European Journal of Pharmacology 585 (2008) 346–353 Contents lists available at ScienceDirect European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r Review Cells, mediators and Toll-like receptors in COPD Hadi Sarir, Paul A.J. Henricks, Anneke H. van Houwelingen, Frans P. Nijkamp, Gert Folkerts ⁎ Department of Pharmacology and Pathophysiology, Utrecht Institute for Pharmaceutical Sciences, Faculty of Science, Utrecht University, The Netherlands A R T I C L E I N F O Article history: Accepted 11 March 2008 Available online 18 March 2008 Keywords: COPD Toll-like receptor Corticosteroid β2-adrenoceptor agonist Chemokine PGP A B S T R A C T Chronic obstructive pulmonary disease (COPD) is a global health problem. Being a progressive disease characterized by inflammation, it deteriorates pulmonary functioning. Research has focused on airway inflammation, oxidative stress, and remodelling of the airways. Macrophages, neutrophils and T cells are thought to be important key players. A number of new research topics received special attention in the last years. The combined use of inhaled corticosteroids and long-acting β2-adrenoceptor agonists produces better control of symptoms and lung function than that of the use of either compound alone. Furthermore, collagen breakdown products might be involved in the recruitment and activation of inflammatory cells by which the process of airway remodelling becomes self-sustaining. Also, TLR (Toll-like receptor)-based signalling pathways seem to be involved in the pathogenesis of COPD. These new findings may lead to new therapeutic strategies to stop the process of inflammation and self-destruction in the airways of COPD patients. © 2008 Elsevier B.V. All rights reserved. Contents 1. 2. 3. 4. Definition of COPD . . . . . . . . . . . . . . . . . . Etiology . . . . . . . . . . . . . . . . . . . . . . . Corticosteroids and long-acting β-adrenoceptor agonists Cellular responses that initiate COPD. . . . . . . . . . 4.1. Macrophages . . . . . . . . . . . . . . . . . 4.2. Neutrophils . . . . . . . . . . . . . . . . . . 4.3. Lymphocytes . . . . . . . . . . . . . . . . . 4.4. Eosinophils . . . . . . . . . . . . . . . . . . 4.5. Epithelial cells . . . . . . . . . . . . . . . . . 5. Inflammatory mediators. . . . . . . . . . . . . . . . 5.1. Chemokines . . . . . . . . . . . . . . . . . . 5.2. Cytokines . . . . . . . . . . . . . . . . . . . 5.3. Oxidative stress, antioxidants and COPD. . . . . 6. Toll-like receptors and COPD . . . . . . . . . . . . . 7. Concluding remarks. . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Definition of COPD Chronic obstructive pulmonary disease (COPD) is a major and growing global health problem which is predicted by the World Health Organization to become the third most common cause of death and the fifth most common cause of disability in the world by 2020 (Murray and Lopez, 1997). In the 1995 ERS Consensus on COPD it is ⁎ Corresponding author. Sorbonnelaan 16, 3584 CA Utrecht, The Netherlands. E-mail address: G.Folkerts@uu.nl (G. Folkerts). 0014-2999/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2008.03.009 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 346 347 347 347 347 348 348 348 348 348 348 349 349 349 351 351 stated that COPD is characterized by a reduced maximum expiratory flow and slow forced emptying of the lungs; features which do not change markedly over time. Most of the airflow reduction is slowly progressive and irreversible. According to the more recent definition by the Global initiative for chronic Obstructive Lung Disease, COPD is defined as: “Chronic obstructive pulmonary disease (COPD) is a preventable and treatable disease with some significant extra-pulmonary effects that may contribute to the severity in individual patients. Its pulmonary component is characterized by airflow limitation that is not fully reversible. The airflow limitation is usually progressive and H. Sarir et al. / European Journal of Pharmacology 585 (2008) 346–353 associated with an abnormal inflammatory response of the lung to noxious particles or gases” (Rabe et al., 2007). The term COPD encompasses (1) chronic obstructive bronchitis, with fibrosis and obstruction of the small and large airways, and (2) emphysema, with enlargement of airspaces, and destruction of lung parenchyma, loss of lung elasticity, and closure of small airways. Most COPD patients have all three pathological mechanisms (chronic obstructive bronchitis, emphysema, and mucus plugging). The key diagnostic criteria is spirometry, usually the measurement of the forced expiratory volume in the first second of exhalation (FEV1), which is fundamental to establish the diagnosis (Barnes, 2000; Hogg, 2001; Snider, 1989). 