AirwayinflammationinCOPD by Asociación Latinoamericana de Tórax ALAT

Airway inflammation in chronic obstructive pulmonary disease Katarzyna Go´rska, Marta Maskey-Warze˛chowska and Rafał Krenke Department of Internal Medicine, Pneumonology and Allergology, Medical University of Warsaw, Warsaw, Poland Correspondence to Katarzyna Go´rska, Banacha 1A, 02-097 Warsaw, Poland Tel: +48 225992562; fax: +48 225991560; e-mail: kpgorska@wp.pl Current Opinion in Pulmonary Medicine 2010, 16:89–96

Purpose of review Understanding the chronic inflammatory process that affects the airways of patients with chronic obstructive pulmonary disease (COPD) is an important clue in the search for new therapeutic options. The main inflammatory cells and mediators involved in COPD pathogenesis have been identified, but there is still little knowledge about their mutual interactions that result in the final outcome, that is, structural airway changes and progressive airflow limitation. Recent findings Recent studies created novel theories on the inflammatory pathway in COPD and focused not only on the influence of cigarette smoke but also on other factors initiating airway inflammation. There is evidence that apart from neutrophils and macrophages, eosinophils may play an important role in the pathogenesis of COPD and patients with eosinophilic inflammation may present a distinct phenotype. This may have therapeutic implications. New cytokines (e.g. interleukin 32) involved in COPD pathogenesis have been identified. The increased number of inflammatory cell subpopulations need not necessarily be associated with their increased activity, suggesting their complex role in inducing/sustaining airway inflammation in COPD. The presence of inflammation in the upper airways in the course of COPD has also been found. Summary There are many questions concerning the pathogenesis of COPD yet to be answered. Results of recently published studies show a new approach to airway inflammation in COPD and indicate new interesting directions in COPD research. Keywords airways, chronic obstructive pulmonary disease, cytokines, inflammation, inflammatory cells Curr Opin Pulm Med 16:89–96 ß 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins 1070-5287

Introduction The pathologic features of chronic obstructive pulmonary disease (COPD) include diffuse persistent inflammation and remodeling of the airways and lung parenchyma. This is caused by an abnormal inflammatory response of the lungs to noxious particles and gases (especially cigarette smoke). The inflammation that occurs in COPD is described as neutrophilic [1]. Together with proteases and oxidant stimuli, which directly affect lung structures, inflammatory cells actively participate in the pathogenesis of the disease and promote biochemical reactions that result in progressive alteration and remodeling of the lower airways. Interesting findings regarding the inflammatory process in COPD show that the airways are not the only site of inflammation induced by cigarette smoke and that chronic inflammation may persist even after smoking cessation [2,3]. Although the pathogenesis of COPD has been extensively studied, the role of 1070-5287 ß 2010 Wolters Kluwer Health | Lippincott Williams & Wilkins

different mechanisms and particular inflammatory cells in sustaining chronic airway inflammation is still unclear. In the past few years, there have been several important advances in our understanding of the pathogenesis of airway inflammation in COPD. This was possible due to studies not only of selected aspects of the inflammatory process but also of a wider spectrum of factors activating and promoting airway inflammation. These include reactive oxygen species (ROS), structural viral/bacterial elements described as pathogen-associated molecular patterns (PAMPs), and, finally, endogenous factors originating from damaged tissue [damage-associated molecular patterns (DAMPs)]. All of these may activate the so-called pattern recognition receptors (PPRs), which have the capability of inducing inflammation and influence destructive/repair processes in the airways (Fig. 1) [4]. DOI:10.1097/MCP.0b013e3283341ba0

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Figure 1 Activation of airway inflammation by exogenous and endogenous factors: a simplified scheme

materials presented above originate from anatomically and functionally different areas of the respiratory tract, they reflect inflammatory phenomena in different compartments of the airways [6].

Inflammatory cells Typically, the cellular composition of the airway infiltrate consists of neutrophils, macrophages, and CD8Ăž T cells [1], nevertheless other cell populations may also participate in the inflammatory response. The main cell types involved in this process are presented below. Neutrophils

There is abundant evidence supporting the neutrophil as the primary effector cell in COPD [1].

Three backgrounds reflect different stages of activation. The large arrows show the pathways of activation, whereas the thin dashed arrows show positive feedback enhancing inflammatory reactions. DAMPs, damage-associated molecular patterns; PAMPs, pathogen-associated molecular patterns; ROS, reactive oxygen species.

Below, we present specific aspects of airway inflammation that have been the subject of research in recent years.

