number of pathological conditions, in particular emphysema. Desmosins, a cross-link amino acid unique to elastin, are excreted in the urine attached to low molecular weight peptides and constitute markers of elastin degradation [176]. It has been demonstrated that urinary desmosin levels are higher in COPD patients with a rapid annual ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262;

14 decline in lung function [177]. In that study no difference in desmosin levels was found between rapid decliners with clear signs of emphysema and those who had less emphysema according to CT [177]. In addition, NE interacts with a number of cell types leading to increased recruitment of neutrophils via enhanced production of CXCL8 [178] and LTB 4 [179]. NE also enhances oxidative stress [165], causes epithelial cell metaplasia and mucus gland hyperplasia [180] and acts as a secretagogue [181], thereby contributing to the development of chronic bronchitis. In a mouse model, IL-13 caused emphysema and induced increased expression of a number of MMPs (-2, -9, -12, -13, -14) and cathepsins (B, S, L, H, K) in the murine lungs [182]. In that study inhibition of MMPs and cathepsin also inhibited the development of emphysema which may constitute a future therapeutic target. Proteinase-3, tryptases and chymases are other serine proteinases that may be of importance in COPD, but this is still unclear. Matrix metalloproteinases degrade extracellular matrix proteins and are therefore of importance for a variety of biological processes such as remodelling and repair processes. Metalloproteinases are mainly produced by macrophages and neutrophil granulocytes [183, 184] but other cells such as eosinophil granulocytes, T lymphocytes, monocytes and fibroblasts are capable of producing certain MMPs. Although not as efficient as NE, there are MMPs such as MMP-2 (gelatinase A), MMP-9 (gelatinase B), MMP-7 (matrilysine) and MMP-12 (macrophage elastase) which exhibit elastolytic activity [185] and there are MMPs such as MMP-1 (interstitial collagenase) and MMP-13 (collagenase 3) that have the capacity to degrade collagen [ ]. It has been demonstrated that the elastolytic activity is enhanced in macrophages from smokers with COPD compared with nonsmokers and smokers who do not suffer from COPD [189]. In that study it was suggested that MMP-9 may be of importance in COPD as the release of MMP-9 was enhanced in macrophages from smokers with COPD in comparison with healthy smokers and controls [189]. Release of MMP-9 and TIMP-1 is stimulated by IL-1b in alveolar macrophages [190]. The importance of IL-1b in this context is supported by the finding that knockout of the IL-1-receptor in mice prevents the animals from emphysema induced by elastase [191]. The findings of elastolytic and collagenolytic activity amongst MMPs indicate that these enzymes may be of importance for the degradation of basement membrane proteins leading to alteration of the airway wall and to development of emphysema. Antiproteinases More than 40 years have passed since Eriksson demonstrated the association between homozygous a 1 -antitrypsin deficiency and pan-acinar emphysema [192]. This finding focused the interest on the association between COPD emphysema and imbalance of the proteinase antiproteinase system. The main task of the antiproteinases is to inactivate, by reversible or irreversible binding, proteolytic activity of the proteinases and thus inhibit degrading and destructive processes. The most extensive studied antiproteinase is a 1 -antitrypsin which is a serine antiproteinase produced in the liver and by certain cell types such as monocytes, macrophages and neutrophil granulocytes [ ]. Proteinases such as trypsin, chymotrypsin, proteinase-3, plasmin and cathepsin G are inhibited by a 1 -antitrypsin but the main inhibitory activity is directed towards NE and the main function is to inhibit proteolytic activity within the lungs. Other antiproteinases are secretory leucoproteinase inhibitor (SLPI), elafin, cystatins and TIMP. The significance of these antiproteinases in COPD is not fully understood. It is known that SLPI, apart from inhibiting NE, also inactivates trypsin and chymotrypsin and cathepsin G [196], and it seems likely that the antibacterial activity of SPLI may be of importance for airway defence mechanisms [197]. The more precise role of SLPI in COPD is, however, not known. This is also the case for elafin which is an inhibitor of NE and proteinase-3 [198, 199]. The major role of TIMPs is to act as inhibitors of MMP activity. The four identified human TIMPs have a variety of effects apart from MMP inhibition but the role in COPD is, to a large extent, unknown. 324 ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262;