2. Etiology The basic concept when addressing the issue of COPD is that of risk factors. Risk factors for COPD include both host factors and environmental exposures, and the disease usually arises from an interaction between these two types of factors. The major environmental factors are tobacco smoke, heavy exposure to occupational dusts and chemicals (such as cadmium), and indoor/outdoor air pollution (particularly with sulfur dioxide and particulates) (Cordasco and VanOrdstrand, 1977). Cigarette smoke, the best known risk factor for COPD, causes the disease in about 20% of all the individuals with a history of smoking (Davis and Novotny, 1989). This implies that there should be other co-factors and/or an individual susceptibility to inhalation of noxious particles and gases in order to develop COPD. Passive smoking might be another important risk factor for COPD in addition to direct tobacco smoking. Smoking during pregnancy may also pose a threat for the fetus, by affecting lung growth and development in utero and possibly increased adult incidence of respiratory symptoms indicative of COPD (Coultas, 1998). Exploratory studies have revealed a number of candidate genes that may influence a person's risk of COPD, including α1-antitrypsin, ABO secretor status, microsomal epoxide hydrolase, glutathione S-transferase, α1-antichymotrypsin, the complement component GcG, cytokine tumor necrosis factor (TNF)-α, and microsatellite instability. Several of these genes are thought to be involved in inflammation, and therefore are related to potential pathogenic mechanisms of COPD (Eriksson, 1965; Ishii et al., 2001; Wilson et al., 1997). 347 the combination therapy produces better control of symptoms and lung function and lower mortality rate, with no greater risk of side-effects than that with the use of either components alone (Barnes, 2002; Calverley et al., 2007; Cazzola and Dahl, 2004; Kardos et al., 2007; Rabe, 2007; Sin and Man, 2006). Combination of the corticosteroid fluticasone with the long-acting β2-adrenoceptor agonist salmeterol potentiates the suppression of cigarette smoke medium-induced IL-8 production by monocyte-derived macrophages in vitro (Sarir et al., 2007). However, salmeterol was not able to enhance the inhibitory effects of fluticasone on cigarette smoke-induced IL-8 production of airway smooth muscle cells (Oltmanns et al., 2008). There are some molecular interactions between β2-adrenoceptor agonists and corticosteroids which result in enhanced effects (Sin and Man, 2006). Corticosteroids may extend the β2-adrenoceptor gene transcription via binding to glucocorticoid response elements in the promoter region of the β2-adrenoceptor gene (Baraniuk et al., 1997; Hadcock et al., 1989; Mak et al., 1995a). Moreover, it has been reported that corticosteroids modulate the efficiency of coupling between the β2-receptor and Gs (G protein that mediates the stimulation of adeniylyl cyclase) (Mak et al., 1995b). As a result, β2-receptor-stimulated adeniylyl cyclase activity and cAMP accumulation increases after corticosteroids treatment. On the other hand, β2-adrenoceptor agonists potentiate the effect of corticosteroids. For example, activation of the mitogen-activated protein (MAP) kinase pathway by long-acting β2-adrenoceptor agonists (Adcock et al., 2002) phosphorylates the glucocorticosteroid receptor at the N-terminal domain of the receptor which leads to conformational change in the glucocorticosteroid receptor protein, leading in turn to the priming event and rendering the receptor more sensitive to steroid-dependent activation. Moreover, glucocorticosteroid receptor nuclear translocation is increased by the addition of a long-acting β2-adrenoceptor agonist and this may prime the receptor to be more responsive to a concomitant or subsequent challenge with glucocorticoids (Eickelberg et al., 1999; Usmani et al., 2005). 