**Meet the editor**

Dr Ravi Mahadeva is Director of the Cambridge COPD Centre and Clinical Director for Respiratory Medicine, Cambridge University Hospitals Foundation Trust and Associate Lecturer, Department of Medicine, University of Cambridge. He trained in pulmonary medicine in London and Cambridge, UK, and was a recipient of a Wellcome Trust Advanced Clinical Fellowship to work

on the pathogenesis of emphysema at Washington University, St Louis and Harvard Medical School, USA. He was awarded the prestigious European Respiratory Society COPD research award 2004.

Contents

**Preface VII** 

Raja T. Abboud

Frank Guarnieri

Chapter 1 **Pathogenic Mechanisms in Emphysema:** 

Sam Alam and Ravi Mahadeva

Chapter 4 **The Dichotomy Between Understanding and Treating Emphysema 69** 

Chapter 6 **Endoscopic Lung Volume Reduction 89** 

**From Protease Anti–Protease Imbalance to Apoptosis 1** 

Chapter 2 **Innate Immunity of Airway Epithelium and COPD 19** 

Chapter 3 **The Role of Alpha–1 Antitrypsin in Emphysema 49** 

Chapter 5 **Combined Pulmonary Fibrosis and Emphysema (CPFE) 79** 

**in Patients with Underlying Emphysema 103**  Boon-Hean Ong, Bien-Keem Tan and Chong-Hee Lim

Keisaku Fujimoto and Yoshiaki Kitaguchi

Daniela Gompelmann and Felix J.F. Herth

Chapter 7 **Surgical Management of Prolonged Air Leak** 

Shyamala Ganesan and Uma S. Sajjan

### Contents

#### **Preface XI**


Chapter 7 **Surgical Management of Prolonged Air Leak in Patients with Underlying Emphysema 103**  Boon-Hean Ong, Bien-Keem Tan and Chong-Hee Lim

Preface

disease.

The last decade has seen the emergence of COPD as a major health problem worldwide. The recognition of this has stimulated the biomedical community to actively research in this area, towards understanding the pathogenesis of this devastating disease. This book contains a mixture of summaries of complex molecular pathogenic mechanisms, emerging new clinical entities and novel treatments. The book begins with sections on pathogenesis, innate immunity, anti-proteinase function and a review of the relationship between hypothesis, basic science and the development of a related treatment. These chapters are followed by description of the newly recognized association between pulmonary fibrosis within COPD and state-of-the art descriptions of novel bronchoscopic treatments and new strategies for the management of the common clinical problem of air leaks. It is currently an exciting time in COPD, and it is hoped that this book will stimulate further interest in this hitherto relatively neglected

Director of the Cambridge COPD Centre and Clinical Director for Respiratory Medicine,

Cambridge University Hospitals Foundation Trust and Associate Lecturer,

Department of Medicine, University of Cambridge

**Dr Ravi Mahadeva** 

United Kingdom

### Preface

The last decade has seen the emergence of COPD as a major health problem worldwide. The recognition of this has stimulated the biomedical community to actively research in this area, towards understanding the pathogenesis of this devastating disease. This book contains a mixture of summaries of complex molecular pathogenic mechanisms, emerging new clinical entities and novel treatments. The book begins with sections on pathogenesis, innate immunity, anti-proteinase function and a review of the relationship between hypothesis, basic science and the development of a related treatment. These chapters are followed by description of the newly recognized association between pulmonary fibrosis within COPD and state-of-the art descriptions of novel bronchoscopic treatments and new strategies for the management of the common clinical problem of air leaks. It is currently an exciting time in COPD, and it is hoped that this book will stimulate further interest in this hitherto relatively neglected disease.

#### **Dr Ravi Mahadeva**

Director of the Cambridge COPD Centre and Clinical Director for Respiratory Medicine, Cambridge University Hospitals Foundation Trust and Associate Lecturer, Department of Medicine, University of Cambridge United Kingdom

**1** 

Raja T. Abboud

 *Canada* 

**Pathogenic Mechanisms in Emphysema:** 

In 1963, Laurel and Erickson reported their discovery of severe α1-antitrypsin (AAT) deficiency and its association with emphysema (Laurell & Erickson, 1963). Soon after, Gross and coworkers reported that emphysema was induced in rats by the intratracheal instillation of a proteolytic enzyme (Gross et al.,1965). These findings led to the proteolytic hypothesis of emphysema (Janoff, 1985) which considers that emphysema develops as a result of the smoking-induced release of proteolytic enzymes from the increased number of neutrophils and macrophages in the lung. Proteolysis of lung connective tissue (more specifically elastin) occurs because the released proteases may not be fully inhibited by antiproteases, resulting in emphysema. However, although proteolysis may have a significant pathogenic role particularly in AAT deficiency, other pathogenic mechanism, such as oxidants either from inhaled smoke or from inflammatory cells, inflammation, T lymphocyte cell mediated

This chapter, based on a previous review article (Abboud & Vimalanathan, 2008), updated and revised following a Pub-Med search, and will cover protease-antiprotease imbalance and apoptosis, as pathogenic mechanisms in emphysema. The pathogenic role of oxidants,

The hypothesis that the main pathogenic mechanism in emphysema in severe AAT deficiency is due to protease-antiprotease , is well supported by evidence since AAT is the main inhibitor of neutrophil elastase. Since this topic will be discussed in detail in another chapter, this paragraph will serve as a brief introduction. In severe AAT deficiency, antielastase protection in the lung interstitium and alveolar space is markedly decreased in proportion to the decreased plasma levels to about 15-20 % of normal, and does not fully protect the lung against released neutrophil elastase. Neutrophil elastase is a potent elastolytic enzyme, which induces emphysema when injected intratracheally in

immunity, and apoptosis have a significant pathogenic role (MacNee, a2005).

inflammatory cells, and cell mediated immunity will be covered in other chapters.

**2. Protease-antiprotease imbalance in severe antitrypsin deficiency** 

**1. Introduction** 

 **From Protease Anti–Protease** 

 *University of British Columbia at Vancouver General Hospital,* 

 **Imbalance to Apoptosis** 

*Division of Respiratory Medicine,* 

 *Seymour Health Centre Vancouver,* 

## **Pathogenic Mechanisms in Emphysema: From Protease Anti–Protease Imbalance to Apoptosis**

Raja T. Abboud *Division of Respiratory Medicine, University of British Columbia at Vancouver General Hospital, Seymour Health Centre Vancouver, Canada* 

#### **1. Introduction**

In 1963, Laurel and Erickson reported their discovery of severe α1-antitrypsin (AAT) deficiency and its association with emphysema (Laurell & Erickson, 1963). Soon after, Gross and coworkers reported that emphysema was induced in rats by the intratracheal instillation of a proteolytic enzyme (Gross et al.,1965). These findings led to the proteolytic hypothesis of emphysema (Janoff, 1985) which considers that emphysema develops as a result of the smoking-induced release of proteolytic enzymes from the increased number of neutrophils and macrophages in the lung. Proteolysis of lung connective tissue (more specifically elastin) occurs because the released proteases may not be fully inhibited by antiproteases, resulting in emphysema. However, although proteolysis may have a significant pathogenic role particularly in AAT deficiency, other pathogenic mechanism, such as oxidants either from inhaled smoke or from inflammatory cells, inflammation, T lymphocyte cell mediated immunity, and apoptosis have a significant pathogenic role (MacNee, a2005).

This chapter, based on a previous review article (Abboud & Vimalanathan, 2008), updated and revised following a Pub-Med search, and will cover protease-antiprotease imbalance and apoptosis, as pathogenic mechanisms in emphysema. The pathogenic role of oxidants, inflammatory cells, and cell mediated immunity will be covered in other chapters.

### **2. Protease-antiprotease imbalance in severe antitrypsin deficiency**

The hypothesis that the main pathogenic mechanism in emphysema in severe AAT deficiency is due to protease-antiprotease , is well supported by evidence since AAT is the main inhibitor of neutrophil elastase. Since this topic will be discussed in detail in another chapter, this paragraph will serve as a brief introduction. In severe AAT deficiency, antielastase protection in the lung interstitium and alveolar space is markedly decreased in proportion to the decreased plasma levels to about 15-20 % of normal, and does not fully protect the lung against released neutrophil elastase. Neutrophil elastase is a potent elastolytic enzyme, which induces emphysema when injected intratracheally in

Pathogenic Mechanisms in Emphysema: From Protease Anti–Protease Imbalance to Apoptosis 3

It is likely that macrophage proteases have a pathogenic role for in human emphysema. Investigators reported that young smokers dying accidentally had an increased number of macrophages in the respiratory bronchioles (Niewoehner et al., 1974), in the same region where centrilobular emphysema develops in smokers without AAT deficiency. Morphometry of resected human lungs indicated that the extent of emphysema was directly related to the numbers of AM but not neutrophils (Finkelstein et al., 1995). These two studies suggested a potential role of macrophages in emphysema. Elastolysis by AM *in vitro* was not inhibited by AAT, while that of neutrophils was inhibited ( Chapman et al., 1984; Chapman & Stone 1984). This finding supported a pathogenic role for AM elastolytic enzymes in emphysema, since these AM enzymes would not be inhibited by AAT, the major protease inhibitor in plasma and interstitial fluid. Subsequently, investigators demonstrated several elastolytic enzymes in human AM: cathepsins L and S (Reilly et al., 1989; Reilly et al.,1991; Shi et al.,1992), the matrix metalloproteases (MMPs) MMP-2 and MMP-9, previously termed 72 & 92 kDa collagenases respectively (Senior et al., 1991) and MMP-12 also named macrophage metallo-elastase (Shapiro et al., 1993). In addition, interstitial collagenase or MMP1, a non-elastolytic enzyme , induced emphysema in transgenic mice expressing MMP1 (D'Armiento et al., 1992; Foronjy et

Several studies support a pathogenic role for AM in human emphysema, by comparing findings in subjects with and without emphysema Cultured AM from patients with emphysema showed increased elastolytic activity compared with that of AM from patients with bronchitis or other lung diseases (Muley et al., 1994). In a study of 34 healthy smokers (mean age 46 yr), there was a significantly greater AM cell counts in BAL in those with emphysema by computed tomography (CT) compared to those without emphysema; this finding indicated a greater AM elastase load in the lungs in those with emphysema, since the AM elastolytic activity/cell was similar in the two groups (Abboud et al., 1998). AM obtained by BAL from 10 emphysema patients, had increased expression of MMP9 and MMP1, when compared with 10 matched controls (Finlay et al., 1997). Emphysematous lung tissue had significantly higher levels of MMP9 and MMP2 compared with control noninvolved lung tissue; and showed elastolytic activity corresponding to MMP2 and MMP9 (Ohnishi et al.,1998). A study using immunohistochemistry of lung tissue, showed increases in MMP1, MMP2, MMP8, and MMP9 in lung tissue from COPD patients compared with controls (Segura-Valdez et al., 2000). There was increased expression of MMP1 in the lungs of patients with emphysema (Imai et al., 2001); however, the MMP1 was localized to the

Cigarette smoke induced emphysema in mice requires MMP12; mice homozygous for a knockout of the MMP12 gene, in contrast to controls, did not develop emphysema in response to cigarette smoke exposure (Hautamaki et al., 1997). However, MMP12 is much more highly expressed in mice compared with humans. A study in COPD patients reported that the number of AM in BAL expressing MMP12 and the level of MMP12 expression was higher in COPD than in controls (Molet et al., 2005). Increased MMP levels by ELISA in induced sputum from 26 stable COPD patients were significantly higher than healthy smokers, never smokers, and former smokers (Demedst et al., 2006); in addition MMP12 enzyme activity in the COPD subjects was markedly increased compared with non-smokers. These two studies support a potential pathogenic role for MMP12 in human emphysema.

**3.2 Potential role of macrophage proteases in emphysema** 

al 2003), by degrading type III collagen ( Shiomy et al., 2003).

type II epithelial cells and not macrophages.

experimental animals (Janoff et al.,1977; Senior et al,1977). Smoking increases the number of neutrophils in the lung, and induces the release of neutrophil elastase (Fera et al., 1986; Abboud et al. 1986). The released neutrophil elastase may not be fully inhibited by the severely deficient AAT levels leading to proteolytic activity and the development of emphysema. The positive correlation between increased leucocyte elastase concentration and severity of emphysema in patients with severe AAT deficiency, supports a pathogenic role for neutrophil elastase in AAT deficient emphysema (Kidokoro et al.,1977).

#### **3. Protease antiprotease imbalance in copd without severe antitrypsin deficiency**

In contrast, in smokers with COPD without AAT deficiency , there is less evidence to support protease antiprotease imbalance as a pathogenic mechanism in emphysema, compared with AAT deficient smokers, because there is no definitive evidence of severe antiprotease deficiency to lead to unopposed proteolysis in the lung. Smoking may cause a protease-antiprotease imbalance in the lung by decreasing the functional activity of AAT and other protease inhibitors in the lung interstitium and "alveolar" lining fluid, and by increasing the amount of elastolytic proteases released in the lung. Some studies reported that smokers had decreased anti-elastase activity of AAT in BAL, compared with nonsmokers (Gadek et al., 1979; Carp et al., 1982). However, this reported degree of inactivation was not confirmed by later studies (Stone et al.,1983; Boudier et al., 1983; Abboud et al., 1985).

#### **3.1 Studies evaluating neutrophil elastase in emphysema**

Cigarette smoking can induce the release of neutrophil elastase (NE) in BAL of healthy volunteers (Fera et al.,1986), and intense smoking can acutely increase plasma NE levels (Abboud et al., 1986). NE released in the lung may be taken up and internalized by alveolar macrophages (AM) (Campbell et al., 1979) . A study evaluating BAL in 28 patients with COPD supported a role for NE and protease-antiprotease imbalance by showing that NE levels in BAL correlated directly and BAL anti-elastase activity correlated inversely with emphysema, assessed by CT scan and carbon monoxide diffusing capacity (Fujita et al.,1990). Another study of older volunteers reported increased levels of NE in AM of smokers with CT scan evidence of emphysema (Betsuyaku et al.,1995), suggesting that NE release in the lung and its uptake by AM could have been a pathogenic factor in emphysema. NE bound to elastin may continue to degrade elastin despite the presence of active AAT in the surrounding medium (Morrison et al., 1990). All these findings support a potential role for NE in the development of human emphysema, despite the lack of severe inactivation of AAT in the lung. The pathogenic role of NE was also confirmed in a mouse NE-knockout exposed to cigarette smoke, where the resulting emphysema was reduced by 59% compared with control smoke-exposed mice (Shapiro et al., 2003). This was not all a direct effect of the absence of NE activity, but partly secondary to decreased macrophage recruitment in the absence of NE; it could be also partly due to the lack of degradation by NE of tissue inhibitors of metalloproteases which inhibit macrophage elastase activity.

#### **3.2 Potential role of macrophage proteases in emphysema**

2 Emphysema

experimental animals (Janoff et al.,1977; Senior et al,1977). Smoking increases the number of neutrophils in the lung, and induces the release of neutrophil elastase (Fera et al., 1986; Abboud et al. 1986). The released neutrophil elastase may not be fully inhibited by the severely deficient AAT levels leading to proteolytic activity and the development of emphysema. The positive correlation between increased leucocyte elastase concentration and severity of emphysema in patients with severe AAT deficiency, supports a pathogenic role for neutrophil elastase in AAT deficient emphysema (Kidokoro et

**3. Protease antiprotease imbalance in copd without severe antitrypsin** 

**3.1 Studies evaluating neutrophil elastase in emphysema** 

metalloproteases which inhibit macrophage elastase activity.

In contrast, in smokers with COPD without AAT deficiency , there is less evidence to support protease antiprotease imbalance as a pathogenic mechanism in emphysema, compared with AAT deficient smokers, because there is no definitive evidence of severe antiprotease deficiency to lead to unopposed proteolysis in the lung. Smoking may cause a protease-antiprotease imbalance in the lung by decreasing the functional activity of AAT and other protease inhibitors in the lung interstitium and "alveolar" lining fluid, and by increasing the amount of elastolytic proteases released in the lung. Some studies reported that smokers had decreased anti-elastase activity of AAT in BAL, compared with nonsmokers (Gadek et al., 1979; Carp et al., 1982). However, this reported degree of inactivation was not confirmed by later studies (Stone et al.,1983; Boudier et al., 1983;

Cigarette smoking can induce the release of neutrophil elastase (NE) in BAL of healthy volunteers (Fera et al.,1986), and intense smoking can acutely increase plasma NE levels (Abboud et al., 1986). NE released in the lung may be taken up and internalized by alveolar macrophages (AM) (Campbell et al., 1979) . A study evaluating BAL in 28 patients with COPD supported a role for NE and protease-antiprotease imbalance by showing that NE levels in BAL correlated directly and BAL anti-elastase activity correlated inversely with emphysema, assessed by CT scan and carbon monoxide diffusing capacity (Fujita et al.,1990). Another study of older volunteers reported increased levels of NE in AM of smokers with CT scan evidence of emphysema (Betsuyaku et al.,1995), suggesting that NE release in the lung and its uptake by AM could have been a pathogenic factor in emphysema. NE bound to elastin may continue to degrade elastin despite the presence of active AAT in the surrounding medium (Morrison et al., 1990). All these findings support a potential role for NE in the development of human emphysema, despite the lack of severe inactivation of AAT in the lung. The pathogenic role of NE was also confirmed in a mouse NE-knockout exposed to cigarette smoke, where the resulting emphysema was reduced by 59% compared with control smoke-exposed mice (Shapiro et al., 2003). This was not all a direct effect of the absence of NE activity, but partly secondary to decreased macrophage recruitment in the absence of NE; it could be also partly due to the lack of degradation by NE of tissue inhibitors of

al.,1977).

**deficiency** 

Abboud et al., 1985).

It is likely that macrophage proteases have a pathogenic role for in human emphysema. Investigators reported that young smokers dying accidentally had an increased number of macrophages in the respiratory bronchioles (Niewoehner et al., 1974), in the same region where centrilobular emphysema develops in smokers without AAT deficiency. Morphometry of resected human lungs indicated that the extent of emphysema was directly related to the numbers of AM but not neutrophils (Finkelstein et al., 1995). These two studies suggested a potential role of macrophages in emphysema. Elastolysis by AM *in vitro* was not inhibited by AAT, while that of neutrophils was inhibited ( Chapman et al., 1984; Chapman & Stone 1984). This finding supported a pathogenic role for AM elastolytic enzymes in emphysema, since these AM enzymes would not be inhibited by AAT, the major protease inhibitor in plasma and interstitial fluid. Subsequently, investigators demonstrated several elastolytic enzymes in human AM: cathepsins L and S (Reilly et al., 1989; Reilly et al.,1991; Shi et al.,1992), the matrix metalloproteases (MMPs) MMP-2 and MMP-9, previously termed 72 & 92 kDa collagenases respectively (Senior et al., 1991) and MMP-12 also named macrophage metallo-elastase (Shapiro et al., 1993). In addition, interstitial collagenase or MMP1, a non-elastolytic enzyme , induced emphysema in transgenic mice expressing MMP1 (D'Armiento et al., 1992; Foronjy et al 2003), by degrading type III collagen ( Shiomy et al., 2003).