Biological material Various types of samples are used to assess the nature and severity of airway inflammation in COPD. The most reliable data are obtained from bronchoscopic sampling, resected lung specimens, and autopsy studies. Numerous studies have investigated techniques for indirect evaluation of airway inflammation, based on samples from the bronchial lumen and/or alveolar space. These include induced sputum and bronchoalveolar lavage fluid (BALF). Exhaled nitric oxide (FENO) and exhaled breath condensate (EBC) are the relatively new, noninvasive tools for the assessment of airway inflammation. Recently, Hens et al. [5 ] reported higher levels of inflammatory mediators [i.e. interferon-g (IFN-g) and granulocyte colony-stimulating factor (G-CSF)] in nasal secretion from patients with COPD when compared to healthy controls. This interesting finding indicates that in COPD, airway inflammation affects not only the lower but also the upper airways and opens new possibilities in the assessment of the inflammatory process in the course of the disease. It is important to note that the methodology of the studies might significantly affect their results. As some of the

Typically, the number of neutrophils is augmented in induced sputum and BALF of COPD patients and this correlates with disease severity [1,7 ]. Moreover, this increase persists even after smoking cessation [3]. Neutrophil recruitment to the airways is induced by chemotactic factors like interleukin (IL)-8 and leukotriene B4 (LTB4), and both these mediators are often elevated in the airways of patients with COPD [8 ,9]. These factors may derive from alveolar macrophages and epithelial cells, but may also be produced by the neutrophil itself [10]. Activated neutrophils may cause tissue damage and mucous hypersecretion by the release of proteins such as neutrophil elastase, matrix metalloproteinases (MMPs), and cathepsin [11 ]. Other markers of neutrophilic inflammation that are increased in COPD include myeloperoxidase (MPO), neutrophils human lipokaine (HNL), and IL-8 [11 ]. Recently, Brown et al. [12 ] showed a significant reduction in the proportion of sputum neutrophils undergoing spontaneous apoptosis in healthy smokers and individuals with COPD compared with nonsmokers. Macrophages

Alveolar macrophages are very important effector cells that play a critical role in the initiation of airway inflammation in COPD. Their number is increased in the airways, lung parenchyma, bronchoalveolar lavage fluid, and sputum of smokers and in patients with COPD (correlating with the disease severity) [13,14 ]. This may be due to enhanced recruitment of monocytes from the circulation in response to monocyte-selective chemokines, an increase in proliferation of monocytes, and prolonged macrophage survival. Cigarette smoke activates macrophages to release inflammatory mediators, such as tumor necrosis factor (TNF)-a, IL-8, and LTB4 [9].

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The macrophages from COPD patients have elevated levels of proteases such as cathepsins K, L, and S, as well as MMP-1, MMP-2, MMP-9, and MMP-12 [15]. Lymphocytes

The total number of T lymphocytes, particularly cytotoxic (Tc, CD8þ) is increased in the lung parenchyma and peripheral and central airways of patients with COPD [16 ]. The number of T lymphocytes in COPD lungs is related to the degree of airflow limitation. Tsuda et al. [17 ], who studied the features of airway inflammation in COPD among smokers and never smokers, showed significantly increased numbers of inflammatory cells, including CD4þ and CD8þ T lymphocytes in airway walls of both study groups. The numbers of CD4þ T cells were greater in never smoking COPD patients when compared with smokers with COPD, but the number of CD8þ T cells was higher among smokers.

serves as a growth factor to promote the activity of the Th17 population, which could result in a selfperpetuating condition of chronic inflammation, explaining the phenomenon of persistent airway inflammation in COPD despite smoking cessation. The observed increased expression of Th17-related cytokines IL-17A, IL-22, and IL-23 in the airways of COPD patients may reflect the involvement of this cell population in driving the chronic inflammation seen in COPD [24 ]. Elevated count of B lymphocytes was also observed in bronchial walls, but their role in the pathogenesis of COPD is not clear [17 ]. Van der Strate et al. [25] hypothesize that B cells contribute to the inflammatory process and/or the development of emphysema by producing antibodies against either tobacco smoke residues or extracellular matrix components. Eosinophils