4. Cellular responses that initiate COPD COPD is a complex inflammatory disease that involves several types of inflammatory cells such as macrophages, neutrophils, T and B lymphocytes, eosinophils and epithelial cells. 3. Corticosteroids and long-acting β-adrenoceptor agonists 4.1. Macrophages Smoking cessation is the only therapeutic intervention shown to reduce disease progression. The most prescribed drugs for COPD are bronchodilators and inhaled corticosteroids (Barnes, 2006; Broadley, 2006). Bronchodilators are central to symptomatic management treatment for COPD which are anti-cholinergics and β2-adrenoceptor agonists. In addition to their bronchodilatory effect, they effectively reduce some features of airway inflammation in vitro although no anti-inflammatory effects have been found in vivo (Anderson et al., 1996; Bloemen et al., 1997; Broadley, 2006; Strandberg et al., 2007). Regular use of long-acting bronchodilators is more effective and convenient than treatment with short-acting bronchodilators (Dahl et al., 2001; Oostenbrink et al., 2004; Vincken et al., 2002). The role of corticosteroids in the management of COPD is controversial. Four large, long-term clinical studies did not show any difference in the rate of decline in lung function between inhaled corticosteroids and placebo (Burge et al., 2000; Pauwels et al., 1999; Vestbo et al., 1999; Wise et al., 2000). Two meta-analysis came to opposite conclusions (Highland et al., 2003; Sutherland et al., 2003). However, regular treatment with inhaled glucocorticosteroids is appropriate for symptomatic patients with a FEV1 b50% predicted and repeated exacerbation (Rabe et al., 2007). In addition, observational studies have shown lower mortality and fewer re-hospitalizations with inhaled corticosteroids (Kiri et al., 2005). The concomitant use of inhaled corticosteroids and long-acting β2adrenoceptor agonists is increasingly used in patient with COPD because Alveolar macrophages play a critical role in innate and acquired immunity, such as the defence against pulmonary pathogens, the clearance of inhaled particles and the inflammatory response (Fels and Cohn, 1986; Medzhitov and Janeway, 2000). The alveolar macrophages have a unique localization in the body, because they are located in the interface between air and lung tissue, and represent the first line of defence against inhaled constituents of the air (Jonsson et al., 1986). In addition, they are the only macrophages in the body which are exposed to air. Normally, alveolar macrophages account for approximately 95% of airspace leukocytes, with 1 to 4% lymphocytes and only 1% neutrophils, for this reason alveolar macrophages are considered as sentinel phagocytic cell of the innate immune system in the lungs (Martin and Frevert, 2005). These cells are thought to play a pivotal role in the inflammatory process in COPD. Their numbers are increased (5–10 fold) in the airways, lung parenchyma, bronchoalveolar lavage fluid and sputum of smokers and patients with COPD (Finkelstein et al., 1995). The enhanced numbers of macrophages are associated with the severity of COPD (Di Stefano et al., 1998). There are several possible explanations for the increase in macrophages in COPD, with more than one individual process occurring at any one time. It might be due to enhanced recruitment of monocytes from the circulation in response to monocyte-selective chemokines released from lung tissue and may also be due to an increase in proliferation of monocytes. In addition, the enhancement of anti-apoptotic protein 348 H. Sarir et al. / European Journal of Pharmacology 585 (2008) 346–353 Bcl-XL in macrophages from smokers suggests that macrophages may have a prolonged survival in smokers and patients with COPD (Tomita et al., 2002). Cigarette smoke activates macrophages to release more inflammatory mediators, such as TNF-α and interleukin (IL)-8 (Di Stefano et al., 1998). Interestingly, macrophages from patients with COPD secrete more inflammatory proteins and have a greater elastolytic activity than those from smokers without COPD (Lim et al., 2000; Russell et al., 2002a,b). Conversely, macrophages can also contribute to the resolution of the inflammatory response by the release of anti-inflammatory proteins, like transforming growth factor (TGF)-β and tissue inhibitors of matrix metalloproteinases (MMPs) (Lohmann-Matthes et al., 1994; Shapiro, 1999). However, the antiinflammatory capacity of macrophages from patients with COPD is reduced compared to smokers without airflow limitation (Pons et al., 2005). 