Several studies support a pathogenic role for AM in human emphysema, by comparing findings in subjects with and without emphysema Cultured AM from patients with emphysema showed increased elastolytic activity compared with that of AM from patients with bronchitis or other lung diseases (Muley et al., 1994). In a study of 34 healthy smokers (mean age 46 yr), there was a significantly greater AM cell counts in BAL in those with emphysema by computed tomography (CT) compared to those without emphysema; this finding indicated a greater AM elastase load in the lungs in those with emphysema, since the AM elastolytic activity/cell was similar in the two groups (Abboud et al., 1998). AM obtained by BAL from 10 emphysema patients, had increased expression of MMP9 and MMP1, when compared with 10 matched controls (Finlay et al., 1997). Emphysematous lung tissue had significantly higher levels of MMP9 and MMP2 compared with control noninvolved lung tissue; and showed elastolytic activity corresponding to MMP2 and MMP9 (Ohnishi et al.,1998). A study using immunohistochemistry of lung tissue, showed increases in MMP1, MMP2, MMP8, and MMP9 in lung tissue from COPD patients compared with controls (Segura-Valdez et al., 2000). There was increased expression of MMP1 in the lungs of patients with emphysema (Imai et al., 2001); however, the MMP1 was localized to the type II epithelial cells and not macrophages.

Cigarette smoke induced emphysema in mice requires MMP12; mice homozygous for a knockout of the MMP12 gene, in contrast to controls, did not develop emphysema in response to cigarette smoke exposure (Hautamaki et al., 1997). However, MMP12 is much more highly expressed in mice compared with humans. A study in COPD patients reported that the number of AM in BAL expressing MMP12 and the level of MMP12 expression was higher in COPD than in controls (Molet et al., 2005). Increased MMP levels by ELISA in induced sputum from 26 stable COPD patients were significantly higher than healthy smokers, never smokers, and former smokers (Demedst et al., 2006); in addition MMP12 enzyme activity in the COPD subjects was markedly increased compared with non-smokers. These two studies support a potential pathogenic role for MMP12 in human emphysema.

Pathogenic Mechanisms in Emphysema: From Protease Anti–Protease Imbalance to Apoptosis 5

there was a significant negative correlation between MMP12 gene expression and carbon monoxide diffusing capacity. These results support a pathogenic role for both MMP1 and MMP12 in human emphysema. A pathogenic role for cathepsin K in the development of emphysema was demonstrated in smoke-exposed guinea pigs compared with controls, and there were also data supporting increased expression of cathepsin K in lungs of emphysema

Fig 1 is a diagram of potential mechanisms leading to protease antiprotease imbalance and

Fig. 1. Diagram showing the pathways leading to smoking-induced protease-antiprotease imbalance in the lung. (Reproduced from Abboud, R. , & Vimanalathan, S. (2008), with

Smoking induces epithelial cells to produce cytokines which stimulate neutrophils and macrophages. Cigarette smoke also acts directly on neutrophils and macrophages to activate them . Cigarette smoke has oxidants which can inactivate antiproteases, in addition to

The stimulated neutrophils and macrophages release proteolytic enzymes. Neutrophil elastase can activate MMPs, while MMPs can inactivate α1-antitrypsin. Not shown in the diagram, is the role of MMP-12 in releasing TNF-α, which amplifies the inflammatory reaction. These processes lead to a protease-antiprotease imbalance, which can degrade lung

antiprotease inactivation by oxidants released by macrophages and neutrophils.

elastin and connective tissue; if sustained, this will lead to emphysema.

permission of the publisher, Int J Tuberc Lung Dis )

patients ( Golovatch et al., 2009).

emphysema.

Smoking and pro-inflammatory stimuli can induce message expression of AM elastases and proteases, which could lead to protease-antiprotease imbalance. Smokers have increased expression of cathepsin L in AM compared to non-smokers (Takahashi et al.,1993), and also increased activity of cathepsin S in AM lysates (Reilly et al., 1991). Pro-inflammatory mediators induce expression of MMPs, such as the marked increase in mRNA for MMP12 in cultured AM by lipopolysaccharide (LPS) (Shapiro et al., 1993). TNF- and IL-1 increased expression of MMP9 by human macrophages without increasing its inhibitor, tissue inhibitor of metalloprotease ( TIMP1) (Saren et al., 1996); these two cytokines , which are increased in COPD, may thus lead to a protease-antiprotease imbalance between MMP9 and its inhibitor. The release of TNF-α in mice by cigarette smoke was dependent on MMP-12 (Churg et al., 2003), and was abolished in MMP12 knockout mice; TNF-α accounted for 70% of the smoke induced emphysema in the mouse (Churg et al., 2004). In-vitro studies showed that AM from patients with COPD released more MMP9 than AM from healthy smokers, and MMP9 release was increased by IL-1, LPS, and cigarette smoke solution (Russell et al., 2002a) . The same investigator reported that MMPs, cysteine and serine proteases contributed to the in-vitro elastolysis by human AM during the 72 hr evaluation (Russell et al., 2002b), indicating the difficulty in implicating a specific protease in lung destruction.

A recent study (Omachi et al.,2011) evaluated plasma MMP9 levels in relation to progression of emphysema over a period of one year, in 126 subjects with severe AAT deficiency who were on placebo treatment in a clinical trial evaluating AAT augmentation therapy. They found that higher baseline plasma MMP-9 levels were associated with lower values of FEV1 and CO diffusing capacity (p=0.03), but not CT scan lung density. Moreover, MMP-9 levels predicted a decline in CO pulmonary diffusing capacity (p=0.04) and worsening lung density by CT scan (p=0.003). This relationship may not apply in human emphysema without severe AAT deficiency. A thorough and elaborate study evaluated the role of MMP9 in cigarette smoke induced emphysema in mice and humans (Atkinson et al., 2011); I will restrict my review to the human findings. Macrophage MMP-9 mRNA isolated by laser capture micro-dissection from 5 human lungs obtained at the time of lung transplantation were similar in areas of lung with and without emphysema. The investigators also enrolled subjects who had completed a National Lung Screening Trial and were free of cancer or an inflammatory or immune disorder into their emphysema biomarker study. Of these 38 had a CT scan emphysema index >10% and were considered to be "emphysema-sensitive", while 47 had an emphysema index of <5% and were "emphysema-resistant" controls. Circulating monocyte MMP9 mRNA showed a positive correlation with emphysema index for all subjects (p=0.02), and a more significant correlation in the "emphysema-sensitive" group (p-0.01), but there was no statistical difference in results between the two groups. There was no correlation of circulating monocyte MMP9 mRNA with the lung injury markers used, Clara cell secretory protein and surfactant protein-D. It would be interesting to check the correlation of emphysema extent with MMP9 plasma levels, which may be a better marker of MMP9 release in the lungs than levels in circulating monocytes.

In studies from my laboratory on alveolar macrophages (AM) lavaged from resected lung specimens, the level of mRNA expression of MMP1 in AM showed a significant positive correlation with the extent of emphysema by CT scan (Wallace et al., 2008). In addition, MMP12 mRNA expression was increased in current smokers vs ex-smokers, and there was

Smoking and pro-inflammatory stimuli can induce message expression of AM elastases and proteases, which could lead to protease-antiprotease imbalance. Smokers have increased expression of cathepsin L in AM compared to non-smokers (Takahashi et al.,1993), and also increased activity of cathepsin S in AM lysates (Reilly et al., 1991). Pro-inflammatory mediators induce expression of MMPs, such as the marked increase in mRNA for MMP12 in cultured AM by lipopolysaccharide (LPS) (Shapiro et al., 1993). TNF- and IL-1 increased expression of MMP9 by human macrophages without increasing its inhibitor, tissue inhibitor of metalloprotease ( TIMP1) (Saren et al., 1996); these two cytokines , which are increased in COPD, may thus lead to a protease-antiprotease imbalance between MMP9 and its inhibitor. The release of TNF-α in mice by cigarette smoke was dependent on MMP-12 (Churg et al., 2003), and was abolished in MMP12 knockout mice; TNF-α accounted for 70% of the smoke induced emphysema in the mouse (Churg et al., 2004). In-vitro studies showed that AM from patients with COPD released more MMP9 than AM from healthy smokers, and MMP9 release was increased by IL-1, LPS, and cigarette smoke solution (Russell et al., 2002a) . The same investigator reported that MMPs, cysteine and serine proteases contributed to the in-vitro elastolysis by human AM during the 72 hr evaluation (Russell et al., 2002b), indicating the difficulty in implicating a specific protease in lung destruction.

A recent study (Omachi et al.,2011) evaluated plasma MMP9 levels in relation to progression of emphysema over a period of one year, in 126 subjects with severe AAT deficiency who were on placebo treatment in a clinical trial evaluating AAT augmentation therapy. They found that higher baseline plasma MMP-9 levels were associated with lower values of FEV1 and CO diffusing capacity (p=0.03), but not CT scan lung density. Moreover, MMP-9 levels predicted a decline in CO pulmonary diffusing capacity (p=0.04) and worsening lung density by CT scan (p=0.003). This relationship may not apply in human emphysema without severe AAT deficiency. A thorough and elaborate study evaluated the role of MMP9 in cigarette smoke induced emphysema in mice and humans (Atkinson et al., 2011); I will restrict my review to the human findings. Macrophage MMP-9 mRNA isolated by laser capture micro-dissection from 5 human lungs obtained at the time of lung transplantation were similar in areas of lung with and without emphysema. The investigators also enrolled subjects who had completed a National Lung Screening Trial and were free of cancer or an inflammatory or immune disorder into their emphysema biomarker study. Of these 38 had a CT scan emphysema index >10% and were considered to be "emphysema-sensitive", while 47 had an emphysema index of <5% and were "emphysema-resistant" controls. Circulating monocyte MMP9 mRNA showed a positive correlation with emphysema index for all subjects (p=0.02), and a more significant correlation in the "emphysema-sensitive" group (p-0.01), but there was no statistical difference in results between the two groups. There was no correlation of circulating monocyte MMP9 mRNA with the lung injury markers used, Clara cell secretory protein and surfactant protein-D. It would be interesting to check the correlation of emphysema extent with MMP9 plasma levels, which may be a better marker

In studies from my laboratory on alveolar macrophages (AM) lavaged from resected lung specimens, the level of mRNA expression of MMP1 in AM showed a significant positive correlation with the extent of emphysema by CT scan (Wallace et al., 2008). In addition, MMP12 mRNA expression was increased in current smokers vs ex-smokers, and there was

of MMP9 release in the lungs than levels in circulating monocytes.

there was a significant negative correlation between MMP12 gene expression and carbon monoxide diffusing capacity. These results support a pathogenic role for both MMP1 and MMP12 in human emphysema. A pathogenic role for cathepsin K in the development of emphysema was demonstrated in smoke-exposed guinea pigs compared with controls, and there were also data supporting increased expression of cathepsin K in lungs of emphysema patients ( Golovatch et al., 2009).

Fig 1 is a diagram of potential mechanisms leading to protease antiprotease imbalance and emphysema.

Fig. 1. Diagram showing the pathways leading to smoking-induced protease-antiprotease imbalance in the lung. (Reproduced from Abboud, R. , & Vimanalathan, S. (2008), with permission of the publisher, Int J Tuberc Lung Dis )

Smoking induces epithelial cells to produce cytokines which stimulate neutrophils and macrophages. Cigarette smoke also acts directly on neutrophils and macrophages to activate them . Cigarette smoke has oxidants which can inactivate antiproteases, in addition to antiprotease inactivation by oxidants released by macrophages and neutrophils.

The stimulated neutrophils and macrophages release proteolytic enzymes. Neutrophil elastase can activate MMPs, while MMPs can inactivate α1-antitrypsin. Not shown in the diagram, is the role of MMP-12 in releasing TNF-α, which amplifies the inflammatory reaction. These processes lead to a protease-antiprotease imbalance, which can degrade lung elastin and connective tissue; if sustained, this will lead to emphysema.

Pathogenic Mechanisms in Emphysema: From Protease Anti–Protease Imbalance to Apoptosis 7

et al., 2006), and validated in two large family-based and case-control association studies ( Zhu et al., 2007). Polymorphism of the SERPINA2 gene was also recently found associated with emphysema in consecutive autopsy cases in Japan (Fujimoto et al., 2010). Decreased activity of the plasminogen activator inhibitor type 1 in the lung can lead to increased activity

As indicated in a previously quoted review article on pathogenesis of COPD (MacNee, a2005), oxidants have a significant pathogenic role in COPD. The gaseous phase of cigarette smoke contains many reactive oxidants such as superoxide anion, nitric oxides and peroxynitrites, as reviewed recently (MacNee b2005; Lin & Thomas 2010). Oxidants and free radicals inhaled in tobacco smoke, can damage airway epithelial cells, and impair antioxidants, such as glutathione to non-reducible glutathione-aldehyde derivatives (van Der Toorn et al., 2007). Oxidants from tobacco smoke may also inactivate antiproteases, predisposing to a protease-antiprotease imbalance from the increased numbers of neutrophils and macrophages in smokers' lungs. Oxidants from cigarette smoke may also directly damage components of the lung connective tissure matrix, and interfere with elasin repair and synthesis (MacNee & Tuder 2009). Neutrophils and macrophages themselves when activated also release oxidants, such superoxides,, and nitric oxides, and contribute to the oxidative burden. Although antioxidants such as glutathione, catalase and superoxide dismutase protect the tissues against oxidants, the oxidant/antioxidant balance may tip in

Patients with COPD have increased levels of hydrogen peroxide and of 8-isoprostane (a peroxidation product of arachidonic acid) in exhaled breath condensates compared with controls (MacNee b2005). Healthy smokers had reduced histone deacetylase activity in bronchial biopsies and in alveolar macrophages obtained by lavage, when compared with age matched nonsmoking controls (Ito, K., et al., 2001). These investigators also demonstrated that smoking resulted in a greater release of TNF-α from the alveolar macrophages when stimulated by IL-1β, which they considered was due to the suppressive effect of smoking on histone deacetylation. This suppressive effect on histone deacetylation results in increased acetylation, causes local unwinding of DNA, and allows increased inflammatory gene expression, which may contribute to the development of COPD. A later study confirmed decreased histone deacetylase acidity in resected lungs of COPD patients, and concluded that there was a progressive decrease in activity with increasing severity of COPD (Ito, K., et al.,

2005). They also reported increased expression of IL-8 mRNA in lung tissue in COPD.

Oxidative stress may be determined non-invasively by measurement of oxidation products in exhaled breath condensates. According to a recent review article, the following markers of oxidative stress have been increased in exhaled breath condensates of subjects with COPD: hydrogen peroxide, nitrite, nitrosothiols, 8-isoprostane, and thiobarbituric acid reactive substances (Lee & Thomas, 2009). Oxidative stress is also indicated by the presence of biomarkers in blood indicative of lipid peroxidation., such as 4-hydroxy-2-nonenal (MacNee & Tuder 2009; Fischer, B.M., et al., 2011). The latter recent review article (Fischer, B.M., et al., 2011) also quoted published reports of increased levels of 4-hydroxy-2-nonenal, in both airways and alveoli of COPD patients, and also increased blood levels of

of plasminogen , which can promote lung matrix degradation (Chapman et al.,1984).

**3.5 Role of oxidants in protease-antiprotease imbalance** 

favor of oxidants leading to oxidative stress.

#### **3.3 Role of polymorphisms in MMPs**

An MMP polymorphism (C-15621) was associated with emphysema by CT scan in one Japanese study (Minematsu et al., 2001) and with upper lobe emphysema in another Japanese study (Ito et al., 2005), and with COPD in a Chinese population (Zhou et al., 2004).

A study from Russia evaluated gene polymorphisms of G(-1607)GG of MMP1, C(-1562)T of MMP9, and A(-82)G of MMP12, and found the frequencies did not differ significantly between 318 COPD patients compared with 319 healthy controls (Korytina et al., 2008). However, the (-1562)T allele of MMP9 was significantly higher in the Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage IV COPD than in stages II and III, indicating that this allele predisposed to severe disease; it also predisposed to early onset of COPD (age < 55 yr).

A multicentre European study determined 26 single nucleotide polymorphisms (SNP)s, covering reported SNP variations, in MMPs- 1, 9 and 12 from 977 COPD patients and 876 non-diseased smokers of European descent and evaluated their association with disease singly and in haplotype combinations (Haq., et al. 2010). They used logistic regression to adjust for age, gender, centre, and smoking history. They reported that the common A-A haplotypes of two SNPs in MMP-12 (rs652438 and rs2276109), were associated with severe or very severe disease ( GOLD Stages III and IV) (p= 0.0039).

This review has focused on neutrophil and macrophages proteases, but proteases from other cells such as lung fibroblasts, and myofibroblasts, and dendritic cells may also be involved.

#### **3.4 Role of the macrophage protease inhibitors TIMPs and cystatin C, and other protease inhibitors in emphysema**

It is likely that it is the balance between macrophage proteases and their respective antiproteases that has a pathogenic role in emphysema. TIMPs are the endogenous inhibitors of MMPs; human AM release TIMP1 and TIMP2 (Shapiro et.,1992). AM from COPD patients release less TIMP1 *in vitro* than those from smokers without COPD and nonsmokers (Pons et al., 2005), predisposing to proteolysis by MMPs. TIMP3 is the only TIMP that binds strongly to the extracellular matrix. TIMP3 knockout mice demonstrate progressive airspace enlargement and enhanced collagen degradation without inflammation or increased elastin breakdown (Leco et al., 2001). However, there are no reported associations between TIMP 3 polymorphisms and COPD. A polymorphism in the TIMP2 gene (G853A) was associated with COPD in a Japanese study (Hirano et al., 2001), and in an Egyptian population (Hegab et al., 2005).

Cystatin C is present in most biological fluids, and is a potent inhibitor of cathepsins. Cystatin C is a major product of AM (Chapman et al., 1990) and is secreted by AM from smokers at higher levels than non-smokers (Warfel et al., 1991) The concentrations of cathepsin L and its inhibitor cystatin C were both significantly increased in BAL fluid from smokers with emphysema compared with those without emphysema; however there was no significant difference in cathepsin L activity in BAL between the two groups (Takeyabu et al., 1998). There are no reports of deficiency or polymorphisms in cystatin C in relation to emphysema or COPD.

Polymorphisms in the Serpina2 gene, which encodes the protease nexin1 ( plasminogen activator inhibitor type 1), were associated with COPD in a Boston population study (Demeo et al., 2006), and validated in two large family-based and case-control association studies ( Zhu et al., 2007). Polymorphism of the SERPINA2 gene was also recently found associated with emphysema in consecutive autopsy cases in Japan (Fujimoto et al., 2010). Decreased activity of the plasminogen activator inhibitor type 1 in the lung can lead to increased activity of plasminogen , which can promote lung matrix degradation (Chapman et al.,1984).