T lymphocytes can cause tissue injuries either by direct cytolytic activity or through the secretion of pro-inflammatory mediators. CD8þ lymphocytes seem to play an important role in the development and progression of COPD [18,19]. Despite their prominence in COPD airways, their precise role still remains speculative. CD8þ lymphocytes secrete a Th1-predominant cytokine pattern including IFN-g, interferon-inducible protein 10 (IP-10; CXCL10), monokine induced by interferongamma (MIG; CXCL9), and cytotoxic mediators (granzyme, perforins) and enhance Fas expression [20 ,21]. Granzyme B and perforins may induce apoptosis of type 1 pneumocytes, thereby contributing to the development of emphysema. T helper cells (Th, CD4þ) affect downstream immune processes by the release of activating cytokines and are important in focusing and amplifying inflammatory responses by other immune effector cells. Some studies have shown an increased number of subepithelial and intraepithelial CD4þ T cells in COPD patients compared with control groups with normal lung function [16 ]. The most prominent CD4þ population that accumulates in the airways and lung are Th1 lymphocytes. The result of a recent study suggests that chronic cigarette exposure results in upregulation of T regulatory cells (a subpopulation of CD4þ T lymphocytes expressing CD25) in the BALF of COPD patients and healthy smokers [22 ]. Th17 cells are a subset of CD4þ T cells that play an important role in inflammatory diseases. Their inflammatory activity is apparently due to the production of a unique set of pro-inflammatory cytokines, including IL-6, IL-17A, IL-17F, granulocyte macrophage colony-stimulating factor (GM-CSF), and TNF-a. These cells also express receptors for both IL-18 and IL-23 [23]. Importantly, IL-23

The role of eosinophils in the pathogenesis of COPD is uncertain. There are some reports of increased eosinophil count in the sputum and BALF of patients with stable COPD [26]. Increased levels of eosinophil basic proteins in BALF and in induced sputum of COPD patients, even in the absence of eosinophils, were observed. This suggests that eosinophil degranulation occurs in COPD [27]. The role of eosinophilic inflammation in COPD is also discussed below. Epithelial cells

Cigarette smoke can activate epithelial cells to secrete a variety of inflammatory mediators and proteases, such as TNF-a, transforming growth factor (TGF)-b, IL-1b, and IL-8, thus promoting inflammation and emphysema [28,29]. Epithelial cells are involved in lymphoid follicle formation in the airways of COPD patients [30 ]. Lymphoid follicles are formed by aggregated inflammatory cells (neutrophils, macrophages as well as CXCR3expressing T lymphocytes and B lymphocytes) and take part in the adaptive immune response. The number of these structures within the airways is reportedly increased in COPD [30 ,31]. Dendritic cells

There is evidence that cigarette smoke exposure may lead to altered maturation and impaired function of airway dendritic cells. Mortaz et al. [32] demonstrated that dendritic cells exposed to cigarette smoke in vitro release chemokines (macrophage inflammatory proteins – MIP1a and MIP-2a), which recruit neutrophils and enhance CD8þ lymphocyte proliferation. Surprisingly, cigarette smoke exposure did not lead to the production of other mediators typical for airway inflammation in COPD (IL-2, IL-6, IL-10, and TNF-a). These results need to be verified in in-vivo models.

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Dendritic cells also participate in lymphoid follicle formation in the airways of COPD patients [30 ].

Inflammatory mediators Many different mediators are known to be involved in COPD-associated inflammatory process, such as interleukins, chemokines, lipid mediators, growth factors, and reactive oxygen and nitrogen species. Inflammatory peptides associated with COPD potentially stimulated by cigarette smoke include IL-1b, IL-8, IL-6, IL-18, IFN-g, TNF-a, TGF-b, and GMCSF [9,20 ,33–35]. Secretion of many cytokines and chemokines is regulated by transcription factors, among which nuclear factor (NF)-kB plays the key role. Chemokines

The most important chemokines associated with the recruitment of inflammatory cells in COPD are IL-8, growth-related oncogene (GRO)-a, epithelial cellderived neutrophil-activating peptide (ENA)-78, monocyte chemoattractant protein (MCP)-1, and MIP-1a [28]. IL-8 (a CXC chemokine) plays the most important role in initiating andamplifying inflammatoryprocesses in COPD. It is secreted by several cell populations such as macrophages, neutrophils, and airway epithelial cells [20 ,36]. IL-8 is a potent neutrophil and lymphocyte chemoattractant that is elaborated by diverse parenchymal and immune effector cells, including monocytes and lymphocytes [20 ]. The concentration of IL-8 is increased in the sputum and BALF of COPD patients and correlates not only with the number of neutrophils [13,37,38] but also with COPD severity [39]. It seems that IL-8 could play a part in the development of emphysema [20 ]. Leukotriene B4, which is released by macrophages, is another important neutrophil chemotactic agent. The elevated LTB4 concentrations and correlation with the number of neutrophils were observed in COPD patients [10].