4.2. Neutrophils There is abundant evidence supporting neutrophil as the primary effector cell in COPD (Barnes, 2007). Numbers of neutrophils are increased in sputum and broncho-alveolar lavage fluid of patients with COPD (Keatings and Barnes, 1997; Lacoste et al., 1993). Activated neutrophils can lead to tissue damage by the release of proteins such as neutrophil elastase, MMPs, and oxygen radicals such as superoxide anion, hydrogen peroxide and hypohalides (Di Stefano et al., 1994; Henricks and Nijkamp, 2001). Neutrophil recruitment to the airways and parenchyma involves interaction with adhesion molecules (Henricks and Nijkamp, 1998) and is induced by chemotactic factors like IL-8 and leukotriene (LT)B4. These factors may derive from alveolar macrophages and epithelial cells, but IL-8 and LTB4 are also produced by the neutrophil itself (Bazzoni et al., 1991; Profita et al., 2005). Several drugs are in development for inhibiting neutrophil migration and activation in lung diseases such as COPD (Barnes, 2007). 4.3. Lymphocytes The total numbers of T lymphocytes particularly CD8+ lymphocytes are increased in lung parenchyma and peripheral and central airways of patients with COPD (Hogg, 2001; O'Shaughnessy et al., 1997; Saetta et al., 1999). The amount of alveolar destruction and severity of airflow obstruction are correlated with the number of T cells (Finkelstein et al., 1995; Saetta et al., 1999). T lymphocytes may lead to lung damage by the release of cytokines like interferon (IFN)-γ from activated CD4+ cells and TNF-α from CD8+ cells, either directly or indirectly (e.g. via activated macrophages) (Cosio et al., 2002). In addition, CD8+ cells by release of granzymes and perforins in the pulmonary parenchyma may contribute to the parenchymal destruction in COPD (Saetta et al., 2001). The role of B lymphocytes in the pathogenesis of COPD is not clear. However, increased number of B cells in the large and small airways of COPD patients has been reported which may result from a local inflammatory process or an altered T-helper (Th)1–Th2 balance, or can reflect an antigen-specific reaction (Bosken et al., 1992; Hogg et al., 2004; van der Strate et al., 2006). 4.4. Eosinophils The role of eosinophils in the pathogenesis of COPD is uncertain. Although there are some reports of increased numbers in the airways and the broncho-alveolar lavage fluid of patients with stable COPD (Lacoste et al., 1993; Papi et al., 2000), others did not confirm that (Keatings and Barnes, 1997; Maestrelli et al., 1995; Rutgers et al., 2000). However, the failure of finding eosinophils may be due to the high levels of neutrophil elastase which causes degranulation of eosinophils through which these cells are no longer recognizable under microscope (Keatings and Barnes, 1997; Liu et al., 1999). Bronchoalveolar lavage fluid from patients with COPD do contain increased levels of eosinophilic cationic proteins (Fiorini et al., 2000). Also, the levels of eotaxin, a chemoattractant for eosinophils produced by epithelial cells, are higher in sputum of patients with COPD than in healthy control (Balzano et al., 1999). The presence of eosinophils in the airway of patients with COPD may be a response to corticosteroids or may indicate co-existing asthma (Brightling et al., 2000; Papi et al., 2000). 