#### **3.5 Role of oxidants in protease-antiprotease imbalance**

6 Emphysema

An MMP polymorphism (C-15621) was associated with emphysema by CT scan in one Japanese study (Minematsu et al., 2001) and with upper lobe emphysema in another Japanese study (Ito et al., 2005), and with COPD in a Chinese population (Zhou et al., 2004). A study from Russia evaluated gene polymorphisms of G(-1607)GG of MMP1, C(-1562)T of MMP9, and A(-82)G of MMP12, and found the frequencies did not differ significantly between 318 COPD patients compared with 319 healthy controls (Korytina et al., 2008). However, the (-1562)T allele of MMP9 was significantly higher in the Global Initiative for Chronic Obstructive Lung Disease (GOLD) stage IV COPD than in stages II and III, indicating that this allele predisposed to severe disease; it also predisposed to early onset of COPD (age < 55 yr). A multicentre European study determined 26 single nucleotide polymorphisms (SNP)s, covering reported SNP variations, in MMPs- 1, 9 and 12 from 977 COPD patients and 876 non-diseased smokers of European descent and evaluated their association with disease singly and in haplotype combinations (Haq., et al. 2010). They used logistic regression to adjust for age, gender, centre, and smoking history. They reported that the common A-A haplotypes of two SNPs in MMP-12 (rs652438 and rs2276109), were associated with severe

This review has focused on neutrophil and macrophages proteases, but proteases from other cells such as lung fibroblasts, and myofibroblasts, and dendritic cells may also be involved.

It is likely that it is the balance between macrophage proteases and their respective antiproteases that has a pathogenic role in emphysema. TIMPs are the endogenous inhibitors of MMPs; human AM release TIMP1 and TIMP2 (Shapiro et.,1992). AM from COPD patients release less TIMP1 *in vitro* than those from smokers without COPD and nonsmokers (Pons et al., 2005), predisposing to proteolysis by MMPs. TIMP3 is the only TIMP that binds strongly to the extracellular matrix. TIMP3 knockout mice demonstrate progressive airspace enlargement and enhanced collagen degradation without inflammation or increased elastin breakdown (Leco et al., 2001). However, there are no reported associations between TIMP 3 polymorphisms and COPD. A polymorphism in the TIMP2 gene (G853A) was associated with COPD in a Japanese study (Hirano et al., 2001), and in an

Cystatin C is present in most biological fluids, and is a potent inhibitor of cathepsins. Cystatin C is a major product of AM (Chapman et al., 1990) and is secreted by AM from smokers at higher levels than non-smokers (Warfel et al., 1991) The concentrations of cathepsin L and its inhibitor cystatin C were both significantly increased in BAL fluid from smokers with emphysema compared with those without emphysema; however there was no significant difference in cathepsin L activity in BAL between the two groups (Takeyabu et al., 1998). There are no reports of deficiency or polymorphisms in cystatin C in relation to

Polymorphisms in the Serpina2 gene, which encodes the protease nexin1 ( plasminogen activator inhibitor type 1), were associated with COPD in a Boston population study (Demeo

**3.4 Role of the macrophage protease inhibitors TIMPs and cystatin C, and other** 

**3.3 Role of polymorphisms in MMPs** 

**protease inhibitors in emphysema** 

Egyptian population (Hegab et al., 2005).

emphysema or COPD.

or very severe disease ( GOLD Stages III and IV) (p= 0.0039).

As indicated in a previously quoted review article on pathogenesis of COPD (MacNee, a2005), oxidants have a significant pathogenic role in COPD. The gaseous phase of cigarette smoke contains many reactive oxidants such as superoxide anion, nitric oxides and peroxynitrites, as reviewed recently (MacNee b2005; Lin & Thomas 2010). Oxidants and free radicals inhaled in tobacco smoke, can damage airway epithelial cells, and impair antioxidants, such as glutathione to non-reducible glutathione-aldehyde derivatives (van Der Toorn et al., 2007). Oxidants from tobacco smoke may also inactivate antiproteases, predisposing to a protease-antiprotease imbalance from the increased numbers of neutrophils and macrophages in smokers' lungs. Oxidants from cigarette smoke may also directly damage components of the lung connective tissure matrix, and interfere with elasin repair and synthesis (MacNee & Tuder 2009). Neutrophils and macrophages themselves when activated also release oxidants, such superoxides,, and nitric oxides, and contribute to the oxidative burden. Although antioxidants such as glutathione, catalase and superoxide dismutase protect the tissues against oxidants, the oxidant/antioxidant balance may tip in favor of oxidants leading to oxidative stress.

Patients with COPD have increased levels of hydrogen peroxide and of 8-isoprostane (a peroxidation product of arachidonic acid) in exhaled breath condensates compared with controls (MacNee b2005). Healthy smokers had reduced histone deacetylase activity in bronchial biopsies and in alveolar macrophages obtained by lavage, when compared with age matched nonsmoking controls (Ito, K., et al., 2001). These investigators also demonstrated that smoking resulted in a greater release of TNF-α from the alveolar macrophages when stimulated by IL-1β, which they considered was due to the suppressive effect of smoking on histone deacetylation. This suppressive effect on histone deacetylation results in increased acetylation, causes local unwinding of DNA, and allows increased inflammatory gene expression, which may contribute to the development of COPD. A later study confirmed decreased histone deacetylase acidity in resected lungs of COPD patients, and concluded that there was a progressive decrease in activity with increasing severity of COPD (Ito, K., et al., 2005). They also reported increased expression of IL-8 mRNA in lung tissue in COPD.

Oxidative stress may be determined non-invasively by measurement of oxidation products in exhaled breath condensates. According to a recent review article, the following markers of oxidative stress have been increased in exhaled breath condensates of subjects with COPD: hydrogen peroxide, nitrite, nitrosothiols, 8-isoprostane, and thiobarbituric acid reactive substances (Lee & Thomas, 2009). Oxidative stress is also indicated by the presence of biomarkers in blood indicative of lipid peroxidation., such as 4-hydroxy-2-nonenal (MacNee & Tuder 2009; Fischer, B.M., et al., 2011). The latter recent review article (Fischer, B.M., et al., 2011) also quoted published reports of increased levels of 4-hydroxy-2-nonenal, in both airways and alveoli of COPD patients, and also increased blood levels of

Pathogenic Mechanisms in Emphysema: From Protease Anti–Protease Imbalance to Apoptosis 9

smokers and normal non-smoking controls, (Keatings, V.M., et al., 1996). The increase in IL-8 was confirmed in a later study evaluating IL-8 in bronchoalveolar lavage fluid of COPD

Cytokine mRNA for IL-8, macrophage inflammatory protein-1α (MIP-1α), and MCP-1 were quantified using laser-capture microdissection of human bronchial epithelial cells and alveolar macrophages (Fuke, S., et al., 2004). The authors found that mRNA levels for IL-8, MIP-1α and MCP-1 were higher in bronchial epithelial cells of smokers with airflow obstruction and/or emphysema, compared with results in smokers without airflow obstruction or emphysema. However, there was no difference in macrophage mRNA levels for these cytokines between the 2 groups. Their findings support the role of the bronchiolar

Although TNF-α has a major pathogenic role in experimental emphysema (Churg, A., et al., 2004), it does not appear to be as implicated in emphysema in human COPD. One study compared gene polymorphism in 169 Dutch COPD patients compared with Dutch controls, and reported an increased frequency of the G/A genotype in patients without radiological emphysema (Kucukaycan, M., et al., 2002). Another study from Italy compared 63 male patients with COPD with 86 healthy controls, and found no difference in gene

It is likely that the pathogenic role of mediators and cytokines will be elucidated in multicenter studies evaluating pathogenetic mechanisms in COPD in association with large

Smokers with symptoms of chronic bronchitis and airflow limitation undergoing lung resection for a localized lesion were found to have increased numbers of CD8+ Tlymphocytes infiltrating the airway wall, which were increased compared with smokers with normal lung function, while the number of neutrophils, macrophages, and CD4+ Tlymphocytes were similar in the two groups (Saetta, M., et al., 1998). This suggested a pathogenic role for CD8+ lymphocytes in the development and progression of COPD. The subject of the role of lymphocytes in COPD is well covered by a recent review article (Gadgil & Duncan 2008). T lymphocytes can cause tissue injury either directly by cytolysis or by secreting pro-inflammatory mediators. Moreover, peripheral T-cells , specially CD8+ cells are activated and secrete mediators ( Gadjil, A., et al., 2006). CD8+ lymphocytes appear to have a role in the development and progression of COPD, as quoted from several references in the review (Gadgil & Duncan 2008). CD8+ T-lymphocytes can mediate cell death directly through secretion of cytotoxins such as granzyme and perforins, as quoted from other

CD4+ T-cells can initiate downstream immune processes by releasing activating cytokines, can amplify inflammatory reactions by other immune cells, and are essential for full adaptive immune cytotoxicity by lowering the threshold of activation and promoting survival of CD8+ T-cells (Gadgil & Duncan 2008). In addition, CD4+ T-cells are important for the activation of antibody producing B-cells. In a previous study, they reported finding circulating IgG autoantibodies against epithelial cells in about 70% of their COPD patients, as compared with 10% of non-smoking controls, and 13% of cigarette smokers without

patients compared with controls (Pesci, A., et al., 1998).

cells as the source of these increased chemokine levels in early COPD.

polymorphisms between the two groups (Ferrarotti, I., et al., 2003).

**3.7 Role of T-lymphocytes and cell mediated immunity** 

longitudonal clinical trials.

references (Gadgil & Duncan 2008).

malondialdehyde (an end product of lipid peroxidation) in COPD due to tobacco smoking as well as wood smoke exposure. 4-hydroxy-2-nonenal can increase gene expression of proinflammatory mediators such as IL-8, monocyte chemoattractant protein-1 (MacNee & Tuder 2009). Reactive oxygen species can also directly or indirectly induce proinflammatory mediators such as IL-1, TNF-α, Il-6, and IL-8 (Rahman & Adcock 2006).

The mRNA of inflammatory cytokines, chemokines, oxidant and antioxidant enzymes, proteases and antiproteases was evaluated in peripheral lung tissues from 14 COPD subjects and compared with 19 subjects without COPD undergoing lung resection for lung cancer (Tomaki, M., et al., 2007). They reported that mRNA, for catalase, two glutathion Stransferases, microsomal epoxide hydrolase, and TIMP2 were significantly decreased in COPD lung tissues compared with the non-COPD controls. On the other hand, the expressions of mRNA for IL-1β, IL-8, and monocyte chemotactic protein-1 (MCP-1) were significantly increased in COPD lungs. Most of these changes were also associated with cigarette smoking. Their data suggest that in addition to the impairment in antioxidant defenses, upregulation of cytokines and chemokines may be involved the development of COPD.

#### **3.6 Role of inflammatory mediators and cytokines in protease-antiprotease imbalance**

The last paragraph of page 4, reviewed the effects of TNF-α and IL-1β, on inducing expression of MMP9 by human macrophages without increasing its inhibitor TIMP1, predisposing to possible protease-antiprotease imbalance. In this section, I will briefly discuss these 2 pro-inflammatory cytokines and an additional one IL-8, which have been included in a review article on inflammatory mediators (Chung, K.F., 2005).

Imbalances between IL-1β and its antagonists in COPD have been reported in 15 patients with stable COPD compared with age matched healthy controls (Sapey, E., et al., 2009). Although mean concentrations of IL-1β in COPD were not different from controls, mean concentrations of their receptor antagonists (IL-RA & IL-1sRII) were markedly reduced, suggesting that IL-1β may have pathogenic role in COPD. In contrast, there were no difference in TNF-α and its antagonists in COPD patients compared with controls. A case control trial in Egyptian subjects over 60 years compared 3 groups of 30 subjects matched by age and sex, consisting of healthy subjects, COPD without any comorbidities, and COPD with cardiovascular disease but no other comorbidities (Amer, M.S., et al., 2010). There was no significant difference in the serum levels of IL-1β, TNF-α, or C reactive protein (CRP) between the control subjects and the COPD subjects with no cardiac disease. The group with cardiovascular disease had increased IL-1β and CRP ( but not TNF-α) levels compared with the other 2 groups. However, the increase in IL-1β and CRP cannot be definitely attributed to the more severe COPD in the 3rd group, since it could be secondary to the cardiovascular comorbidity.

A study from Korea evaluated four potentially functional polymorphisms in the IL-1β in 311 COPD patients and 386 healthy controls and found polymorphisms that significantly increased the odds ratio of developing COPD (Lee, J.M., et al., 2008). In addition, they reported that a polymorphism in the Il-1β receptor antagonist gene IL-1RN afforded some protection.

Induced sputum from patients with moderate to severe COPD, had increased neutrophils, and increased levels of IL-8 and TNF-α, when compared with that of healthy cigarette

malondialdehyde (an end product of lipid peroxidation) in COPD due to tobacco smoking as well as wood smoke exposure. 4-hydroxy-2-nonenal can increase gene expression of proinflammatory mediators such as IL-8, monocyte chemoattractant protein-1 (MacNee & Tuder 2009). Reactive oxygen species can also directly or indirectly induce proinflammatory mediators such as IL-1, TNF-α, Il-6, and IL-8 (Rahman & Adcock 2006).

The mRNA of inflammatory cytokines, chemokines, oxidant and antioxidant enzymes, proteases and antiproteases was evaluated in peripheral lung tissues from 14 COPD subjects and compared with 19 subjects without COPD undergoing lung resection for lung cancer (Tomaki, M., et al., 2007). They reported that mRNA, for catalase, two glutathion Stransferases, microsomal epoxide hydrolase, and TIMP2 were significantly decreased in COPD lung tissues compared with the non-COPD controls. On the other hand, the expressions of mRNA for IL-1β, IL-8, and monocyte chemotactic protein-1 (MCP-1) were significantly increased in COPD lungs. Most of these changes were also associated with cigarette smoking. Their data suggest that in addition to the impairment in antioxidant defenses, upregulation of

**3.6 Role of inflammatory mediators and cytokines in protease-antiprotease imbalance**  The last paragraph of page 4, reviewed the effects of TNF-α and IL-1β, on inducing expression of MMP9 by human macrophages without increasing its inhibitor TIMP1, predisposing to possible protease-antiprotease imbalance. In this section, I will briefly discuss these 2 pro-inflammatory cytokines and an additional one IL-8, which have been

Imbalances between IL-1β and its antagonists in COPD have been reported in 15 patients with stable COPD compared with age matched healthy controls (Sapey, E., et al., 2009). Although mean concentrations of IL-1β in COPD were not different from controls, mean concentrations of their receptor antagonists (IL-RA & IL-1sRII) were markedly reduced, suggesting that IL-1β may have pathogenic role in COPD. In contrast, there were no difference in TNF-α and its antagonists in COPD patients compared with controls. A case control trial in Egyptian subjects over 60 years compared 3 groups of 30 subjects matched by age and sex, consisting of healthy subjects, COPD without any comorbidities, and COPD with cardiovascular disease but no other comorbidities (Amer, M.S., et al., 2010). There was no significant difference in the serum levels of IL-1β, TNF-α, or C reactive protein (CRP) between the control subjects and the COPD subjects with no cardiac disease. The group with cardiovascular disease had increased IL-1β and CRP ( but not TNF-α) levels compared with the other 2 groups. However, the increase in IL-1β and CRP cannot be definitely attributed to the more severe COPD in the 3rd group, since it could be secondary to the cardiovascular

A study from Korea evaluated four potentially functional polymorphisms in the IL-1β in 311 COPD patients and 386 healthy controls and found polymorphisms that significantly increased the odds ratio of developing COPD (Lee, J.M., et al., 2008). In addition, they reported that a polymorphism in the Il-1β receptor antagonist gene IL-1RN afforded some protection. Induced sputum from patients with moderate to severe COPD, had increased neutrophils, and increased levels of IL-8 and TNF-α, when compared with that of healthy cigarette

cytokines and chemokines may be involved the development of COPD.

included in a review article on inflammatory mediators (Chung, K.F., 2005).

comorbidity.

smokers and normal non-smoking controls, (Keatings, V.M., et al., 1996). The increase in IL-8 was confirmed in a later study evaluating IL-8 in bronchoalveolar lavage fluid of COPD patients compared with controls (Pesci, A., et al., 1998).

Cytokine mRNA for IL-8, macrophage inflammatory protein-1α (MIP-1α), and MCP-1 were quantified using laser-capture microdissection of human bronchial epithelial cells and alveolar macrophages (Fuke, S., et al., 2004). The authors found that mRNA levels for IL-8, MIP-1α and MCP-1 were higher in bronchial epithelial cells of smokers with airflow obstruction and/or emphysema, compared with results in smokers without airflow obstruction or emphysema. However, there was no difference in macrophage mRNA levels for these cytokines between the 2 groups. Their findings support the role of the bronchiolar cells as the source of these increased chemokine levels in early COPD.

Although TNF-α has a major pathogenic role in experimental emphysema (Churg, A., et al., 2004), it does not appear to be as implicated in emphysema in human COPD. One study compared gene polymorphism in 169 Dutch COPD patients compared with Dutch controls, and reported an increased frequency of the G/A genotype in patients without radiological emphysema (Kucukaycan, M., et al., 2002). Another study from Italy compared 63 male patients with COPD with 86 healthy controls, and found no difference in gene polymorphisms between the two groups (Ferrarotti, I., et al., 2003).

It is likely that the pathogenic role of mediators and cytokines will be elucidated in multicenter studies evaluating pathogenetic mechanisms in COPD in association with large longitudonal clinical trials.

#### **3.7 Role of T-lymphocytes and cell mediated immunity**

Smokers with symptoms of chronic bronchitis and airflow limitation undergoing lung resection for a localized lesion were found to have increased numbers of CD8+ Tlymphocytes infiltrating the airway wall, which were increased compared with smokers with normal lung function, while the number of neutrophils, macrophages, and CD4+ Tlymphocytes were similar in the two groups (Saetta, M., et al., 1998). This suggested a pathogenic role for CD8+ lymphocytes in the development and progression of COPD. The subject of the role of lymphocytes in COPD is well covered by a recent review article (Gadgil & Duncan 2008). T lymphocytes can cause tissue injury either directly by cytolysis or by secreting pro-inflammatory mediators. Moreover, peripheral T-cells , specially CD8+ cells are activated and secrete mediators ( Gadjil, A., et al., 2006). CD8+ lymphocytes appear to have a role in the development and progression of COPD, as quoted from several references in the review (Gadgil & Duncan 2008). CD8+ T-lymphocytes can mediate cell death directly through secretion of cytotoxins such as granzyme and perforins, as quoted from other references (Gadgil & Duncan 2008).