O’Reilly et al. [40 ] showed that also collagen fragments, like proline–glycine–proline (PGP) and N-a-PGP are able to induce neutrophil chemotaxis. They were detected in COPD sputum samples and may represent novel diagnostic tests and biomarkers for COPD.

to produce several MMPs, and transcription of inflammatory genes through the activation of NF-kB and p38 mitogen-activated protein kinase (MAPK) [41 ]. Increased levels of TNF-a in sputum and BALF of patients with COPD were documented [1,42]. Recently, Bathoorn et al. [43] showed that induced sputum TNF-a concentration might be a sensitive and specific marker of COPD exacerbations caused by bacterial infection. IFN-g is a pro-inflammatory cytokine produced by lymphocytes and natural killer cells. It plays a crucial role in the activation of macrophages and in the response to viral and bacterial infections. It has been shown to be upregulated in lymphocytes isolated from emphysematous lung tissue samples [21], bronchoalveolar lavage fluid [19], and sputum of patients with COPD [44]. IL-1b is a potent stimulator of alveolar macrophages to secrete inflammatory factors. Positive correlations between sputum concentration of IL-1b, sputum neutrophils, and macrophages count were found in COPD patients [45]. The precise role of IL-6 in the development of COPD is still unclear. Increased IL-6 levels have been found in induced sputum, exhaled breath condensate, and BALF from COPD patients. IL-6 is a part of the acute phase immune response and appears to be a potent inducer of C-reactive protein expression in the liver [46]. An interesting finding is that inflammation, most likely involving IL-6, may substantially contribute to pulmonary hypertension complicating COPD [47]. IL-18, a member of the IL-1 family, plays an important role in Th1 polarization and was described as an important regulator of the innate and adaptive immunity. Hoshino et al. [48] reported that constitutive IL-18 overproduction in the lungs of mice induces emphysema, suggesting its important role in the pathogenesis of COPD. The overexpression of IL-18 in the lungs of COPD patients was demonstrated, and it is possible that IL-18 blockade may be a feasible anti-inflammatory treatment for these patients [34]. Calabrese et al. [49 ] were the first to demonstrate an increased expression of IL-32 in the lung tissue of patients with COPD, which correlated with the level of TNF-a and the degree of airflow obstruction. This suggests that IL-32 is involved in the specific immune response observed in COPD and may have an impact on disease progression.

Cytokines

TNF-a is a proapoptotic cytokine, which is mainly produced by macrophages, but also by T and B cells. This cytokine has multiple pro-inflammatory actions, including neutrophil degranulation, activation of macrophages

Enzymes

Pro-inflammatory cytokines (such as IL-8, TNF-a) and IL-1b activate the p38 subgroup of MAPKs. The p38 subgroup is involved in mediating pro-inflammatory

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responses and seems to play a prominent role in pathogenesis of COPD. A recent study by Renda et al. [41 ] showed an increase in the phosphorylated form of p38 MAPK in the alveolar macrophages and the alveolar walls of smokers with severe and mild-to-moderate COPD. Phosphor-p38 expression was related to the degree of lung function impairment and the number of CD8þ T cells infiltrating the alveolar walls. Matrix metalloproteinases

MMPs are proteolytic enzymes involved in lung tissue destruction. They can be detected in the lung tissue, BALF, and induced sputum from COPD patients. Interestingly, elevated sputum MMP levels do not necessarily correlate with their activity [50]. This warrants further studies on the role of MMPs in COPD pathogenesis. Lipid mediators

We have already mentioned the pro-inflammatory activity of LTB4. Prostaglandin (PG)E2 is another lipid mediator that is reported to play a role in airway inflammation. In COPD, the sputum level of PGE2 is elevated and correlates with the degree of airflow limitation [51,52]; this may be related to impaired fibroblast tissue repair and may enhance emphysema formation [52]. Growth factors

In COPD, GM-CSF is released by alveolar macrophages and may be important for increased survival of neutrophils and macrophages in the airways [53]. The sputum concentrations of GM-CSF were recently found to be elevated in moderate and severe COPD [54]. TGF-b is a multifunctional growth factor that plays an important role in bronchial airway remodeling, inducing the proliferation of fibroblasts and airway smooth muscle cells. TGF-b also has immunoregulatory effects by CD25þ regulatory T cells suppressing Th1 and Th2 cells. In COPD, increased expression of TGF-b in small airway epithelium was found and TGF-b mRNA levels correlated with the extent of smoking history and the degree of airway obstruction [55]. Increased TGF-b production by fibroblasts that is observed in COPD is, however, associated with a reduced response of these cells to this growth factor [52]. A simplified scheme which summarizes the most important inflammatory cell and mediator interactions in the airways of COPD patients is presented in Fig. 2.