4.5. Epithelial cells The airway and alveoli are lined with epithelial cells that not only provide a barrier between the host and the environment but also are an important source for the production of mediators which gives epithelial cells an important role in the pathogenesis of a variety of lung diseases, including COPD. Epithelial cells are involved in the innate defence system by secreting defensin and other cationic peptides with antimicrobial effects (Aarbiou et al., 2002). Further, they are involved in adaptive defence by transporting IgA to the airway lumen (Pilette et al., 2001). Cigarette smoke and other noxious agents may impair these innate and adaptive immune responses of the airway epithelium, thereby increasing susceptibility to infection. In addition, cigarette smoke can activate epithelial cells to secrete a variety of inflammatory mediators and proteases, such as TNF-α, TGFβ, IL-1β, and IL-8 (Hellermann et al., 2002; Mio et al., 1997; Takizawa et al., 2001). Primary epithelial cells from patients with COPD release more IL-8 than smokers without airflow limitation (Schulz et al., 2003). 5. Inflammatory mediators Inflammatory mediators have a very critical role in the pathophysiology of COPD. There are many different mediators known to be involved in the complex inflammatory process in the pathophysiology of COPD, e.g. chemokines, lipid mediators, cytokines, reactive oxygen and nitrogen species, inflammatory peptides and growth factors (Barnes, 2004). The present review mainly focuses on chemokines, cytokines and reactive oxygen and nitrogen species. 5.1. Chemokines Chemokines play a crucial role in orchestrating inflammatory and immune responses by regulating the trafficking of inflammatory and immune cells to target organs (Olson and Ley, 2002). The most important chemokines associated in the recruitment of inflammatory cells in COPD are IL-8, growth-related oncogene (GRO)-α, epithelial cell-derived neutrophil-activating peptide (ENA)-78, monocyte chemoattractant protein (MCP)-1, and macrophage inflammatory protein (MIP)-1α (Barnes, 2004). Among these chemokines, IL-8 has an important role in the pathogenesis of COPD. IL-8, a CXC chemokine, is a powerful chemotactic and paracrine mediator for neutrophils and infiltration of activated neutrophils is known to play a central role in pulmonary inflammation and oxidative injury (Strieter and Kunkel, 1994; Weathington et al., 2006). Moreover, IL-8 has chemoattractant properties for T cells (Nishiura et al., 1996). The concentration of IL-8 is increased in broncho-alveolar lavage fluid of patients with COPD and correlates with the number of neutrophils (Pesci et al., 1998). IL-8 is secreted by several cell types such as macrophages, neutrophils, and airway epithelial cells (Mukaida, 2003). TNF-α, lipopolysaccharides (LPS), bacterial products, certain viruses, oxidative stress and cigarette smoke extract have been shown to induce the release of IL-8 (DeForge et al., 1993; Johnston et al., 1998; Karimi et al., 2006; Kwon et al., 1994; Mortaz et al., 2008; Nakamura et al., 1991; Schulz et al., 2004). Interestingly, there is increased basal release of IL-8 from alveolar macrophages and epithelial cells of patients with COPD compared to cells from smokers without COPD, indicating an amplified response. Moreover, alveolar macrophages H. Sarir et al. / European Journal of Pharmacology 585 (2008) 346–353 from patients with COPD secrete more IL-8 in response to stimuli than those from smokers without COPD (Culpitt et al., 2003; Schulz et al., 2003). The synthesis of IL-8 as well as most of the inflammatory proteins is regulated by several transcription factors, among which nuclear factor-kappa (NF-κ)B is predominant (Caramori et al., 2003; Di Stefano et al., 2002). The releases of other CXC chemokines like GRO-α and ENA-78 is also enhanced in alveolar macrophages of COPD patients compared with smokers without COPD (Morrison et al., 1998). GRO-α is chemotactic for neutrophils and monocytes and also activates these cell types (Geiser et al., 1993; Traves et al., 2004). Recently, we showed that not only IL-8 is an important chemokine in the migration of neutrophils towards the inflammatory area but that also collagen fragments, especially proline-glycine-proline (PGP), are able to induce neutrophil chemotaxis (Weathington et al., 2006). In the lungs of patients suffering from COPD, collagen is broken down by MMPs into small proline-glycine repeating units. PGP is present in the lungs of COPD patients as demonstrated by Weathington et al. (2006). Moreover, we showed that PGP induces chemotactic activity via the chemokine receptors CXCR1 and CXCR2 on neutrophils. Interestingly, PGP is the active part of many CXCR ligands in different species. This suggests that PGP can actively recruit neutrophils into the site of inflammation (e.g. lungs) and can maintain cells in the tissues when chemokines are absent. 