CD4+ T-cells can initiate downstream immune processes by releasing activating cytokines, can amplify inflammatory reactions by other immune cells, and are essential for full adaptive immune cytotoxicity by lowering the threshold of activation and promoting survival of CD8+ T-cells (Gadgil & Duncan 2008). In addition, CD4+ T-cells are important for the activation of antibody producing B-cells. In a previous study, they reported finding circulating IgG autoantibodies against epithelial cells in about 70% of their COPD patients, as compared with 10% of non-smoking controls, and 13% of cigarette smokers without

Pathogenic Mechanisms in Emphysema: From Protease Anti–Protease Imbalance to Apoptosis 11

correlation of apoptosis with lung surface area. They also evaluated cell proliferation by immunostaining for proliferating cell nuclear antigen (PCNA)., and reported that it was increased but was not correlated with apoptosis index or lung surface area. Other investigators evaluated apoptosis by flow cytometry in cells obtained by bronchoalveolar lavage in subjects with COPD, and compared results in 16 exsmokers with 13 current smokers, and 20 nonsmoking volunteers (Hodge et al., 2005). There was a mean 87% increase in apoptotic airway epithelial cells in COPD subjects, and a mean doubling of apoptosis by airway T lymphocytes compared with non-smoking volunteers, but there was no difference between COPD subjects still smoking and those who had quit. They concluded that this increased airway cell apoptosis

A study from Japan sought to evaluate the turnover of alveolar wall cells in emphysema by comparing lung tissue specimens from 13 patients with emphysema who had lung volume reduction surgery, 7 asymptomatic smokers and 9 non-smokers undergoing lung resection for solitary lung cancers (Yokohori et., 2004). They reported that the percentages of alveolar wall cells undergoing apoptosis and proliferation were higher in the emphysema patients than asymptomatic smokers or non-smokers. They concluded that emphysema is a dynamic process in which both alveolar cell wall apoptosis and proliferation are recurring. The same investigators also demonstrated that activated caspase 3 (an enzyme inducing apostosis) when instilled into the lungs of mice resulted in alveolar wall destruction and emphysema (Aoshiba et al., 2003). A study of 16 end-stage lungs from subjects undergoing lung transplantation for advanced emphysema (7 were due to AAT deficiency) were compared with 6 unused donor lungs (Calabrese et al., 2005). The apoptotic index was significantly increased in the emphysema lungs compared with controls, but the alveolar proliferation was similar in emphysema and control lungs. They concluded that there was a marked imbalance between alveolar apoptosis and alveolar proliferation in advanced emphysema. In a study in patients undergoing lobectomy for lung cancer, there was increased apoptosis of alveolar walls by TUNEL assay and increased proliferation of alveolar cells in 10 subjects with emphysema, when compared with lungs from 10 asymptomatic smokers, and 10 nonsmokers (LIU et al. 2009). They also demonstrated increased apoptosis and decreased

As a result of previous studies showing increased apoptosis in human lungs with emphysema (Yokohori et al., 2004), and induction of apoptosis by caspase 3 in mice (Aoshiba et al., 2003), these investigators (Aoshiba & Nagai, 2009) proposed a senescence hypothesis as a pathogenic mechanism in emphysema. They speculated that cellular senescence was the cause of the insufficient cellular proliferation in emphysema, and found that senescence markers were increased in emphysema lungs. They considered that smoking and aging caused alveolar and airway cells to senesce , and senescence decreased

Protease-antiprotease imbalance is likely to have a major pathogenic role in the development of emphysema in severe AAT deficiency. However the case in non-AAT deficient smokers is not firmly established, but is supported by several studies showing associations of emphysema with proteolyic enzyme levels or message expression, and by the

in COPD persists despite smoking cessation.

numbers of Type II epithelial cells in the lungs with emphysema.

tissue repair resulting in reduced cell numbers .

**5. Conclusions** 

evidence of lung disease (Feghali-Bostwick, C.A., et al., 2008). There was also immune complex deposition in six end stage explanted lungs. These autoantibodies may have a pathogenic role in airway epithelial injury in COPD. Also, a number of studies indicate that the lymphocyte proliferations in COPD are driven by peptide antigens, and consider various possibilities such as microbial peptide antigens, adenoviral antigens, tobacco smoke related peptides, elastin peptides, and auto-antigens from apoptotic cells and cellular debris (Gadgil & Duncan 2008).

#### **4. Apoptosis and emphysema**

This is an exciting new area of intense investigation which will further elucidate pathogenetic mechanisms in emphysema and is likely to lead to specific therapies in the future. Apoptosis refers to programmed cell death, affecting the endothelial capillaries and the alveolar epithelium leading to the development of emphysema. This area of investigation was initiated by the landmark study reporting that chronic blockade of Vascular Endothelial Growth Factor (VEGEF) receptors in rats by a chemical SU5416, induced alveolar septal apoptosis and enlargement of the air spaces indicating emphysema (Kasahara et al., 2000). The apoptosis was mediated by caspase 3, a proteolytic enzyme inducing apoptosis, and was prevented by treatment with a caspase inhibitor. The topic of apoptosis is covered by recent reviews (Demedts et al., 2006; Tuder et al., 2006, Morissette et al., 2009, Macnee & Tuder 2009). Additionally, specific sections about alveolar cell apoptosis and proliferation, aging and senescence, as well as mediators and signaling pathways, are also covered in a comprehensive review article about the pathobiology of cigarette smokeinduced COPD (Yoshida & Tuder, 2008). The pathways in apoptosis are involved, but may be simplified to an extrinsic and intrinsic pathway, The extrinsic pathway is activated by extracellular death ligands, such as those related to TNF-α which result in activation of caspases (proteolytic enzymes involved in apoptosis). The intrinsic pathway is triggered by cellular or DNA injury leading to the release of cytochrome C and apoptosis.

#### **4.1 Human studies**

Investigators studying human lung specimens to evaluate MMPs by immunohistochemistry in lungs with emphysema compared with controls, also evaluated apoptosis by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assays, and were the first to report increased endothelial cell apoptosis and, to a lesser extent alveolar epithelial apoptosis in emphysema (Segura-Valdez et al., 2000). In 2001, the investigators who showed that VEGEF blockade in rats induced apoptosis , reported results from human lungs (Kasahara et al., 2001). The number of apoptotic epithelial and endothelial cells in alveolar septa of emphysema lungs per unit of lung tissue nucleic acid was about double in emphysema compared with normal lungs. In addition, VEGF, its receptor protein and mRNA expression were reduced in emphysema lungs, suggesting that apoptosis due to a decrease in endothelial maintenance factors may have a pathogenic role in emphysema, However another study reported no significant difference in apoptotic index in the lungs of 10 smokers with emphysema compared with 5 smokers without emphysema (Majo et a. 2001). Another group reported increased apoptosis of alveolar epithelial and endothelial cells as well as mesenchymal cells in lung tissue from 10 emphysema patients, compared with 6 controls without emphysema (Imai et al.,2005), and there was significant inverse correlation of apoptosis with lung surface area. They also evaluated cell proliferation by immunostaining for proliferating cell nuclear antigen (PCNA)., and reported that it was increased but was not correlated with apoptosis index or lung surface area. Other investigators evaluated apoptosis by flow cytometry in cells obtained by bronchoalveolar lavage in subjects with COPD, and compared results in 16 exsmokers with 13 current smokers, and 20 nonsmoking volunteers (Hodge et al., 2005). There was a mean 87% increase in apoptotic airway epithelial cells in COPD subjects, and a mean doubling of apoptosis by airway T lymphocytes compared with non-smoking volunteers, but there was no difference between COPD subjects still smoking and those who had quit. They concluded that this increased airway cell apoptosis in COPD persists despite smoking cessation.

A study from Japan sought to evaluate the turnover of alveolar wall cells in emphysema by comparing lung tissue specimens from 13 patients with emphysema who had lung volume reduction surgery, 7 asymptomatic smokers and 9 non-smokers undergoing lung resection for solitary lung cancers (Yokohori et., 2004). They reported that the percentages of alveolar wall cells undergoing apoptosis and proliferation were higher in the emphysema patients than asymptomatic smokers or non-smokers. They concluded that emphysema is a dynamic process in which both alveolar cell wall apoptosis and proliferation are recurring. The same investigators also demonstrated that activated caspase 3 (an enzyme inducing apostosis) when instilled into the lungs of mice resulted in alveolar wall destruction and emphysema (Aoshiba et al., 2003). A study of 16 end-stage lungs from subjects undergoing lung transplantation for advanced emphysema (7 were due to AAT deficiency) were compared with 6 unused donor lungs (Calabrese et al., 2005). The apoptotic index was significantly increased in the emphysema lungs compared with controls, but the alveolar proliferation was similar in emphysema and control lungs. They concluded that there was a marked imbalance between alveolar apoptosis and alveolar proliferation in advanced emphysema.

In a study in patients undergoing lobectomy for lung cancer, there was increased apoptosis of alveolar walls by TUNEL assay and increased proliferation of alveolar cells in 10 subjects with emphysema, when compared with lungs from 10 asymptomatic smokers, and 10 nonsmokers (LIU et al. 2009). They also demonstrated increased apoptosis and decreased numbers of Type II epithelial cells in the lungs with emphysema.

As a result of previous studies showing increased apoptosis in human lungs with emphysema (Yokohori et al., 2004), and induction of apoptosis by caspase 3 in mice (Aoshiba et al., 2003), these investigators (Aoshiba & Nagai, 2009) proposed a senescence hypothesis as a pathogenic mechanism in emphysema. They speculated that cellular senescence was the cause of the insufficient cellular proliferation in emphysema, and found that senescence markers were increased in emphysema lungs. They considered that smoking and aging caused alveolar and airway cells to senesce , and senescence decreased tissue repair resulting in reduced cell numbers .

#### **5. Conclusions**

10 Emphysema

evidence of lung disease (Feghali-Bostwick, C.A., et al., 2008). There was also immune complex deposition in six end stage explanted lungs. These autoantibodies may have a pathogenic role in airway epithelial injury in COPD. Also, a number of studies indicate that the lymphocyte proliferations in COPD are driven by peptide antigens, and consider various possibilities such as microbial peptide antigens, adenoviral antigens, tobacco smoke related peptides, elastin peptides, and auto-antigens from apoptotic cells and cellular debris

This is an exciting new area of intense investigation which will further elucidate pathogenetic mechanisms in emphysema and is likely to lead to specific therapies in the future. Apoptosis refers to programmed cell death, affecting the endothelial capillaries and the alveolar epithelium leading to the development of emphysema. This area of investigation was initiated by the landmark study reporting that chronic blockade of Vascular Endothelial Growth Factor (VEGEF) receptors in rats by a chemical SU5416, induced alveolar septal apoptosis and enlargement of the air spaces indicating emphysema (Kasahara et al., 2000). The apoptosis was mediated by caspase 3, a proteolytic enzyme inducing apoptosis, and was prevented by treatment with a caspase inhibitor. The topic of apoptosis is covered by recent reviews (Demedts et al., 2006; Tuder et al., 2006, Morissette et al., 2009, Macnee & Tuder 2009). Additionally, specific sections about alveolar cell apoptosis and proliferation, aging and senescence, as well as mediators and signaling pathways, are also covered in a comprehensive review article about the pathobiology of cigarette smokeinduced COPD (Yoshida & Tuder, 2008). The pathways in apoptosis are involved, but may be simplified to an extrinsic and intrinsic pathway, The extrinsic pathway is activated by extracellular death ligands, such as those related to TNF-α which result in activation of caspases (proteolytic enzymes involved in apoptosis). The intrinsic pathway is triggered by

cellular or DNA injury leading to the release of cytochrome C and apoptosis.

Investigators studying human lung specimens to evaluate MMPs by immunohistochemistry in lungs with emphysema compared with controls, also evaluated apoptosis by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) assays, and were the first to report increased endothelial cell apoptosis and, to a lesser extent alveolar epithelial apoptosis in emphysema (Segura-Valdez et al., 2000). In 2001, the investigators who showed that VEGEF blockade in rats induced apoptosis , reported results from human lungs (Kasahara et al., 2001). The number of apoptotic epithelial and endothelial cells in alveolar septa of emphysema lungs per unit of lung tissue nucleic acid was about double in emphysema compared with normal lungs. In addition, VEGF, its receptor protein and mRNA expression were reduced in emphysema lungs, suggesting that apoptosis due to a decrease in endothelial maintenance factors may have a pathogenic role in emphysema, However another study reported no significant difference in apoptotic index in the lungs of 10 smokers with emphysema compared with 5 smokers without emphysema (Majo et a. 2001). Another group reported increased apoptosis of alveolar epithelial and endothelial cells as well as mesenchymal cells in lung tissue from 10 emphysema patients, compared with 6 controls without emphysema (Imai et al.,2005), and there was significant inverse

(Gadgil & Duncan 2008).

**4.1 Human studies** 

**4. Apoptosis and emphysema** 

Protease-antiprotease imbalance is likely to have a major pathogenic role in the development of emphysema in severe AAT deficiency. However the case in non-AAT deficient smokers is not firmly established, but is supported by several studies showing associations of emphysema with proteolyic enzyme levels or message expression, and by the

Pathogenic Mechanisms in Emphysema: From Protease Anti–Protease Imbalance to Apoptosis 13

Campbell, E.J., White, R.R., Senior, R.M., Rodriguez, R.J., & Kuhn, C. (1979). Receptor-

Chapman, H.A.Jr., Stone, O.L., & Vavrin, Z. (1984). Degradation of fibrin and elastin by

Chapman, H.A.Jr., & Stone, O.L. (1984). Comparison of live human neutrophil and alveolar

Chapman, H.A.Jr., Reilly, J.J.Jr., Yee, R., & Grubb, A. (1990). Identification of cystatin C, a

Churg, A., Wang, R.D., Tai, H., Wang, X., Xie, C., & Wright, J.L. (2004). Tumor necrosis

D'Armiento, J., Dalal, S.S., Okada, Y., Berg, R.A., & Chada, K. (1992). Collagenase expression

Demedts, I.K., Morel-Montero, A., Lebecque, S., Pacheco, Y., Cataldo, D., Joos, G.F.,

Demedts, I.K., Demoor, T., & Bracke, K.R. et al., (2006). Role of apoptosis in the pathogenesis of COPD and pulmonary emphysema. *Respir Res,* Vol. 7, No. 53, (March 2006) Demeo, D.L., Mariani, T.J., & Lange, C. et al., (2006). The Serpine2 gene is associated with

Feghali-Bostwick, C.A., Gadjil, A.S., Otterbein, L.E., et al., (2008). Autoantibodies in patients

Fera, T., Abboud, R.T., Richter, A., & Johal, S. (1986). Acute effect of smoking on elastase-like

*Rev Respir Dis,* Vol. 133, Vol. 4, (April 1986), pp. (568-573)

*Respir Crit Care Med,* Vol. 167, No. 8, (April 2003), pp. (1083-1089)

*Respir Crit Care Med*, Vol. 170, No. 5, (September 2004), pp. (492-498) Churg, A., Cosio, M., & Wright, J.L. (2008). Mechanisms of cigarette smoke-induce COPD:

*Sci USA,* Vol. 79, No. 6, (March 1982), pp. (2041-2045)

No. 5, (November 1984), pp. (1693-1700)

(April 2008), pp. (L612-L631)

(December 1992), pp. (955-961)

(February 2006), pp. (253-264)

No. 2, (January 2008), pp. (156-163)

1984), pp. 806-815)

mediated binding and internalization of leukocyte elastase by alveolar macrophages in vitro. *J Clin Invest,* Vol. 64, No. 3, (September 1979), pp. 824-833) Carp, H., Miller, F., Hoidal, J.R., & Janoff, A. (1982). Potential mechanism of emphysema:

alpha 1-proteinase inhibitor recovered from lungs of cigarette smokers contains oxidized methionine and has decreased elastase inhibitory capacity. *Proc Natl Acad* 

intact human alveolar macrophages in vitro. Characterization of a plasminogen activator and its role in matrix degradation. *J Clin Invest,* Vol. 73, No. 3, (March

macrophage elastolytic activity in vitro. Relative resistance of macrophage elastolytic activity to serum and alveolar proteinase inhibitors. *J Clin Invest,* Vol. 74,

cysteine proteinase inhibitor, as a major secretory product of human alveolar macrophages in vitro. *Am Rev Respir Dis,* Vol. 141, No. 3, (March 1990), pp. (698-705) Chung, K.F. (2005). Inflammatory mediators in chronic obstructive pulmonary disease. *Curr Drug Targets Inflamm Allergy,* Vol. 4, No. 6, (December 2005), pp. (619-625) Churg, A., Wang, R.D., & Tai, H. et al., (2003). Macrophage metalloelastase mediates acute

cigarette smoke-induced inflammation via tumor necrosis factor-α release. *Am J* 

factor-alpha drives 70% of cigarette smoke-induced emphysema in the mouse. *Am J* 

insights from animal models. *Am J Physiol Lung Cell Mol Physiol,* Vol. 294, No. 4,

in the lungs of transgenic mice causes pulmonary emphysema. *Cell,* Vol. 71, No. 6,

Pauwels, R.A., & Brusselle, G.G. (2006). Elevated MMP-12 protein levels in induced sputum from patients with COPD. *Thorax,* Vol. 61, No. 3, (Mar 2006), pp. (196–201)

chronic obstructive pulmonary disease . *Am J Hum Genetics,* Vol. 78, No. 2,

with chronic obstructive pulmonary disease. *Am J Respir Crit Care Med*, Vol. 177,

activity and immunologic neutrophil elastase levels in bronchoalveolar lavage. *Am* 

association of polymorphisms with decline in lung function. It is also supported by a review of animal models of cigarette smoke-induced COPD, where the opening sentence of the Abstract supports the protease-antiprotease hypothesis of emphysema (Churg, A., et al., 2008). However there are other mechanisms that play a pathogenic role such as oxidants, inflammation, and T lymphocyte induced immunity. Apoptosis is likely to have a significant pathogenic role in emphysema and may be amenable to therapy in the future.

#### **6. Acknowledgements**

I thank Andrew Sandford, Allison Wallace, Hong Li, Takeo Ishii, and Selvarani Vimanathalan for their collaboration in studies on alveolar macrophage proteases and antiproteases in relation to emphysema.

#### **7. References**


association of polymorphisms with decline in lung function. It is also supported by a review of animal models of cigarette smoke-induced COPD, where the opening sentence of the Abstract supports the protease-antiprotease hypothesis of emphysema (Churg, A., et al., 2008). However there are other mechanisms that play a pathogenic role such as oxidants, inflammation, and T lymphocyte induced immunity. Apoptosis is likely to have a significant

I thank Andrew Sandford, Allison Wallace, Hong Li, Takeo Ishii, and Selvarani Vimanathalan for their collaboration in studies on alveolar macrophage proteases and

Abboud, R. , & Vimanalathan, S. (2008). Pathogenesis of COPD. Part I. The role of protease-

Abboud, R.T., Fera, T., Richter, A., Tabona, M.Z., & Johal, S. (1985). Acute effect of smoking

Abboud, R.T., Fera, T., Johal, S., Richter, A., & Gibson, N. (1986). Effect of smoking on

Abboud, R.T., Ofulue, A.F., Sansores, R.H., & Muller, N.L. (1998). Relationship of alveolar

evidence of emphysema. *Chest,* Vol. 113, No. 5, (May 1998), pp. (1257-1263) Amer, M.S., Wahba, H.M., Ashmawi, S.S., et al., (2010). Proinflammatory cytokines in

Aoshiba, K., Yokohori, N., & Nagai, A. (2003). Alveolar wall apoptosis causes lung

Aoshiba, K., & Nagai A. (2009). Senescence hypothesis for the pathogenetic mechanism of

Atkinson, J.J., Lutey, B.A., & Suzuki, Y. et al., (2011). The role of matrix metalloproteinase-9

Betsuyaku, T., Yoshioka, A., & Nishimura, M. et al., (1995). Neutrophil elastase associated

Boudier, C., Pelletier, A., Pauli, G., & Bieth, J.G. (1983). The functional activity of a1-

fluid. *Am Rev Respir Dis,* Vol. 131, No. 1, (January 1985), pp. (79-85)

antiprotease imbalance in emphysema. *Int J Tuberc Lung Dis*, Vol. 12, No. 4, (April

on the functional activity of alpha1-protease inhibitor in bronchoalveolar lavage

plasma neutrophil elastase levels. *J Lab Clin Med,* Vol. 108, No. 4, (October 1986),

macrophage plasminogen activator and elastase activities to lung function and CT

Egyptian elderly with chronic obstructive pulmonary disease. *Lung India,* Vol. 27,

destruction and emphysematous changes. *Am J Respir Cell Mol Biol,* Vol. 28, No. 5,

chronic obstructive pulmonary disease. *Proc Am Thorac Soc,* Vol. 6, No. 7,

in cigarette smoke-induced emphysema. *Am J Respir Crit Care Med,* Vol. 183, No. 7,

with alveolar macrophages from older volunteers. *Am J Respir Crit Care Med,* Vol.

proteinase inhibitor in bronchoalveolar lavage fluids from healthy human smokers and non-smokers. *Clin Chim Acta,* Vol. 132, No. 3, (August 1983), pp. (309-315) Calabrese, F., Giacometti, C., & Beghe, B. et al., (2005). Marked alveolar

apoptosis/proliferation imbalance in end-stage emphysema. *Respir Res,* Vol. 6, No.

pathogenic role in emphysema and may be amenable to therapy in the future.