Neutrophilic vs. eosinophilic phenotype of airway inflammation in chronic obstructive pulmonary disease Airway neutrophilia and CD8þ T lymphocyte infiltration have been the hallmarks of COPD inflammation, but

some studies in COPD have also shown the presence of eosinophilic inflammation. Increased numbers of eosinophils with increased levels of eosinophil cationic protein (ECP) have been reported in induced sputum, BALF, and airway wall [18,56]. Patients with eosinophilic airway inflammation may represent a distinct subgroup of COPD. Identifying this subgroup may offer new insights into the pathogenesis of the disease and have implications for disease management. Increased sputum eosinophils have been found not only in acute COPD exacerbation but also in patients with stable mild-to-moderate COPD [56]. In the study by Bartoli et al. [11 ], nearly 26% COPD patients had sputum eosinophil percentages greater than 3%. In our own study [56], as many as 15 of 17 patients with mild-to-moderate COPD presented with sputum eosinophilia. Animal model studies revealed an increased influx not only of neutrophils but also of eosinophils to the airways after persistent exposure to tobacco smoke. This inflammatory cell migration was observed mainly in the bronchial lumen, not in the alveoli [33]. A recent study reported that a preferential distribution of eosinophils toward the airway lumen characterized patients with COPD with symptoms of chronic bronchitis compared with those without these symptoms [57 ]. Bafadhel et al. [26] reported higher levels of IL-5 in sputum samples from COPD patients with sputum eosinophilia when compared with those with a normal sputum eosinophil count. Importantly, they also found a correlation between sputum IL-5 concentration and sputum eosinophil count. The importance of sputum eosinophilia in COPD patients is not known, but it has been associated with a better response to corticosteroids [26] and a reduction in severe exacerbation rates [58]. It is possible that eosinophilic airway inflammation in COPD patients reflects a different inflammatory pathway in this disease. The increased frequency of eosinophilic inflammation in mild COPD (mainly in the chronic bronchitis phenotype) [56] may suggest that eosinophilic infiltration could initiate the chronic inflammation with neutrophil predominance seen in more advanced disease stages. However, as Gibson and Simpson [59] noted in their recent review, there is a subgroup of patients with an overlap syndrome of asthma and COPD, and patients from this subgroup could have affected the results of the above-mentioned studies. The characteristics of airway inflammation in the overlap syndrome are poorly described, as most studies exclude smokers labeled as asthmatics or atopic COPD patients.

94 Obstructive, occupational and environmental diseases Figure 2 Airway inflammation in chronic obstructive pulmonary disease: basic pathways of the inflammatory cell and mediator interactions

CCL2 (MCP-1, monocyte chemoattractant protein); CXCL, CXC chemokine ligand; CXCL10 (IP-10, interferon-inducible protein); CXCL11 (IP-9); CXCL9 (MIG, monokine induced by interferon); ECP, eosinophil cationic protein; IFN, interferon; IL, interleukin; LT, leukotriene; MIP, macrophage inflammatory proteins; MMP, matrix metalloproteinase; NE, neutrophil elastase; PAF, platelet-activating factor; PE, prostaglandin; Tc, T-cytotoxic cell; TGF, transforming growth factor; Th, T helper cell; TNF, tumor necrosis factor.

Conclusion There has been a notable progress in the knowledge on airway inflammation in COPD. The role of various inflammatory cells and mediators has been elucidated. It seems that a change in the approach to this issue is observed and this includes the acknowledgement of airway inflammation as one of the most important but, however, not the sole element of inflammation triggered by cigarette smoke and other noxious agents, identifying the pro-inflammatory pathways triggered by ROS, PAMPs, and DAMPs, and a tendency to focus on the cell interaction and cytokine network rather than specific cell populations or mediators in research studies. Due to volume restrictions, certain important issues were not addressed. These include potential differences in the airway inflammatory pattern in COPD with chronic bronchitis and emphysema predominance and COPD in

smokers and never smokers. Comparing airway inflammation in smokers and never smokers as well as in COPD induced by cigarette smoke and that induced by airway pollution/occupational exposure seems to be a promising new direction for COPD research, as the number of nonsmoking patients with COPD is increasing [60 ]. Also, it seems that airway inflammation, which occurs in the overlap syndrome of COPD and asthma, requires special attention, as this may have therapeutic and prognostic implications. Another interesting issue is COPDrelated inflammation in the upper airways. This may offer a powerful tool in the assessment of airway inflammation in COPD, as the upper airways are easily accessible and require relatively noninvasive methods of investigation. In summary, despite numerous studies on COPD, many questions concerning its pathogenesis are yet to be answered. Results of recently published studies indicate new interesting directions in COPD research.

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References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: of special interest of outstanding interest Additional references related to this topic can also be found in the Current World Literature section in this issue (pp. 163–164).

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