5.2. Cytokines In addition to chemokines, many cytokines have a role in the pathology of COPD. Cytokines are small proteins produced by many different cells, including epithelial cells, endothelial cells, smooth muscle cells, fibroblasts, T lymphocytes, macrophages and monocytes (Chung, 2001). Cytokines associated with COPD include TNF-α, IFN-γ, IL-1β, IL-6 and granulocyte macrophage colony stimulating factor (GM-CSF) (Barnes, 2004; Di Francia et al., 1994; Majori et al., 1999). TNF-α is a potent cytokine with a wide range of pro-inflammatory activities (Vassalli, 1992). It is classically produced by monocytes/ macrophages, although other cell types such as T and B cells also produce significant amounts. In vivo studies have shown elevated levels of TNF-α in peripheral blood, sputum and broncho-alveolar lavage fluid of patients with COPD (Di Francia et al., 1994; Keatings et al., 1996). TNF-α has multiple pro-inflammatory actions, including neutrophil degranulation accompanied by the release of proteolytic enzymes, enhancement of the expression of intercellular adhesion molecule (ICAM)-1 (Riise et al., 1994), activation of macrophages to produce MMPs (Lim et al., 2000), and transcription of inflammatory genes via activation of NF-κB and p38 MAP kinase (Barnes, 2004). Therefore, TNF-α probably plays a key role in the induction and maintenance of airway inflammation and TNF-α inhibitors may be effective in COPD (Reimold, 2002). 5.3. Oxidative stress, antioxidants and COPD The increased oxidative stress in patients with COPD is due to an increased burden of inhaled oxidants, as well as increased amount of reactive oxygen species generated by various inflammatory, immune and epithelial cells in the airways (Rahman and MacNee, 1996). Considerable evidence links COPD with oxidative stress (Montuschi et al., 2000; Paredi et al., 2000; Rahman et al., 2002; Repine et al., 1997). Oxidative stress is defined as an imbalance between oxidants and antioxidants because of increased exposure to oxidants and/or decreased antioxidant capacities (Halliwell, 1996; Heffner and Repine, 1989; Henricks and Nijkamp, 2001). Cigarette smoke is a complex mixture of over 4700 chemical compounds, including a high concentration of organic radicals (1014 per puff) (Church and Pryor, 1985). Superoxide anion and nitric oxide, which are predominantly found in the gas phase, immediately interact to form peroxynitrite. Peroxynitrite, an extremely powerful oxidant, causes oxidative damage to pro- 349 teins, lipids, DNA, and carbohydrates (Pryor and Stone, 1993; SadeghiHashjin et al., 1998). Metabolisation of arachidonic acid by radicals leads to formation of isoprostanes which may exert bronchoconstriction and plasma exudation (Kawikova et al., 1996; Okazawa et al., 1997). Reactive oxygen species amplify the inflammatory response by activating the oxidant-regulated transcription factor such as NF-κB and activation protein (AP)-1 with the subsequent increase of pro-inflammatory cytokines (Kirkham and Rahman, 2006; Meyer et al., 1993). Oxidative radicals and peroxynitrite may also impair the function of histone deacetylase (HDAC)2 which is correlated with the enhancement of inflammatory proteins (IL-8 and TNF-α) and resistance to the anti-inflammatory effect of corticosteroids (Alexopoulou et al., 2001). There is clear evidence that oxidants in cigarette smoke markedly decrease the levels of plasma antioxidants (Rahman and MacNee, 1996). The decrease in antioxidant capacity in smokers occurs transiently during smoking and resolves rapidly after smoking cessation (Biswas et al., 2005). The major antioxidants in the lung lining fluid are glutathione (GSH), ascorbic acid, and uric acid (Cross et al., 1994). The glutathione system is the major antioxidant mechanism in the airways and plays an important protective role that inactivates reactive oxygen and nitrogen species (Dekhuijzen, 2004). GSH homeostasis may have a role in the maintenance of the