**6. Acknowledgements** 

**7. References** 

antiproteases in relation to emphysema.

2008), pp. (361-367).

No.4, (October 2010), pp. (225-229)

(May 2003), pp. 555-562)

(December 2009), pp. (596-601)

151, No. 2, (February 1995), pp. (436-442)

(April 2011), pp. (876-884)

14, (February 2005)

pp. (294-300)


Pathogenic Mechanisms in Emphysema: From Protease Anti–Protease Imbalance to Apoptosis 15

Hirano, K., Sakamoto, T., Uchida, Y. et al., (2001). Tissue inhibitor of metalloproteinases-2

Hodge, S., Hodge, G., Holmes, M., & Reynolds, P.M. (2005). Increased airway epithelial and

Imai, K., Dalal, S.S., Chen, E.S. et al., (2001). Human collagenase (matrix metalloproteinase-

Imai, K., Mercer, B.A., & Schulman, L.L. et al., (2005). Correlation of lung surface area to

Ito, I., Nagai, S., & Handa, T. et al., (2005). Matrix Metalloproteinase-9 Promoter

Ito, K., Lim, S., Caramori, K.F., et al., (2001). Cigarette smoking reduces histone deacetylase 2

Janoff, A., Sloan, B., & Weinbaum, G. et al., (1977). Experimental emphysema induced with

Janoff A. (1985). Elastases and emphysema. Current assessment of the protease-antiprotease hypothesis. *Am Rev Respir Dis,* Vol. 132 No. 2, (August 1985), pp. (417-433) Kasahara, Y., Tuder, R.M., & Taraseeviciene-Stewart, L. et al., (2000). Inhibition of VEGF

Kasahara, Y., Tuder, R.M., & Vool, C.D. et al., (2001). Endothelial cell death and decreased

Kidokoro, Y., Kravis, T.C., Moser, K.M., Taylor, J.C., & Crawford, I.P. (1977). Relationship of

Korytina, G.F., Akhmadishina, L.Z., Ianbaeva, D.G. et al., (2008). Polymorphism in promoter

Kucukayan, M., Van Kugten, M., Pennings, H.J., et al., (2002). Tumor necrosis factor-alpha

Laurell, C.B., & Eriksson, S. (1963). The electrophoretic alpha-1-globulin pattern of serum in α1-antitrypsin deficiency. *Scand J Clin Invest,* Vol. 15, (1963), pp. (132-140) Leco, K.J., Waterhouse, P., Sanchez, O.H. et al., (2001). Spontaneous air space enlargement in

*Respir Crit Care Med,* Vol. 163, No. 3, (March 2001), pp. (737-744)

*Crit Care Med*, Vol. 172, No. 11, (December 2005), pp. (1378-1382)

*Rev Respir Dis,* Vol. 115, No. 3, (March 1977), pp. (461-478)

18, No. 5, (November 2001), pp. (748-52)

Vol. 163, No. 3, (March 2001), pp. (786-791)

No. 3, (March 2005), pp. (447-454)

(February 2005), pp. (250-258)

(December 2000), pp. (1311-1319)

44, No. 2, (February 2008), pp. 242-249)

disease. *Respir Res,* 2002; 3:29. Epub 2002, November 29

*Invest*, Vol. 108, No. 6, (September 2001), pp. (817-829)

(1967-1976)

(793-803)

gene polymorphisms in chronic obstructive pulmonary disease. *Eur Respir J,* Vol.

T-cell apoptosis in COPD remains despite smoking cessation. *Eur Respir J,* Vol. 25,

1) expression in the lungs of patients with emphysema. *Am J Respir Crit Care Med,*

apoptosis and proliferation in human emphysema. *Eur Respir J,* Vol. 25, No. 2,

Polymorphism Associated with Upper Lung Dominant Emphysema. *Am J Respir* 

expression, enhances cytokine expression, and inhibits glucocorticoid actions in alveolar macrophages. *FASEB J,* Vol. 15, (February 5, 2001), Published on line Ito, K., Ito, M., Elliot, W.M., et al., (2005). Decreased histone deacetylase activity in chronic

obstructive pulmonary disease. *N Engl J Med,* Vol. 352, No. 19, (May 2005) pp.

purified human neutrophil elastase: tissue localization of the instilled protease. *Am* 

receptors causes lung cell apoptosis and emphysema. *J Clin Invest,* Vol. 106, No. 11,

expression of of vascular endothelial growth factor receptor 2 in emphysema. *Am J* 

leukocyte elastase concentration to severity of emphysema in homozygous alpha1 antitrypsin-deficient persons. *Am Rev Respir Dis,* Vol. 115, No. 5, (May 1977), pp.

regions of matrix metalloproteinases (MMP1, MMP9, and MMP12) in chronic obstructive pulmonary disease patients [Russian, English Abstract]. *Genetika,* Vol.

+489G/A gene polymorphisms is associated with chronic obstructive pulmonary

the lungs of mice lacking tissue inhibitor of metalloproteinases-3 (TIMP-3). *J Clin* 


Ferrarotti, I., Zorzetto, M., Beccaria, M., et al., (2003). Tumour necrosis factor family genes in

Finkelstein, R., Fraser, R.S., Ghezzo, H., & Cosio, M.G. (1995). Alveolar inflammation and its

Finlay, G.A., O'Driscoll, L.R., & Russell, K.J. et al., (1997). Matrix metalloproteinase

Fischer, B.M., Pavlisko, E., Voynow, J.A. (2011). Pathogenic Triad in COPD: oxidative stress,

Foronjy, R.F., Okada, Y., Cole, R., & D'Armiento, J. (2003). Progressive adult-onset

Fujimoto, K., Ikeda, S., & Arai, T. et al., (2010). Polymorphism of Serpina 2 gene is associated

Fujita, J., Nelson, N.L., & Daughton, D.M. et al., (1990). Evaluation of elastase and

Gadek, J.E., Fells, G.A,. & Crystal, R.G. (1979). Cigarette smoking induces functional

Gadgil, A., & Duncan, S.R. (2008).Role of T-lymphocytes and pro-inflammatory mediators in

Gadgil, A., Zhu, X., Sciurba, F.C., Duncan, S.R. (2006). Altered T-cell phenotypes in chronic

Golovatch, P., Mercer, B.A., Lemaitre, V. et al., (2009). Role for cathepsin K in emphysema in

Gross, P., Pfitzer, E.A., Toker, A. et al., (1965). Experimental emphysema: its production

Haq, I., Chappell, S., Johnson, S.R. et al., (2010). Association of MMP-12 polymorphisms

Hautamaki, R.D., Kobayashi, D.K., Senior, R.M., & Shapiro, S.D. (1997). Requirement for

Hegab, A.E., Sakamoto, T., Uchida, Y. et al., (2005). Association analysis of tissue inhibitor of

European population. *BMC Med Genet,* Jan 15; 11: 7, (January 2010)

emphysema. *Am Rev Respir Dis,* Vol. 142, No. 1, (July 1990), pp. 57-62) Fuke, S., Betsuyaku, T., Nasuhara, Y., et al., (2004). Chemokines in bronchiolar epithelium in

*Lung Cell Mol Physiol*, Vol. 284, No. 5, (May 2003), pp. (L727-737)

*Crit Care Med,* Vol. 156, No. 1, (July 1997), pp. (240-247)

(March 2003), pp. (444-449)

11, 159, (November 2010)

(487-488)

pp. (50-58)

645)

(November 1995), pp. (1666-1672)

*Dis,* Vol. 6, (August 2011), pp. (413-421)

*Biol,* Vol. 31, No. 4, (June 2004), pp. (405-412)

No. 4424, (December 1979), pp. (1315-1316)

*Pulmon Dis,* Vol. 3, No. 4, (2008), pp. (531-541)

277, No. 5334, (September 1997), pp. (2002-2004)

99, No. 1, (January 2005), pp. (107-110)

a phenotype of COPD associated with emphysema. *Eur Respir J,* Vol. 21, No. 3,

relation to emphysema in smokers. *Am J Respir Crit Care Med,* Vol. 152, No. 5,

expression and production by alveolar macrophages in emphysema. *Am J Respir* 

protease-antiprotease imbalance, and inflammation. *Int J Chron Obstruct Pulmon* 

emphysema in transgenic mice expressing human MMP-1 in the lung. *Am J Physiol* 

with pulmonary emphysema in consecutive autopsy cases. *BMC Med Genet,* Nov;

antielastase balance in patients with chronic bronchitis and pulmonary

the development of chronic obstructive pulmonary disease. *Am J Respir Cell Mol* 

antiprotease deficiency in the lower respiratory tract of humans. *Science,* Vol. 206,

the pathogenesis of chronic obstructive pulmonary disease. *Int J Chron Obstruct* 

obstructive pulmonary disease. *Proc Am Thorac Soc,* Vol. 3, No. 6. (August 2006) pp.

smoke exposed guinea pigs. *Exp Lung Res,* Vol. 38, No. 8, (October 2009), pp. (631-

with papain in normal and silicotic rats. *Arch Environ Health,* Vol. 11, (July 1965),

with severe and very severe COPD: a case control study of MMPs-1, 9 and 12 in a

macrophage elastase for cigarette smoke-induced emphysema in mice. *Science,* Vol.

metalloproteinase2 gene polymorphisms with COPD in Egyptians. *Respir Med,* Vol.


Pathogenic Mechanisms in Emphysema: From Protease Anti–Protease Imbalance to Apoptosis 17

Park, J.W., Ryter, S.W., & Choi, A.M. (2007). Functional significance of apoptosis in chronic

Pons, A.R., Sauleda, J., & Noguera, A. et al., (2005). Decreased macrophage release of TGF-

Rahman, I., & Adcock, I.M. (2006). Oxidative stress and redox regulation of lung inflammation in COPD. Eur Respir J, Vol. 28, No. 1, (July 2006), pp. (219-242) Reilly, J.J., Mason, R.W., & Chen, P. et al., (1989). Synthesis and processing of cathepsin L, an

Reilly, J.J., Chen, P., & Sailor, L.Z. et al., (1991). Cigarette smoking induces an elastolytic

Russell, R.E., Culpitt, S.V., & DeMatos, C. et al., (2002). Release and activity of` matrix

Russell, R.E., Thorley, A., & Murray, R. et al., (2002). Alveolar macrophage-mediated

Sapey, E. Ahmad, A., Bayley, D., et al., (2009).Imbalances between interleukin-1 and tumor

Segura-Valdez, L., Pardo, A., & Gaxiola, M. et al., (2000). Upregulation of gelatinases A and

Senior, R.M., Tegner, H., & Kuhn, C. et al., (1977). The induction of pulmonary emphysema

Senior, R.M., Griffin, G.L., & Fliszar, C.J. et al., (1991). Human 92- and 72-kilodalton type IV

Shapiro, S.D., Kobayashi, D.K., & Welgus, H.G. (1992). Identification of TIMP-2 in human

Shapiro, S.D., Kobayashi, D., Pentland, A.P., & Welgus, H.G. (1993). Induction of

Shapiro, S.D., Kobayashi, D.K., & Ley, T.J. (1993). Cloning and characterization of a unique

Shapiro, S.D., Goldstein, N.M., & Houghton, A.M. et al., (2003). Neutrophil elastase

*Chem,* Vol. 268, No. 11, (April 1993), pp. (8170-8175)

No. 6, (December 2003), pp. (2329-2335)

*Chem,* Vol. 268, No. 32, (November 1993), pp. (23824-23829)

*Respir Cell Mol Biol,* Vol. 26, No. 5, (May 2002), pp. (602-609)

No. 1, (July 2005), pp. (60-66)

261, No. 2, (August 1991), pp. (L41-48)

(November 1996), pp. (4159-4165)

No.4, (July 2009), pp. (508-516)

117, No. 3, (March 2000), pp. (684-694)

pp. 493-498)

(469-475)

(13890-13894)

obstructive pulmonary disease. *COPD,* Vol. 4, No. 4, (December 2007), pp. (347-353)

beta and TIMP-1 in chronic obstructive pulmonary disease. *Eur Respir J,* Vol. 26,

elastase, by human alveolar macrophages. *Biochem J,* Vol. 257, No. 2, (January 1989),

cysteine proteinase in macrophages distinct from cathepsin L. *Am J Physiol*, Vol.

metalloproteinase-9 and tissue inhibitor of metalloproteinase-1 by alveolar macrophages from patients with chronic obstructive pulmonary disease. *Am J* 

elastolysis: roles of matrix metalloproteinases, cysteine, and serine proteases. *Am J Physiol Lung Cell Mol Physiol,* Vol. 283, No. 4, (October 2002), pp. (L867-L873) Saren, P., Welgus, H.G., & Kovanen, P.T. (1996). TNF-alpha and IL-1beta selectively induce

expression of 92-kDa gelatinase by human macrophages. *J Immunol,* Vol. 157, No. 9,

necrosis factor agonists and antagonists in stable COPD. *J Clin Immunol,* Vol. 29,

B, collagenases 1 and 2, and increased parenchymal cell death in COPD. *Chest,* Vol.

with leukocyte elastase. *Am Rev Respir Dis,* Vol. 116, No. 3, (September 1977), pp.

collagenases are elastases. *J Biol Chem,* Vol. 266, No. 12, (April 1991), pp. (7870-7875)

alveolar macrophages. Regulation of biosynthesis is opposite to that of metalloproteinases and TIMP-1. *J Biol Chem,* Vol. 267, No. 20, (July 1992), pp.

macrophage metalloproteinases by extracellular matrix. Evidence for enzyme- and substrate-specific responses involving prostaglandin-dependent mechanisms. *J Biol* 

elastolytic metalloproteinase produced by human alveolar macrophages. *J Biol* 

contributes to cigarette smoke-induced emphysema in mice. *Am J Pathol*, Vol. 163,


Lee, J.M., Kang, Y.R., Park, S.H., et al., (2008). Polymorphisms in interleukin-1β and its

Lee, W., & Thomas, P.S. (2009). Oxidative stress in COPD and its measurement through exhaled breath condensate. *Clin Transl Sci,* Vol. 2, No. 2, (April 2009), pp. (150-155) Lin, J-L., & Thomas, P.S. (2010). Current perspectives of oxidative stress and its

Liu, H., Ma, L., Wu, J., Wang, K., & Chen, X.J. (2009). Apoptosis of alveolar wall cells in

MacNee, W. (a2005). Pathogenesis of Chronic Obstructive Pulmonary Disease. *Proc Am* 

MacNee, W. (b2005). Pulmonary and systemic oxidant/antioxidant imbalance in chronic

MacNee, W., & Tuder, R.M. (2009). New paradigms in the pathogenesis of COPD I. *Proc Am* 

Majo, J., Ghezzo, H., & Cosio, M.G. (2001). Lymphocyte population and apoptosis in the

Minematsu, N., Nakamura, H., Tateno, H., Nakajima, T., & Yamaguchi, K. (2001). Genetic

Morissette, M.C., Parent, J., & Milot, J. (2009). Alveolar epithelial and endothelial cell

Morrison, H.M., Welgus, H.G., Stockley, R.A., Burnett, D., & Campbell, E.J. (1990). Inhibition

Muley, T., Wiebel, M., Schulz, V., & Ebert, W. (1994). Elastinolytic activity of alveolar

Niewoehner, D.E., Kleinerman, J., & Rice, D.B. (1974). Pathologic changes in the peripheral

Ohnishi, K., Takagi, M., Kurokawa, Y., Satomi, S., & Konttinen, Y.T. (1998). Matrix

*Biophys Res Commun,* Vol. 289, No. 1, (November 2001), pp. (116-119) Molet, S., Belleguic, C., & Lena, H. et al., (2005). Increase in macrophage elastase (MMP-12)

2008), pp. (1311-1320)

pp. (50-60)

(August 2010), pp. (291-306)

No. 4, (August 2009), pp. (466-469)

2001), pp. (946-953), ISSN 0903-1936

Vol. 54, No. 1, (January 2005), pp. 31-36)

No. 4, (March 1994), pp. (269-276)

1990), pp. (263-269)

pp. (755-758)

(March 2011)

*Obstruct Pulmon Dis,* Vol. 4, (April 2009), pp. (19-31)

*Thoracic Soc,* Vol. 2, No. 4, (2005), pp. (258-266)

*Thoracic Soc*, Vol. 6, No. 6, (September 2009), pp. (527-531)

receptor antagonist genes and the risk of chronic obstructive pulmonary disease in a Korean population: a case control study. *Respir Med*, Vol. 102, No. 9, (September

measurement in chronic obstructive pulmonary disease. *COPD:* Vol. 7, No. 4,

chronic obstructive pulmonary disease patients with pulmonary emphysema is involved in emphysematous changes. *Huazhong Univ Sci Technolog Med Sci,* Vol. 29,

obstructive pulmonary disease. Proc Am Thoracic Soc, Vol. 2, No. 1, (January 2005),

lungs of smokers and their relation to emphysema. *Eur Respir J,* Vol. 17, No. 5, (May

polymorphism in matrix metalloproteinase-9 and pulmonary emphysema. *Biochem* 

in lungs from patients with chronic obstructive pulmonary disease. *Inflamm Res,*

apoptosis in emphysema: what we know and what we need to know. *Int J Chron* 

of human leucocyte elastase bound to elastin: relative ineffectiveness and two mechanisms of inhibitory activity. *Am J Respir Cell Mol Biol,* Vol. 2, No. 3, (March

macrophages in smoking-associated pulmonary emphysema. *Clin Investig,* Vol. 72,

airways of young cigarette smokers. *N Engl J Med,* Vol. 291, No. 15, (October 1974),

metalloproteinase-mediated extracellular matrix protein degradation in human pulmonary emphysema. *Lab Invest,* Vol. 78, No. 9, (September 1998), pp. (1077-1087) Omachi, T.A., Eisner, M.D., & Rames, A. et al., (2011). Matrix metalloproteinase-9 predicts

pulmonary status declines in α1-antitrypsin deficiency. *Respir Res,* Vol. 12, No. 35,