15 Remodelling The inflammatory and destructive airways processes which are in progress during the development of COPD are characterized by air space enlargement with alveolar destruction, peribronchiolar fibrosis, increased number of inflammatory cells, increased mucus secretion due to increased number of epithelial secretory cells, plasma leakage and expansion of the submucous glands [200]. All these processes contribute to the decline in lung function and to the progress of the disease. The restructuring, or remodelling, of the airways in COPD has attracted great interest in recent years. The mechanism of airway remodelling in COPD is incompletely understood. The repair and regeneration processes start with cell migration in a provisional extracellular matrix formed by plasma exudation. Basal cells interact with the extracellular matrix following actin polymerization. The polymerization of actin seems to be of importance for wound repair as actin polymerization blockers (e.g. chalasin B) have a negative influence on tissue repair [201, 202]. TGF-b is produced by a variety of cells, e.g. fibroblasts, myofibroblasts, airway epithelial cells, macrophages and neutrophils, and constitutes an important factor for epithelial wound repair by interacting with the composition of the provisional extracellular matrix facilitating cell migration. MMPs play a role in the TGF-b-induced tissue repair and MMP-2, which is up-regulated by TGF-b, is a potent promoter of wound repair [203]. In a recent study Hogg et al. demonstrated that the progression of COPD was related to remodelling and deposition of connective tissue in the small airway wall and to accumulation of inflammatory exudates in the airway lumen [204]. The remodelling process in the small airways is closely related to the decline in lung function in patients with COPD [204]. Myofibroblasts are present in the human lung [205] and stellate myofibroblasts, located in the alveolar wall interstitium, seem to play an important role in the inflammatory reaction in small airways [ ]. Myofibroblasts are active in producing cytokines and other mediators [209, 210], and are activated by a number of cytokines and growth factors. TGF-b, today identified as three isoforms (TGF-b 1, TGF-b 2 and TGF-b 3 ), is an important mediator for the development of tissue fibrosis and is a stimulus for the activation of fibroblasts and stellate myofibroblasts into activated myofibroblasts [ ]. TGF-b also induces expression of extracellular matrix proteins in mesenchymal cells and induces production of proteinase inhibitors capable of preventing enzymatic breakdown of the extracellular matrix proteins [214]. The TGF-b-induced synthesis of extracellular matrix proteins is mediated by the induction of connective tissue growth factor (CTGF) [214]. It has also been demonstrated that the fibrogenic effects of TGF-b may be mediated through CTGF [215]. In this context it is interesting that the mrna expression of TGF-b is lower in human alveolar macrophages than in blood monocytes [216]. Recent data indicate that TGF-b may be of importance in the initial stages of COPD but this mechanism seems to be of less importance as the disease becomes more severe. In COPD there is no thickening of the sub-basement located lamina reticularis which is a typical feature of asthma [217, 218]. Epidermal growth factor signalling may be of importance for goblet cell metaplasia which is inhibited by EGF-blocking antibodies and catalase, a substance which inhibits kallikrein-induced EGF activation [219]. In animal models IL-13 has been demonstrated to increase mucus cell metaplasia [ ] and in human bronchial epithelial cells, IL-13 and IL-4 have been shown to increase goblet cell density [223]. The finding of enhanced IL-13 expression in CD4 + lymphocytes from smokers [224] and farmers [225, two groups of individuals with a high prevalence of chronic bronchitis and airflow limitation [226, 227], is interesting in this context. It has also been shown that IL-13 induces TGF-b in macrophages [69, 228]. It thus seems as if IL-13 has more than one role in the remodelling processes as it both stimulates goblet metaplasia and TGF-b mediated fibrosis. These findings seem contradictory to the results of a recent study in which IL-13 mrna expression in lung tissue obtained at lung surgery was reduced in patients with severe emphysema who did not have a 1 -antitrypsin deficiency compared with patients with emphysema ª 2007 Blackwell Publishing Ltd Journal of Internal Medicine 262;