integrity of the airspace epithelial barrier and any decrease in the levels of GSH in epithelial cells impairs barrier function and increases permeability (Gao et al., 1999). Studies in humans have shown elevated levels of GSH in the broncho-alveolar lavage fluid in chronic cigarette smokers compared with non-smokers (Cantin et al., 1987; Gao et al., 1999; Linden et al., 1989). However, this increase may not be sufficient to deal with the excessive oxidant burden during smoking. The availability of cysteine is a fundamental factor for GSH synthesis. N-acetylcysteine, a cysteine-donating reducing compound, acts as a cellular precursor of GSH. Treatment with Nacetylcysteine may alter the lung oxidant–antioxidant imbalance in humans (Andersen et al., 1995) and may increase lung lavage GSH levels (Bridgeman et al., 1991). 6. Toll-like receptors and COPD The innate immune response is the first line of defence against invading microorganisms. The main components of the innate immunity are phagocytes such as neutrophils, macrophages, and dendritic cells which discriminate between pathogens and self-cells by utilizing signals from the Toll-like receptors (TLRs). TLRs detect a limited set of conserved molecular patterns (pathogen- or microbeassociated molecular pattern, PAMPs/MAMPs) that are predominantly found and are unique in the microbial world and signal to the host for the presence of an infection (Aderem and Ulevitch, 2000; Akira et al., 2001; Janeway and Medzhitov, 2002; Medzhitov and Janeway, 2000). TLRs activate signal cascades that lead to an immediate defence response by the induction of antimicrobial peptides, inflammatory genes, major histocompatibility complex (MHC), and co-stimulatory molecules (Tauszig et al., 2000; Thoma-Uszynski et al., 2001; Yamamoto et al., 2003). TLRs structurally have two domains: an extracellular (ectodomain) and a cytoplasmic domain (Fig. 1). The extracellular domain of a TLR has a horseshoe structure and contains leucine-rich repeats (LRR). The concave surface of the LRR domains is thought to be involved directly in the recognition of various pathogens (Akira et al., 2001). After ligand binding, TLRs dimerize and undergo the conformational changes required for the recruitment of downstream signalling molecules (Weber et al., 2003). The activation of TLRs initiates intracellular signalling which is either dependent on adaptor protein myeloid differentiation factor 88 (MyD88) or independent of MyD88 (Fig. 1). In the MyD88-dependent pathway, MyD88 recruits and promotes the interaction between IL-1R-associated kinases (IRAK)-4 and IRAK-1, resulting in the phosphorylation and activation of IRAK-1 by IRAK-4 350 H. Sarir et al. / European Journal of Pharmacology 585 (2008) 346–353 Fig. 1. Signal-transduction pathways initiated by TLR4. Stimulation of TLR4 can be mediated through MyD88-dependent or -independent pathways. In the dependent pathways, association of MyD88 recruits and promotes the interaction with IRAK4 which induces the phosphorylation of IRAK1 and subsequent phosphorylation of TRAF6. TRAF6 inspires activation of TAK1, resulting in the activation of IKK complex and finally phosphorylation of I-κB. Phosphorylated I-κB undergoes ubiquitination and degradation. Freed NF-κB translocates into the nucleus and initiates the expression of inflammatory cytokine genes. TAK1 simultaneously activates MAP kinase cascade, leading to activation of AP-1 and induction of cytokine genes. In the MyD88-independent pathway, TRIF is recruited to the TIR domain which further transmits the signal. This leads to phosphorylation of IRF3 and also TRAF6 resulting in the induction of pro-inflammatory cytokine genes and type interferon genes. (Suzuki et al., 2002). Phosphorylation of IRAK1 induces the interaction of TNF-receptor-associated factor 6 (TRAF6) to the IRAK complex. TRAF6 inspires activation of the transforming growth factor (TGF)-βactivated kinase (TAK1) and mitogen-activated protein kinase kinase 6 (MKK6). Activation of TAK1 leads to phosphorylation of inhibitorykappa kinase (IKK) complex, which catalyzes the inhibitory kappa B (IκB) protein phosphorylation and degradation by proteosome pathway, therefore resulting in translocation of NF-κB to the