**1. Introduction** 

**2** 

*USA* 

**Innate Immunity** 

*University of Michigan,* 

**of Airway Epithelium and COPD** 

The mammalian immune system consists of two branches- innate and adaptive immune systems and together they provide protection against infection. Innate immunity is a first line of host defense and is responsible for immediate recognition of pathogens to prevent microbial invasion. In addition innate immune responses also stimulate adaptive immune system (Medzhitov and Janeway, 1997). Cellular components of innate immune system include mucosal epithelial cells, macrophages, neutrophils, natural killer cells, basophils, eosinophils and others. The airway mucosa represents the body's largest mucosal surface and is the first point of contact for inhaled microorganisms, environmental pollutants, airborne allergens and cigarette smoke (Diamond et al., 2000). Airway mucosa provides protection against potentially hazardous inhaled factors by multiple mechanisms. For instance, mucus secreted by the airway epithelium covers the apical surface of airway epithelium and traps inhaled microorganisms, allergens and particulate material. The trapped material is then cleared by mucociliary escalator away from lungs and towards the pharynx. Tight junctions between the polarized airway epithelial cells restrict the paracellular movement of solutes and ions, and prevent pathogens from gaining access to the submucosal compartment. In addition to its role as a physical barrier between environmental factors and internal milieu, airway epithelial cells also play a critical role in bridging innate and adaptive immune defenses (Hammad and Lambrecht, 2011; Kato and Schleimer, 2007). Airway epithelial cells express number of innate immune receptors also known as pattern recognition molecules, which recognizes pathogen-associated molecular patterns (PAMPS) or danger-associated molecular patterns (DAMPS) to initiate appropriate innate defense mechanisms. This includes elaboration of antimicrobial molecules, proinflammatory cytokines and chemokines that recruits and activates other mucosal innate immune cells. The responses of activated innate immune cells lead to recruitment of immune cells into epithelium or airway lumen and initiate adaptive immune responses. Continuous exposure to environmental stimuli, such as cigarette smoke, noxious gases or other environmental hazards may lead to prolonged and aberrant activation of airway epithelial cells resulting in excessive expression of pro-inflammatory cytokines and chemokines that recruit large number of inflammatory cells into airway lumen. This in turn leads to persistent inflammation, airway damage and abnormal repair, impaired innate immune responses. There are reports suggesting that exposure to cigarette smoke also

Shyamala Ganesan and Uma S. Sajjan


## **Innate Immunity of Airway Epithelium and COPD**

Shyamala Ganesan and Uma S. Sajjan *University of Michigan, USA* 

#### **1. Introduction**

18 Emphysema

Shi, G.P., Munger, J.S., Meara, J.P., Rich, D.H., & Chapman, H.A. (1992). Molecular cloning

cysteine protease. *J Biol Chem,* Vol. 267, No. 11, (April 1992), pp. (7258-7262) Shiomi, T., Okada, Y., & Foronjy, R. et al., (2003). Emphysematous changes are caused by

Stone, P.J., Calore, J.D., McGowan, S.E., Bernardo, J., Snider, G.L., & Franzblau, C. (1983).

Takeyabu, K., Betsuyaku, T., & Nishimura, M. et al., (1998). Cysteine proteinases and

emphysema. *Eur Respir J,* Vol. 12, No. 5, (November 1998), pp. (1033-1039) Tomaki, M., Sugiura, H., Koarai A., et al., (2007). Decreased expression of antioxidant

Tuder, R.M., Yoshida, T., Arap, W., Pasqualini, R., & Petrache, I. (2006). State of the art.

Van der Toorn, M., Smit-de Vries, M.P., Slebos, D.J., et al., (2007). Cigarette smoke

Wallace, A.M., Sandford, A.J., & English, J.C., et al., (2008). Matrix metalloproteinase

Warfel, A.H., Cardozo, C., Yoo, O.H., & Zucker-Franklin D. (1991). Cystatin C and cathepsin

Yokohori, N., Aoshiba, K., & Nagai, A. (2004). Increased levels of cell death and proliferation

Yoshida, T., & Tuder, R.M. (2007). Pathobiology of cigarette smoke-induced chronic

Zhou, M., Huang, S.G., & Wan, H.Y. et al., (2004). Genetic polymorphism in matrix

Zhu, G., Warren, L., & Aponte, J. et al., (2007). The SERPINE2 gene is associated with

*Mol Physiol,* Vol.293, No. 5, (November 2007), pp. (L1156-1162)

*Res*, Vol. 29, No. 1, (Jan-Feb 2003), pp. (1-15)

*Dis,* Vol. 147, No. 6, (June 1993), pp. (1562-1568)

*Ther,* Vol.20, No. 5, (July 2007), pp. (596-605)

5, No. 1, (February 2008), pp. (13-23)

( February 2004), pp. (626-632)

(October 2004), pp. (1481-1484)

176, No. 2, (July 2007), pp. (167-173)

*Biol*, Vol. 49, No. 1, (January 1991), pp. (41-47)

510)

and expression of human alveolar macrophage cathepsin S, an elastinolytic

degradation of type III collagen in transgenic mice expressing MMP-1. *Exp Lung* 

Functional a1-protease inhibitor in the lower respiratory tract of cigarette smokers is not decreased. *Science,* Vol. 221, No. 4616, (September 1983), pp. (1187-1189) Takahashi, H., Ishidoh, K., & Muno, D. et al., (1993). Cathepsin L activity is increased in

alveolar macrophages and bronchoalveolar lavage fluid of smokers. *Am Rev Respir* 

cystatin C in bronchoalveolar lavage fluid from subjects with subclinical

enzymes and increased expression of chemokines in COPD lung. *Pulm Pharmacol* 

Cellular and molecular mechanisms of alveolar destruction in emphysema: an evolutionary perspective. *Proc Am Thorac Soc,* Vol. 3, No. 6, (August 2006), pp. (503-

irreversibly modifies glutathione in airway epithelial cells. *Am J Physiol Lung Cell* 

expression by human alveolar macrophages in relation to emphysema. *COPD,* Vol.

B production by alveolar macrophages from smokers and nonsmokers. *J Leukoc* 

in alveolar wall cells in patients with pulmonary emphysema. *Chest,* Vol. 125, No. 2

obstructive pulmonary disease. *Physiol Rev*, Vol. 87, No. 3, (July 2007), pp. (1047-1082)

metalloproteinase-9 and the susceptibility to chronic obstructive pulmonary disease in Han population of south China. *Chin Med J (Engl),* Vol. 117, No. 10,

chronic obstructive disease in two large populations. *Am J Respir Crit Care Med,* Vol.

The mammalian immune system consists of two branches- innate and adaptive immune systems and together they provide protection against infection. Innate immunity is a first line of host defense and is responsible for immediate recognition of pathogens to prevent microbial invasion. In addition innate immune responses also stimulate adaptive immune system (Medzhitov and Janeway, 1997). Cellular components of innate immune system include mucosal epithelial cells, macrophages, neutrophils, natural killer cells, basophils, eosinophils and others. The airway mucosa represents the body's largest mucosal surface and is the first point of contact for inhaled microorganisms, environmental pollutants, airborne allergens and cigarette smoke (Diamond et al., 2000). Airway mucosa provides protection against potentially hazardous inhaled factors by multiple mechanisms. For instance, mucus secreted by the airway epithelium covers the apical surface of airway epithelium and traps inhaled microorganisms, allergens and particulate material. The trapped material is then cleared by mucociliary escalator away from lungs and towards the pharynx. Tight junctions between the polarized airway epithelial cells restrict the paracellular movement of solutes and ions, and prevent pathogens from gaining access to the submucosal compartment. In addition to its role as a physical barrier between environmental factors and internal milieu, airway epithelial cells also play a critical role in bridging innate and adaptive immune defenses (Hammad and Lambrecht, 2011; Kato and Schleimer, 2007). Airway epithelial cells express number of innate immune receptors also known as pattern recognition molecules, which recognizes pathogen-associated molecular patterns (PAMPS) or danger-associated molecular patterns (DAMPS) to initiate appropriate innate defense mechanisms. This includes elaboration of antimicrobial molecules, proinflammatory cytokines and chemokines that recruits and activates other mucosal innate immune cells. The responses of activated innate immune cells lead to recruitment of immune cells into epithelium or airway lumen and initiate adaptive immune responses. Continuous exposure to environmental stimuli, such as cigarette smoke, noxious gases or other environmental hazards may lead to prolonged and aberrant activation of airway epithelial cells resulting in excessive expression of pro-inflammatory cytokines and chemokines that recruit large number of inflammatory cells into airway lumen. This in turn leads to persistent inflammation, airway damage and abnormal repair, impaired innate immune responses. There are reports suggesting that exposure to cigarette smoke also

Innate Immunity of Airway Epithelium and COPD 21

mucus hypersecretion by increasing expression of hypoxia-induced factor 1 and growth factors such as TGF-β, and EGF ligands (Yu et al., 2011a, b). Smokers with COPD also show goblet cell metaplasia and submucosal gland hypertrophy (Innes et al., 2006). Increased EGF receptor expression and activation and increased expression of platelet activating factor caused by cigarette smoke are thought to play a role in development of goblet cell metaplasia (Curran and Cohn, 2010; Komori et al., 2001; O'Donnell et al., 2004). Cigarette smoke decreases water and ion transport by inhibiting apical chloride channel and basolaterally located potassium channel in primary human and mouse airway epithelial cells(Cohen et al., 2009; Savitski et al., 2009). This essentially reduces the periciliary liquid layer in which cilia can beat rapidly and also increases the viscosity of mucus resulting in reduced clearance of mucus from the airways. In addition, respiratory epithelial cells exposed to cigarette smoke extract or condensate showed 70% less cilia and shorter cilia compared to control cells (Tamashiro et al., 2009). Mice exposed to cigarette smoke although showed slight increase in ciliary beat frequency at 6 weeks and 3 months, it was significantly reduced at 6 months and these mice also showed significant loss of tracheal ciliated cells (Simet et al., 2010). Decreased number of cilia, reduced ciliary function combined with hypersecretion of mucin, increased viscoelasticity of secreted mucus in COPD patients can lead to airways obstruction and promote persistence of trapped pathogens in the airways(Rose and Voynow, 2006; Voynow et al., 2006). Persistence of bacteria or viruses can

further increase production of mucus in the airways (Baginski et al., 2006).

Normal

COPD

Fig. 1. Airway epithelial cells isolated from COPD patient cultured at air/liquid interface show more goblet cells (arrows) than the similarly grown normal airway epithelial cells.

barrier function and persistence of inhaled pathogens.

Another feature that is frequently noted in airways of COPD patients is squamous metaplasia (Araya et al., 2007) and it correlates with the severity of airway obstruction (Cosio et al., 1978). The airway epithelium exposed to cigarette smoke responds by secreting TGF-β (de Boer et al., 1998), which is required for repair of injured epithelium and maintain homeostasis. However, chronic exposure to cigarette smoke can induce sustained production of TGF-β and increased TGF-β activation leading to expression of the β6 integrin, a TGF-β responsive gene (Wang et al., 1996). This in turn contributes to a phenotypic switch from columnar ciliated to squamous epithelium (Masui et al., 1986a; Masui et al., 1986b). Squamous epithelial cells secrete increased amounts of IL-1β, which acts as a paracrine factor with adjacent airway fibroblasts to further activate TGF-β (Araya et al., 2006), thereby increasing squamous metaplasia and further contributing to impaired

dampens the needed innate immune responses to infection, thereby promoting the persistence of infecting organism. This may result in delayed but sustained inflammation that can lead to progression of lung disease. In this chapter, we will discuss how the impaired innate immune defense mechanisms fail to provide protection against invading pathogens and its impact on progression of lung disease in patients with chronic obstructive pulmonary disease (COPD).

#### **2. Barrier function of airway epithelium**

Airway epithelium lines the entire airway mucosa. In normal adult human, the large airways are cartilaginous and mainly made up of ciliated cells, mucus producing goblet cells, undifferentiated columnar cells and basal cells with a capacity to multiply and differentiate into ciliated or goblet cells. Large airways are also surrounded by submucosal and serous glands. As the large airways branches out, it gradually becomes noncartilaginous, loses surrounding submucosal and serous glands, the cells become more columnar and cuboidal, and Clara secreting cells replace goblet cells in the small airways. Airway epithelium also consists of other minor cell types such as neuroendocrine cells, dendritic cells and others.

The three essential components that contributes to barrier function of airway epithelium are mucociliary apparatus (Knowles and Boucher, 2002), intercellular tight and adherens junctions (Pohl et al., 2009) that regulates epithelial paracellular permeability, and secreted antimicrobial products that kill the inhaled pathogens (Bals and Hiemstra, 2004).

#### **2.1 Mucociliary clearance**

The primary players of mucociliary apparatus are mucus produced by goblet cells and submucosal glands that overlay the airway epithelium and cilia. Mucociliary dysfunction results in recurrent and persistent respiratory infections as evidenced in patients with cystic fibrosis, ciliary dyskinesia and COPD (Bhowmik et al., 2009; Jansen et al., 1995; Livraghi and Randell, 2007; Sethi, 2000). In COPD patients, the dysfunction of mucociliary clearance is due to combined effect of mucus hypersecretion, increased viscosity of mucus and dysfunction or loss of cilia (Mehta et al., 2008). The airway mucus is a viscoelastic gel and contains more than 200 proteins, and it is secreted by goblet cells that are present in the airway epithelium and by submucosal glands. The main components of airway mucus are mucins, which are high molecular weight glycoproteins and cross link to form structural framework of mucus barrier (Rose et al., 2001; Thornton et al., 2008). At least 12 mucins are detected in human lungs, of these MUC5AC and MUC5B are the predominant mucins in normal airways (Rose and Voynow, 2006). Airways infection with virus or bacteria, exposure to toxic agents such as cigarette smoke and pollutants that induce airway inflammation and oxidative stress have been shown to upregulate expression of MUC5AC and MUC5B (Borchers et al., 1999; Casalino-Matsuda et al., 2009; Dohrman et al., 1998; Gensch et al., 2004; Haswell et al., 2010; Shao et al., 2004). Cigarette smoke induces expression of number of inflammatory mediators including IL-1β, IL-8, TNF-α, MCP-1, leukotrienes through oxidative stress-related pathways from airway epithelial cells, resident macrophages and infiltrated neutrophils, which can increase mucus secretion (Adcock et al., 2011; Choi et al., 2010; Cohen et al., 2009; Mebratu et al., 2011). Cigarette smoke also causes

dampens the needed innate immune responses to infection, thereby promoting the persistence of infecting organism. This may result in delayed but sustained inflammation that can lead to progression of lung disease. In this chapter, we will discuss how the impaired innate immune defense mechanisms fail to provide protection against invading pathogens and its impact on progression of lung disease in patients with chronic obstructive

Airway epithelium lines the entire airway mucosa. In normal adult human, the large airways are cartilaginous and mainly made up of ciliated cells, mucus producing goblet cells, undifferentiated columnar cells and basal cells with a capacity to multiply and differentiate into ciliated or goblet cells. Large airways are also surrounded by submucosal and serous glands. As the large airways branches out, it gradually becomes noncartilaginous, loses surrounding submucosal and serous glands, the cells become more columnar and cuboidal, and Clara secreting cells replace goblet cells in the small airways. Airway epithelium also consists of other minor cell types such as neuroendocrine cells,

The three essential components that contributes to barrier function of airway epithelium are mucociliary apparatus (Knowles and Boucher, 2002), intercellular tight and adherens junctions (Pohl et al., 2009) that regulates epithelial paracellular permeability, and secreted

The primary players of mucociliary apparatus are mucus produced by goblet cells and submucosal glands that overlay the airway epithelium and cilia. Mucociliary dysfunction results in recurrent and persistent respiratory infections as evidenced in patients with cystic fibrosis, ciliary dyskinesia and COPD (Bhowmik et al., 2009; Jansen et al., 1995; Livraghi and Randell, 2007; Sethi, 2000). In COPD patients, the dysfunction of mucociliary clearance is due to combined effect of mucus hypersecretion, increased viscosity of mucus and dysfunction or loss of cilia (Mehta et al., 2008). The airway mucus is a viscoelastic gel and contains more than 200 proteins, and it is secreted by goblet cells that are present in the airway epithelium and by submucosal glands. The main components of airway mucus are mucins, which are high molecular weight glycoproteins and cross link to form structural framework of mucus barrier (Rose et al., 2001; Thornton et al., 2008). At least 12 mucins are detected in human lungs, of these MUC5AC and MUC5B are the predominant mucins in normal airways (Rose and Voynow, 2006). Airways infection with virus or bacteria, exposure to toxic agents such as cigarette smoke and pollutants that induce airway inflammation and oxidative stress have been shown to upregulate expression of MUC5AC and MUC5B (Borchers et al., 1999; Casalino-Matsuda et al., 2009; Dohrman et al., 1998; Gensch et al., 2004; Haswell et al., 2010; Shao et al., 2004). Cigarette smoke induces expression of number of inflammatory mediators including IL-1β, IL-8, TNF-α, MCP-1, leukotrienes through oxidative stress-related pathways from airway epithelial cells, resident macrophages and infiltrated neutrophils, which can increase mucus secretion (Adcock et al., 2011; Choi et al., 2010; Cohen et al., 2009; Mebratu et al., 2011). Cigarette smoke also causes

antimicrobial products that kill the inhaled pathogens (Bals and Hiemstra, 2004).

pulmonary disease (COPD).

dendritic cells and others.

**2.1 Mucociliary clearance** 

**2. Barrier function of airway epithelium** 

mucus hypersecretion by increasing expression of hypoxia-induced factor 1 and growth factors such as TGF-β, and EGF ligands (Yu et al., 2011a, b). Smokers with COPD also show goblet cell metaplasia and submucosal gland hypertrophy (Innes et al., 2006). Increased EGF receptor expression and activation and increased expression of platelet activating factor caused by cigarette smoke are thought to play a role in development of goblet cell metaplasia (Curran and Cohn, 2010; Komori et al., 2001; O'Donnell et al., 2004). Cigarette smoke decreases water and ion transport by inhibiting apical chloride channel and basolaterally located potassium channel in primary human and mouse airway epithelial cells(Cohen et al., 2009; Savitski et al., 2009). This essentially reduces the periciliary liquid layer in which cilia can beat rapidly and also increases the viscosity of mucus resulting in reduced clearance of mucus from the airways. In addition, respiratory epithelial cells exposed to cigarette smoke extract or condensate showed 70% less cilia and shorter cilia compared to control cells (Tamashiro et al., 2009). Mice exposed to cigarette smoke although showed slight increase in ciliary beat frequency at 6 weeks and 3 months, it was significantly reduced at 6 months and these mice also showed significant loss of tracheal ciliated cells (Simet et al., 2010). Decreased number of cilia, reduced ciliary function combined with hypersecretion of mucin, increased viscoelasticity of secreted mucus in COPD patients can lead to airways obstruction and promote persistence of trapped pathogens in the airways(Rose and Voynow, 2006; Voynow et al., 2006). Persistence of bacteria or viruses can further increase production of mucus in the airways (Baginski et al., 2006).

Fig. 1. Airway epithelial cells isolated from COPD patient cultured at air/liquid interface show more goblet cells (arrows) than the similarly grown normal airway epithelial cells.

Another feature that is frequently noted in airways of COPD patients is squamous metaplasia (Araya et al., 2007) and it correlates with the severity of airway obstruction (Cosio et al., 1978). The airway epithelium exposed to cigarette smoke responds by secreting TGF-β (de Boer et al., 1998), which is required for repair of injured epithelium and maintain homeostasis. However, chronic exposure to cigarette smoke can induce sustained production of TGF-β and increased TGF-β activation leading to expression of the β6 integrin, a TGF-β responsive gene (Wang et al., 1996). This in turn contributes to a phenotypic switch from columnar ciliated to squamous epithelium (Masui et al., 1986a; Masui et al., 1986b). Squamous epithelial cells secrete increased amounts of IL-1β, which acts as a paracrine factor with adjacent airway fibroblasts to further activate TGF-β (Araya et al., 2006), thereby increasing squamous metaplasia and further contributing to impaired barrier function and persistence of inhaled pathogens.

Innate Immunity of Airway Epithelium and COPD 23

has been shown to induce disassembly of tight junction complex in endothelial cells by suppressing PTEN activity (Barbieri et al., 2008). Chen et al showed that cigarette smoke also alters epithelial permeability by disrupting cell polarity via activation of EGFR, dissociation of β-catenin and E-cadherin from adherence junctional complex and redistribution of apical MUC1 membrane bound mucin to cytoplasm (Chen et al., 2010). In a homestatic epithelium, β-catenin cooperates with E-cadherin to form apical junctional complex and maintain cell polarity (Xu and Kimelman, 2007). In airway regeneration or oncogenic formation β-catenin translocates to nucleus, and activates canonical Wnt signaling pathway (Mazieres et al., 2005; Tian et al., 2009). Similar to β-catenin, the cytoplasmic tail of MUC1 also supports structural barrier during homeostasis (Chen et al., 2010). Since cigarette smoke causes aberrant activation of both EGFR and canonical Wnt/βcatenin signaling (Khan et al., 2008; Lemjabbar et al., 2003), it is plausible that chronic cigarette smoke exposure decreases barrier function and promote microbial invasion of

In addition to acting as a physical barrier, airway epithelial cells also secrete antimicrobial substances, which include enzymes, protease inhibitors, oxidants and antimicrobial peptides. Lysozyme is an enzyme found in airway epithelial secretions and exerts antimicrobial effect against wide range of gram-positive bacteria by degrading peptidoglycan layer (Ibrahim et al., 2002). Lysozyme is also effective against gramnegative bacteria in the presence of lactoferrin, which disrupts the outer membrane allowing lysozyme to gain access to peptidoglycan layer (Ellison and Giehl, 1991). Lactoferrin is an iron-chelator and inhibit microbial growth by sequestering iron which is essential for microbial respiration (Ganz, 2002). Lactoferrin also display antiviral activity against both RNA and DNA viruses either by inhibiting binding of virus to host cells or by binding to virus itself (van der Strate et al., 2001; Laube et al., 2006). Lactoferrin levels increase in response to bacterial and viral infections. Epithelial cells produce protease inhibitors, such as secretory leukoprotease inhibitor (SLPI), elastase inhibitor, α1 antiprotease and antichymotrypsin. These protease inhibitors mitigate the effects of proteases expressed by pathogens and recruited innate immune cells. Administration of SLPI decreased the levels of IL-8 and elastase activity in airway secretion of cystic fibrosis

Human beta defensins (hBD) are the most abundant antimicrobial peptides expressed on the surface of airway epithelium and are effective against wide range of bacteria and viruses (Ganz, 2003; Kota et al., 2008; McCray and Bentley, 1997). While hBD1 is constitutively expressed, hBD2 to hBD4 expression is induced by LPS via NF-κB activation and also by IL-1 (Becker et al., 2000; Singh et al., 1998). hBD2 is induced by *P. aeruginosa* infection in normal but not in cystic fibrosis airway epithelia (Dauletbaev et al., 2002). Environmental factors such as air pollutants decrease defensin gene expression in the airways (Laube et al., 2006). In CF airway epithelia activity of hBD2 is also decreased due to increased salt concentration (Goldman et al., 1997). Cathelicidins are another class of antimicrobial peptides and LL37 is the only human cathelicidin identified to date. LL37 bind to LPS and inactivate its biological function. Overexpression of human LL37 in CF mouse model increased killing of *P. aeruginosa* and reduced the ability of this bacterium to colonize the airways (Bals et al., 1998).

airway epithelium.

patients (McElvaney et al., 1992).

**2.3 Antimicrobial products of airway epithelium** 

In our laboratory, we observed that cultured airway epithelial cells isolated from COPD patients show goblet cell metaplasia, decreased number of ciliated cells (Figure 1), and increased MMP activity suggesting that epigenetic changes that occur *in vivo* are maintained even when cells are expanded *ex vivo* (Schneider et al., 2010). COPD epithelial cells also showed increased viral load following rhinovirus challenge compared to normal cells. Similarly, we also found that elastase/LPS exposed mice which show typical features of COPD, including emphysema, airway remodeling, diffuse lung inflammation and goblet cell hypertrophy, also showed increased persistence of virus compared to normal mice following rhinovirus challenge and majority of the virus particles were observed in the airway epithelium (Sajjan et al., 2009). Rinovirus infection increased mucin expression further in these mice. Since goblet cells are the target for rhinovirus infection (Lachowicz-Scroggins et al., 2010) we suggest that COPD airway epithelial cultures which have increased number of goblet cells are more susceptible to rhinovirus infection than the controls. Patients with COPD, cystic fibrosis and asthma show goblet cell metaplasia and this may be one of the reasons these patients are more susceptible to rhinovirus infection. In addition, airway epithelial mucins also interact with several other respiratory pathogens including *Pseudomonas aeruginosa*, *Staphylococcus aureus*, *Heamophilus influenza*, *Streptococcus pneumonia*, *Burkholderia cenocepacia*, influenza virus, adenovirus and coronavirus (Landry et al., 2006; Matrosovich and Klenk, 2003; Plotkowski et al., 1993; Ryan et al., 2001; Sajjan and Forstner, 1992; Sajjan et al., 1992; Walters et al., 2002). The bound pathogens which are cleared under normal conditions, persist in the airway lumen when the mucociliary clearance is impaired and initiate inflammatory response and damage the airway epithelium.

#### **2.2 Junctional adherens complexes and airway epithelial permeability**

Epithelial permeability is maintained through the cooperation of two mutually exclusive structural components: Tight junctions and adherence junctions on the lateral membranes (Pohl et al., 2009). While tight junctions regulate the transport of solutes and ions across epithelia, adherence junctions mediate cell to cell adhesion (Hartsock and Nelson, 2008; Schneeberger and Lynch, 2004; Shin et al., 2006). Under homeostatic conditions, these intercellular junctions prevent inhaled pathogens and also serve as signaling platforms that regulate gene expression, cell proliferation and differentiation (Balda and Matter, 2009; Koch and Nusrat, 2009). Therefore disassociation or sustained insult that affects junctional complex will disrupt not only barrier function, but also prevent normal repair of airway epithelium. Compared to control nonsmokers, airway epithelium is leaky, hyperproliferative and abnormally differentiated in smokers (Hogg and Timens, 2009). Consistent with this observation, various *in vivo* and *in vitro* studies showed that cigarette smoke increases airway epithelial permeability (Boucher et al., 1980; Gangl et al., 2009; Olivera et al., 2007; Serikov et al., 2006). Recently, transcriptome analysis of airway epithelial cells from normal and COPD patients revealed global down-regulation of physiological tight junction complex gene expression (Shaykhiev et al., 2011). Further, normal airway epithelial cells exposed to cigarette smoke extract also showed similar down-regulation of genes related to tight junction complex. This was associated with decreased expression of PTEN and FOXO3A, a transcriptional factor in the PTEN pathway, suggesting that cigarette smoke down-regulates expression of apical junctional complex genes by modulating PTEN signaling pathway. Consistent with this notion, cigarette smoke in combination with IL-1β

In our laboratory, we observed that cultured airway epithelial cells isolated from COPD patients show goblet cell metaplasia, decreased number of ciliated cells (Figure 1), and increased MMP activity suggesting that epigenetic changes that occur *in vivo* are maintained even when cells are expanded *ex vivo* (Schneider et al., 2010). COPD epithelial cells also showed increased viral load following rhinovirus challenge compared to normal cells. Similarly, we also found that elastase/LPS exposed mice which show typical features of COPD, including emphysema, airway remodeling, diffuse lung inflammation and goblet cell hypertrophy, also showed increased persistence of virus compared to normal mice following rhinovirus challenge and majority of the virus particles were observed in the airway epithelium (Sajjan et al., 2009). Rinovirus infection increased mucin expression further in these mice. Since goblet cells are the target for rhinovirus infection (Lachowicz-Scroggins et al., 2010) we suggest that COPD airway epithelial cultures which have increased number of goblet cells are more susceptible to rhinovirus infection than the controls. Patients with COPD, cystic fibrosis and asthma show goblet cell metaplasia and this may be one of the reasons these patients are more susceptible to rhinovirus infection. In addition, airway epithelial mucins also interact with several other respiratory pathogens including *Pseudomonas aeruginosa*, *Staphylococcus aureus*, *Heamophilus influenza*, *Streptococcus pneumonia*, *Burkholderia cenocepacia*, influenza virus, adenovirus and coronavirus (Landry et al., 2006; Matrosovich and Klenk, 2003; Plotkowski et al., 1993; Ryan et al., 2001; Sajjan and Forstner, 1992; Sajjan et al., 1992; Walters et al., 2002). The bound pathogens which are cleared under normal conditions, persist in the airway lumen when the mucociliary clearance is impaired and initiate inflammatory response and damage the airway

**2.2 Junctional adherens complexes and airway epithelial permeability** 

Epithelial permeability is maintained through the cooperation of two mutually exclusive structural components: Tight junctions and adherence junctions on the lateral membranes (Pohl et al., 2009). While tight junctions regulate the transport of solutes and ions across epithelia, adherence junctions mediate cell to cell adhesion (Hartsock and Nelson, 2008; Schneeberger and Lynch, 2004; Shin et al., 2006). Under homeostatic conditions, these intercellular junctions prevent inhaled pathogens and also serve as signaling platforms that regulate gene expression, cell proliferation and differentiation (Balda and Matter, 2009; Koch and Nusrat, 2009). Therefore disassociation or sustained insult that affects junctional complex will disrupt not only barrier function, but also prevent normal repair of airway epithelium. Compared to control nonsmokers, airway epithelium is leaky, hyperproliferative and abnormally differentiated in smokers (Hogg and Timens, 2009). Consistent with this observation, various *in vivo* and *in vitro* studies showed that cigarette smoke increases airway epithelial permeability (Boucher et al., 1980; Gangl et al., 2009; Olivera et al., 2007; Serikov et al., 2006). Recently, transcriptome analysis of airway epithelial cells from normal and COPD patients revealed global down-regulation of physiological tight junction complex gene expression (Shaykhiev et al., 2011). Further, normal airway epithelial cells exposed to cigarette smoke extract also showed similar down-regulation of genes related to tight junction complex. This was associated with decreased expression of PTEN and FOXO3A, a transcriptional factor in the PTEN pathway, suggesting that cigarette smoke down-regulates expression of apical junctional complex genes by modulating PTEN signaling pathway. Consistent with this notion, cigarette smoke in combination with IL-1β

epithelium.

has been shown to induce disassembly of tight junction complex in endothelial cells by suppressing PTEN activity (Barbieri et al., 2008). Chen et al showed that cigarette smoke also alters epithelial permeability by disrupting cell polarity via activation of EGFR, dissociation of β-catenin and E-cadherin from adherence junctional complex and redistribution of apical MUC1 membrane bound mucin to cytoplasm (Chen et al., 2010). In a homestatic epithelium, β-catenin cooperates with E-cadherin to form apical junctional complex and maintain cell polarity (Xu and Kimelman, 2007). In airway regeneration or oncogenic formation β-catenin translocates to nucleus, and activates canonical Wnt signaling pathway (Mazieres et al., 2005; Tian et al., 2009). Similar to β-catenin, the cytoplasmic tail of MUC1 also supports structural barrier during homeostasis (Chen et al., 2010). Since cigarette smoke causes aberrant activation of both EGFR and canonical Wnt/βcatenin signaling (Khan et al., 2008; Lemjabbar et al., 2003), it is plausible that chronic cigarette smoke exposure decreases barrier function and promote microbial invasion of airway epithelium.

#### **2.3 Antimicrobial products of airway epithelium**

In addition to acting as a physical barrier, airway epithelial cells also secrete antimicrobial substances, which include enzymes, protease inhibitors, oxidants and antimicrobial peptides. Lysozyme is an enzyme found in airway epithelial secretions and exerts antimicrobial effect against wide range of gram-positive bacteria by degrading peptidoglycan layer (Ibrahim et al., 2002). Lysozyme is also effective against gramnegative bacteria in the presence of lactoferrin, which disrupts the outer membrane allowing lysozyme to gain access to peptidoglycan layer (Ellison and Giehl, 1991). Lactoferrin is an iron-chelator and inhibit microbial growth by sequestering iron which is essential for microbial respiration (Ganz, 2002). Lactoferrin also display antiviral activity against both RNA and DNA viruses either by inhibiting binding of virus to host cells or by binding to virus itself (van der Strate et al., 2001; Laube et al., 2006). Lactoferrin levels increase in response to bacterial and viral infections. Epithelial cells produce protease inhibitors, such as secretory leukoprotease inhibitor (SLPI), elastase inhibitor, α1 antiprotease and antichymotrypsin. These protease inhibitors mitigate the effects of proteases expressed by pathogens and recruited innate immune cells. Administration of SLPI decreased the levels of IL-8 and elastase activity in airway secretion of cystic fibrosis patients (McElvaney et al., 1992).

Human beta defensins (hBD) are the most abundant antimicrobial peptides expressed on the surface of airway epithelium and are effective against wide range of bacteria and viruses (Ganz, 2003; Kota et al., 2008; McCray and Bentley, 1997). While hBD1 is constitutively expressed, hBD2 to hBD4 expression is induced by LPS via NF-κB activation and also by IL-1 (Becker et al., 2000; Singh et al., 1998). hBD2 is induced by *P. aeruginosa* infection in normal but not in cystic fibrosis airway epithelia (Dauletbaev et al., 2002). Environmental factors such as air pollutants decrease defensin gene expression in the airways (Laube et al., 2006). In CF airway epithelia activity of hBD2 is also decreased due to increased salt concentration (Goldman et al., 1997). Cathelicidins are another class of antimicrobial peptides and LL37 is the only human cathelicidin identified to date. LL37 bind to LPS and inactivate its biological function. Overexpression of human LL37 in CF mouse model increased killing of *P. aeruginosa* and reduced the ability of this bacterium to colonize the airways (Bals et al., 1998).

Innate Immunity of Airway Epithelium and COPD 25

the toll/interleukin-1 receptor (TIR) homology domain (Hoffmann, 2003) (Figure 2). To date thirteen TLRs have been identified in mammalian system. Only TLRs1 to 10 are expressed in humans. TLRs1, -2, -4, -5 and -6 are expressed on the cell surface and TLRs3, -7,- 8 and -9 are expressed in the endosomes, lysozomes and the endoplastic reticulum. (Kawai and Akira, 2009). TLRs recognize a wide range of PAMPS- lipoproteins by TLRs 1, -2, and -6 (Aliprantis et al., 1999; Schwandner et al., 1999; Takeuchi et al., 2001; Takeuchi et al., 2002), LPS by TLR4 (Poltorak et al., 1998), flagella by TLR5 (Hayashi et al., 2001), DNA by TLR9 (Hemmi et al., 2000), and RNA by TLR3, -7 and -8 (Alexopoulou et al., 2001; Diebold et al., 2004; Heil et al.,

Increased neutrophils

T L R 4 T I R A P M y D 8 8

T L R 4 T I R A P M y D 8 8

T L R 4 T I R A P M y D 8 8

> IRAK-1 IRAK-2 IRAK- 4

> > TAK1

IKKβ IKK<sup>α</sup> NEMO

**MAPK activation NF-B**

Increased CXCL-8, IL-6, IL-1β

2004). TLR4 also recognizes respiratory syncytial virus (Kurt-Jones et al., 2000).

Decreased neutrophils

T L R 4 T I R A P M y D 8 8

T L R 4 T I R A P M y D 8 8

**A B C**

IRAK-1 IRAK-2 IRAK- 4

TAK1

AP-1 AP-1 AP-1

IKKβ IKK<sup>α</sup> NEMO

**MAPK activation NF-B**

Fig. 2. Impact of cigarette smoke on persistence of bacteria and inflammation. Under homeostasis, TLR4 recognizes infecting bacteria and activates both MAP kinase and NF-kB pathway to stimulate normal levels of CXCL-8, IL-6 and IL-1β to recruit neutrophils, which clear bacteria. Decreased expression of TLR4 caused by acute exposure to cigarette smoke attenuates release of CXCL-8, IL-6 and IL-1β, there by decreasing the neutrophil infiltration and increasing the bacterial persistence. Under chronic exposure as noted in COPD patients, if the TLR4 expression is increased, then chemokine and cytokine expression is increased

leading to decreased bacteria coupled with increased inflammation.

Decreased CXCL-8, IL-6, IL-1β

TLRs initiate signaling by MyD (myeloid differentiation primary-response protein) 88 dependent and –independent pathways. Except for TLR3, all TLRs initiate signaling by MyD-88-depnedent pathway to activate NF-κB. MyD88 is located in the cytoplasm and is similar to

Neutrophils

T L R 4 T I R A P M y D 8 8

IRAK-1 IRAK-2 IRAK- 4

T L R 4 T I R A P M y D 8 8

TAK1

IKKβ IKK<sup>α</sup> NEMO

**MAPK activation NF-B**

CXCL-8, IL-6, IL-1β

Airway epithelial cells also generate oxidants such as nitric oxide (NO) and hydrogen peroxide. Three NO synthases contribute to production of NO in airway epithelia: the constitutively expressed NOS1 and NOS3 and inducible NOS2. Viral infections and proinflammatory cytokines induce expression of NOS2 and defective NOS2 expression is responsible for increased viral replication in cystic fibrosis and overexpression of NOS2 provides protection against viral infection (Zheng et al., 2003; Zheng et al., 2004). Hydrogen peroxide is produced by dual oxidase 1 and 2. These belong to a family of NADPH oxidases and are located in the plasma membrane and secrete hydrogen peroxide to extracellular milieu. The dual oxidase-generated hydrogen peroxide in combination with thiocyanate and lactoperoxidase generates the microbicidal oxidant hypothiocyanite , which effectively kills both gram positive and gram negative bacteria and this innate defense mechanism is defective in cystic fibrosis airway epithelium due to impaired transport of thiocyanate (Moskwa et al., 2007).