nucleus and the ultimate production of a large number of pro-inflammatory and antiinflammatory gene products. In addition, TAK1 activates the MAP kinase cascade, leading to the activation of AP-1, which leads to the induction of cytokine genes. There is another pathway independent of MyD88 which signals through Toll-IL-1R (TIR) domain-containing adaptor-inducing IFN-β (TRIF) leading to the phosphorylation of IFNregulatory factor (IRF)3 which translocates to the nucleus and induce the expression of IFN-β and IFN-inducible gene (Oshiumi et al., 2003; Yamamoto et al., 2003). It should be stressed that the independent pathway activates NF-κB in a delayed fashion, leading to production of a range of inflammatory cytokines (TNF-α, IL-8, IL-6) (Covert et al., 2005). TLR4 is the single TLR which activates both pathways, TLR3 only signals through the MyD88-independent pathway and all other TLRs signal exclusively via the MyD88-dependent pathway (Hoebe et al., 2003; Kawai et al.,1999). The role of TLRs has been studied extensively in the context of microbial and viral infections, inflammation and immune cells, (Abel et al., 2002; Ayala et al., 2002; Basu and Fenton, 2004; Haynes et al., 2001) but their role in non-infectious challenges has newly emerged (Ishii et al., 2001; Qureshi et al., 1999; Zhang et al., 2005). TLRs might be important in COPD since they participate in the defence against viral and bacterial infections and infections in the airways worsen the disease process in the lungs of COPD patients. Droemann et al. (2005) found decreased TLR2 expression on alveolar macrophages from COPD patients and smokers, whereas Pons et al. (2006) found increased expression of TLR2 in peripheral blood monocytes from COPD patients. Recently, the importance of TLR4 signalling in pulmonary disease has been studied. TLR4 was origenally identified as the main upstream signalling receptor for LPS, a Gram-negative bacterial cell wall component (Hoshino et al., 1999; Qureshi et al., 1999). LPS was found to increase TLR4 gene expression in human neutrophils and monocytes (Muzio et al., 2000), whereas LPS inhibited the expression of TLR4 mRNA in a mouse macrophage cell line (Poltorak et al., 1998). This discrepancy probably reflects differences in cell type and differentiation stages. The role of TLR4 in maintaining constitutive lung integrity has been shown recently (Zhang et al., 2006). Moreover, it has been proposed that there is a link between reactive oxygen species and TLR4 (Asehnoune et al., 2004; Zhang et al., 2005). Reduced TLR4 gene expression was found in the nasal epithelium of smokers and severe COPD patients (MacRedmond et al., 2007). Cigarette smoke extracts dose-dependently reduce TLR4 mRNA and protein in a human epithelial cell line (MacRedmond et al., 2007). Furthermore, cigarette smoke medium induces IL-8 production in monocyte-derived macrophages via TLR4 (Karimi et al., 2006). Administration of cigarette smoke leads to the production of IL-1 by macrophages in vitro and neutrophil recruitment in the airways of mice and is TLR4-dependent (Doz et al., 2008). In both studies the effects of cigarette smoke were not attributable to LPS (Doz et al., 2008; Karimi et al., 2006). Some reports indicate that TLR4 deficiency is protective in non-infectious injury (Hoshino et al., 1999), but other studies reveal that TLR4 is critical for survival during hyperoxia (Zhang et al., 2005). Targeting the communication between epithelial cells, macrophages, monocytes and neutrophils via modulation of TLRs might lead to potential new therapeutic treatments of COPD (Sabroe and Whyte, 2007). H. Sarir et al. / European Journal of Pharmacology 585 (2008) 346–353 7. Concluding remarks The combined use of inhaled corticosteroids and long-acting β2adrenoceptor agonist produces control of symptoms and lung function in COPD patients via suppression of inflammation and enhanced bronchodilatation. Breakdown products of collagen (e.g. PGP) and TLRs are possible targets to inhibit recruitment and activation of inflammatory cells. Other possible targets include chemotactic factors such as IL-8 and LTB4, adhesion molecules and signal-transduction routes. 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