In COPD patients, levels of lysozyme and SLPI decrease with bacterial infection, while lactoferrin levels remain unchanged (Parameswaran et al., 2011). Lower levels of salivary lysozyme in clinically stable COPD patients correlated with increased risk of exacerbations (Taylor et al., 1995). Reduced lysozyme levels in COPD is thought to be due to degradation by proteases elaborated by bacterial pathogens or neutrophils(Jacquot et al., 1985; Taggart et al., 2001). These proteases also inactivate SLPI (Parameswaran et al., 2009). In addtion, SLPI forms complexes with neutrophil elastase and binds to negatively charged membranes, thus decreasing the levels of SLPI further in the airway secretions during infection. In clinically stable patients however, the levels of SLPI were increased compared to smokers without COPD and never smokers (Tsoumakidou et al., 2010). In contrast, hBD2 was absent in COPD patients. Herr et al showed that hBD2 is significantly reduced in pharyngeal wash and suptum of current or former smokers compared to non-smokers, and exposure of airway epithelium to cigarette smoke *in vitro* inhibited induction of HBD2 by bacteria (Herr et al., 2009). Recently, we showed that COPD airway epithelial cells show a trend in decreased expression of NOS2 and Duox oxidases and this was associated with impaired clearance of rhinovirus (Schneider et al., 2010).

#### **3. Innate immune receptors of airway epithelium**

Airway epithelium in addition to providing a physical barrier, it also plays a pivotal role in recognition of pathogens and releasing appropriate chemokine and cytokines to initiate an inflammatory response. This inflammatory response includes recruitment of phagocytes to clear pathogens that are not cleared by barrier function of epithelium, and immune cells, such as dendritic cells and lymphocytes that initiate adaptive immune response. Airway epithelium recognizes pathogens or pathogen associated molecular patterns (PAMPS) by innate immune receptors also known as pattern recognition receptors (PRRs), which are germ-line encoded receptors. One of best characterized PRRs are Toll-like receptors (TLRs)(Akira et al., 2001; Medzhitov, 2001).

#### **3.1 Toll-like receptors**

TLRs are type I transmembrane receptors with an extracellular domain that contains leucine-rich-repeat motifs, a transmembrane domain and a cytoplasmic domain known as

Airway epithelial cells also generate oxidants such as nitric oxide (NO) and hydrogen peroxide. Three NO synthases contribute to production of NO in airway epithelia: the constitutively expressed NOS1 and NOS3 and inducible NOS2. Viral infections and proinflammatory cytokines induce expression of NOS2 and defective NOS2 expression is responsible for increased viral replication in cystic fibrosis and overexpression of NOS2 provides protection against viral infection (Zheng et al., 2003; Zheng et al., 2004). Hydrogen peroxide is produced by dual oxidase 1 and 2. These belong to a family of NADPH oxidases and are located in the plasma membrane and secrete hydrogen peroxide to extracellular milieu. The dual oxidase-generated hydrogen peroxide in combination with thiocyanate and lactoperoxidase generates the microbicidal oxidant hypothiocyanite , which effectively kills both gram positive and gram negative bacteria and this innate defense mechanism is defective in cystic fibrosis airway epithelium due to impaired transport of thiocyanate

In COPD patients, levels of lysozyme and SLPI decrease with bacterial infection, while lactoferrin levels remain unchanged (Parameswaran et al., 2011). Lower levels of salivary lysozyme in clinically stable COPD patients correlated with increased risk of exacerbations (Taylor et al., 1995). Reduced lysozyme levels in COPD is thought to be due to degradation by proteases elaborated by bacterial pathogens or neutrophils(Jacquot et al., 1985; Taggart et al., 2001). These proteases also inactivate SLPI (Parameswaran et al., 2009). In addtion, SLPI forms complexes with neutrophil elastase and binds to negatively charged membranes, thus decreasing the levels of SLPI further in the airway secretions during infection. In clinically stable patients however, the levels of SLPI were increased compared to smokers without COPD and never smokers (Tsoumakidou et al., 2010). In contrast, hBD2 was absent in COPD patients. Herr et al showed that hBD2 is significantly reduced in pharyngeal wash and suptum of current or former smokers compared to non-smokers, and exposure of airway epithelium to cigarette smoke *in vitro* inhibited induction of HBD2 by bacteria (Herr et al., 2009). Recently, we showed that COPD airway epithelial cells show a trend in decreased expression of NOS2 and Duox oxidases and this was associated with impaired

Airway epithelium in addition to providing a physical barrier, it also plays a pivotal role in recognition of pathogens and releasing appropriate chemokine and cytokines to initiate an inflammatory response. This inflammatory response includes recruitment of phagocytes to clear pathogens that are not cleared by barrier function of epithelium, and immune cells, such as dendritic cells and lymphocytes that initiate adaptive immune response. Airway epithelium recognizes pathogens or pathogen associated molecular patterns (PAMPS) by innate immune receptors also known as pattern recognition receptors (PRRs), which are germ-line encoded receptors. One of best characterized PRRs are Toll-like receptors

TLRs are type I transmembrane receptors with an extracellular domain that contains leucine-rich-repeat motifs, a transmembrane domain and a cytoplasmic domain known as

(Moskwa et al., 2007).

clearance of rhinovirus (Schneider et al., 2010).

(TLRs)(Akira et al., 2001; Medzhitov, 2001).

**3.1 Toll-like receptors** 

**3. Innate immune receptors of airway epithelium** 

the toll/interleukin-1 receptor (TIR) homology domain (Hoffmann, 2003) (Figure 2). To date thirteen TLRs have been identified in mammalian system. Only TLRs1 to 10 are expressed in humans. TLRs1, -2, -4, -5 and -6 are expressed on the cell surface and TLRs3, -7,- 8 and -9 are expressed in the endosomes, lysozomes and the endoplastic reticulum. (Kawai and Akira, 2009). TLRs recognize a wide range of PAMPS- lipoproteins by TLRs 1, -2, and -6 (Aliprantis et al., 1999; Schwandner et al., 1999; Takeuchi et al., 2001; Takeuchi et al., 2002), LPS by TLR4 (Poltorak et al., 1998), flagella by TLR5 (Hayashi et al., 2001), DNA by TLR9 (Hemmi et al., 2000), and RNA by TLR3, -7 and -8 (Alexopoulou et al., 2001; Diebold et al., 2004; Heil et al., 2004). TLR4 also recognizes respiratory syncytial virus (Kurt-Jones et al., 2000).

Fig. 2. Impact of cigarette smoke on persistence of bacteria and inflammation. Under homeostasis, TLR4 recognizes infecting bacteria and activates both MAP kinase and NF-kB pathway to stimulate normal levels of CXCL-8, IL-6 and IL-1β to recruit neutrophils, which clear bacteria. Decreased expression of TLR4 caused by acute exposure to cigarette smoke attenuates release of CXCL-8, IL-6 and IL-1β, there by decreasing the neutrophil infiltration and increasing the bacterial persistence. Under chronic exposure as noted in COPD patients, if the TLR4 expression is increased, then chemokine and cytokine expression is increased leading to decreased bacteria coupled with increased inflammation.

TLRs initiate signaling by MyD (myeloid differentiation primary-response protein) 88 dependent and –independent pathways. Except for TLR3, all TLRs initiate signaling by MyD-88-depnedent pathway to activate NF-κB. MyD88 is located in the cytoplasm and is similar to

Innate Immunity of Airway Epithelium and COPD 27

chemokine responses further. Stimulation of TLR2 or TLR3 also induces mucin expression by activating MAP kinases and inducing EGF receptor signaling (Chen et al., 2004; Kohri et al., 2002; Li et al., 1997; Zhu et al., 2009). MUC1, a transmembrane mucin is a negative regulator of TLRs and therefore may play an important role in limiting TLR- induced

There are conflicting reports with regards to expression of TLRs and their role in innate immune responses in patients with COPD. Airway epithelial cells from patients with severe COPD showed decreased expression of TLR4, but not TLR2 (MacRedmond et al., 2007). In contrast, recently Pace et al observed increased neutrophils and decreased apoptosis of neutrophils in the bronchoalveolar lavage and increased expression of TLR4 in airway epithelium of COPD patients providing evidence that increased TLR4 may contribute to airway neutrophilia in COPD (Pace et al., 2011). Pace et al also demonstrated increased TLR4 expression and concurrent increased CXCL-8 in response to LPS challenge in cigarette smoke exposed airway epithelial cells(Pace et al., 2008), while other investigators showed decreased TLR4 expression which was associated with reduced CXCL-8 and hBD2 production (Kulkarni et al., 2010; MacRedmond et al., 2007). Our preliminary studies involving primary airway epithelial cells from COPD patients suggested heightened expression of CXCL-8 in responses to *P. aeruginosa* infection compared to normal airway epithelial cells (Ganesan and Sajjan, unpublished results). However, role of TLR in this context is yet to be established. Whether TLR4 expression is decreased or increased it has important implications in COPD airway inflammation and obstruction (Figure 2). The decreased expression of TLR4 may lead to decreased innate immune responses and increased persistence of infecting organism. On the other hand increased expression of TLR4 increases neutrophil recruitment and mucus production in response to bacterial or viral

infection, thereby leading to increased airways inflammation and obstruction.

Another family of PRRs that play a role in innate defense mechanisms of airway epithelial cells is retinoic acid inducible (RIG)-I like receptors (RLR). This family of PRRs includes RIG-I, MDA-5 (melanoma differentiation associated protein 5) and LGP-2 (Laboratory of genetics and physiology 2). RLRs are the primary sensor molecules for detection of viral RNA in the cytoplasm (Meylan and Tschopp, 2006; Sun et al., 2006). Both RIG-I and MDA-5 contain a caspase recruitment domain (CARD) and a RNA helicase domain (Kang et al., 2002; Yoneyama et al., 2005; Yoneyama et al., 2004). On the other hand, LPG-2 has only RNA helicase domain but not CARD domain, which is required for recruiting adaptor protein MAVS (also known as VISA, Cardiff)(Yoneyama et al., 2005). Therefore recognition of viral RNA by RIG-I and MDA-5 leads to IFN or chemokine response, and LPG-2 suppresses this response (Yoneyama et al., 2005). RIG-I and MDA-5 recognize different RNA species. RIG-I recognizes single stranded (ss)RNA viruses, such as influenza virus, paramyxoviruses and deficiency in RIG-I increases the susceptibility of mice to RNA viruses (Kato et al., 2005). RIG-I specifically binds to the 5'-triphosphate moiety, the signature of which is exposed in the process of viral entry or replication. The host RNA which loses 5'triphosphate moiety during processing is therefore not recognized by RIG-I preventing cytokine and chemokine response due to self-recognition. RIG-I also recognizes short dsRNA (<1 kb) in 5'triphosphate-

inflammatory responses (Ueno et al., 2008).

**3.2 RIG-I like receptors** 

TLR in structure and has an N-terminal death domain, an intermediary domain and Cterminal TIR domain. Upon recognition of PAMPs by TLRs, the TIR domain of TLR interacts with TIR domain of MyD88 directly or indirectly via MyD88-adaptor like protein (MAL)/TIR adaptor protein (TIRAP)(Horng et al., 2002; Li et al., 2005). TLR5, -7, -8 and -9 does not require TIRAP to initiate signaling events that leads to NF-κB activation (Horng et al., 2002). Association of MyD88 to TLR leads to recruitment of IL-1R associated kinase (IRAK)-4, IRAK-1, TNFR-associated factor 6 (TRAF6), which then through a number of kinases activates NF-κB and AP-1 and stimulates expression of CXCL-8, IL-6, IL-1β and TNF-α (Adachi et al., 1998; Mukaida et al., 1990; Jeong and Lee, 2011). TLR4 also signals via MyD88-independent pathway and the first supporting evidence came from the studies on MyD88 knockout mice, which failed to respond normally to TLR2, -5, -7 and -9 ligands, but not to TLR4 (Kawai et al., 1999). Later TLR4 endocytosed upon binding to LPS was shown to signal through TIR-domaincontaining adapter-inducing interferon (IFN)-β (TRIF) pathway similar to TLR3 (Alexopoulou et al., 2001; Hoebe et al., 2003; Kagan et al., 2008). TLR2 was shown to be internalized and stimulate type I interferon (IFN) response by MyD88-dependent pathway in virus-, but not bacteria infected inflammatory monocytes (Barbalat et al., 2009).

The airway epithelium expresses all 10 TLRs, but the expression of TLR2 to TLR6 is stronger than the others. Expression of TLRs7 through -10 is variable depending on type of cells used (Mayer et al., 2007; Platz et al., 2004; Sha et al., 2004). Expression of TLRs 1 through -6 and -9 on the cell surface was confirmed by flow cytometry (Greene et al., 2005). However the signaling from these TLRs depends on the expression of adaptor molecules and coreceptors. Primary airway epithelial cells are hyporesponsive to LPS despite expressing TLR4 and this is because of reduced surface expression of co-receptor CD14 and low expression levels of co-stimulatory molecule MD2 (Jia et al., 2004). This may be necessary to restrict TLR4 activation under unstimulated conditions to prevent chronic inflammation of airways that is constantly exposed to inhaled bacteria and endotoxin. On the contrary, LPS was shown to activate TLR4 signaling in small airway and alveolar epithelial cells even though the TLR4 was localized to cytoplasmic compartment (Guillot et al., 2004). More recently John et al attributed chronic colonization of bacteria in CF airways to decreased expression of TLR4 in CF airway epithelial cells (John et al., 2010). TLR2, which is expressed on the apical surface of polarized airway cells is mobilized into an apical lipid raft receptor complex following *P. aeruginosa* infection and initiate signalling (Soong et al., 2004). TLR5 recgonizes flagella of *P. aeruginosa* and *Burkholderia cenocepacia* and activate NF-κB (Adamo et al., 2004; Urban et al., 2004; Zhang et al., 2005). *Haemophilus infIuenzae* traverses polarized airway epithelial cells by interacting with TLR2, which then activates p38 mitogen activated protein (MAP) kinase and TGF-β Signalling(Beisswenger et al., 2007). TLR3 recognizes double stranded (ds)-RNA, an intermediate generated during RNA virus replication and elicits chemokine and type I IFN responses by MyD88- independent signaling mechanism (Gern et al., 2003; Wang et al., 2009). Upon ligation of ds-RNA, TRIF and TRAM (TRIFrelated adaptor molecule) are recruited to TIR domain of TLR3 and TRAM acts as a bridge between TLR and TRIF and this allows activation of TRIF-dependent signaling leading to activation of IRF3 via IKKε/TBK-1 to stimulate IFN production or activation of NF-κB via IKKα/IKKβ to stimulate CXCL-8 expression (Kawai and Akira, 2008). The recognition of double-stranded RNA by TLR3 also increases expression of hBD2 (Duits et al., 2003). Viral or bacterial infection transcriptionally upregulates TLR3 expression (Liu et al., 2007; Sajjan et al., 2006; Wang et al., 2009; Xing et al., 2011), thereby increasing viral induced cytokine and

TLR in structure and has an N-terminal death domain, an intermediary domain and Cterminal TIR domain. Upon recognition of PAMPs by TLRs, the TIR domain of TLR interacts with TIR domain of MyD88 directly or indirectly via MyD88-adaptor like protein (MAL)/TIR adaptor protein (TIRAP)(Horng et al., 2002; Li et al., 2005). TLR5, -7, -8 and -9 does not require TIRAP to initiate signaling events that leads to NF-κB activation (Horng et al., 2002). Association of MyD88 to TLR leads to recruitment of IL-1R associated kinase (IRAK)-4, IRAK-1, TNFR-associated factor 6 (TRAF6), which then through a number of kinases activates NF-κB and AP-1 and stimulates expression of CXCL-8, IL-6, IL-1β and TNF-α (Adachi et al., 1998; Mukaida et al., 1990; Jeong and Lee, 2011). TLR4 also signals via MyD88-independent pathway and the first supporting evidence came from the studies on MyD88 knockout mice, which failed to respond normally to TLR2, -5, -7 and -9 ligands, but not to TLR4 (Kawai et al., 1999). Later TLR4 endocytosed upon binding to LPS was shown to signal through TIR-domaincontaining adapter-inducing interferon (IFN)-β (TRIF) pathway similar to TLR3 (Alexopoulou et al., 2001; Hoebe et al., 2003; Kagan et al., 2008). TLR2 was shown to be internalized and stimulate type I interferon (IFN) response by MyD88-dependent pathway in virus-, but not

The airway epithelium expresses all 10 TLRs, but the expression of TLR2 to TLR6 is stronger than the others. Expression of TLRs7 through -10 is variable depending on type of cells used (Mayer et al., 2007; Platz et al., 2004; Sha et al., 2004). Expression of TLRs 1 through -6 and -9 on the cell surface was confirmed by flow cytometry (Greene et al., 2005). However the signaling from these TLRs depends on the expression of adaptor molecules and coreceptors. Primary airway epithelial cells are hyporesponsive to LPS despite expressing TLR4 and this is because of reduced surface expression of co-receptor CD14 and low expression levels of co-stimulatory molecule MD2 (Jia et al., 2004). This may be necessary to restrict TLR4 activation under unstimulated conditions to prevent chronic inflammation of airways that is constantly exposed to inhaled bacteria and endotoxin. On the contrary, LPS was shown to activate TLR4 signaling in small airway and alveolar epithelial cells even though the TLR4 was localized to cytoplasmic compartment (Guillot et al., 2004). More recently John et al attributed chronic colonization of bacteria in CF airways to decreased expression of TLR4 in CF airway epithelial cells (John et al., 2010). TLR2, which is expressed on the apical surface of polarized airway cells is mobilized into an apical lipid raft receptor complex following *P. aeruginosa* infection and initiate signalling (Soong et al., 2004). TLR5 recgonizes flagella of *P. aeruginosa* and *Burkholderia cenocepacia* and activate NF-κB (Adamo et al., 2004; Urban et al., 2004; Zhang et al., 2005). *Haemophilus infIuenzae* traverses polarized airway epithelial cells by interacting with TLR2, which then activates p38 mitogen activated protein (MAP) kinase and TGF-β Signalling(Beisswenger et al., 2007). TLR3 recognizes double stranded (ds)-RNA, an intermediate generated during RNA virus replication and elicits chemokine and type I IFN responses by MyD88- independent signaling mechanism (Gern et al., 2003; Wang et al., 2009). Upon ligation of ds-RNA, TRIF and TRAM (TRIFrelated adaptor molecule) are recruited to TIR domain of TLR3 and TRAM acts as a bridge between TLR and TRIF and this allows activation of TRIF-dependent signaling leading to activation of IRF3 via IKKε/TBK-1 to stimulate IFN production or activation of NF-κB via IKKα/IKKβ to stimulate CXCL-8 expression (Kawai and Akira, 2008). The recognition of double-stranded RNA by TLR3 also increases expression of hBD2 (Duits et al., 2003). Viral or bacterial infection transcriptionally upregulates TLR3 expression (Liu et al., 2007; Sajjan et al., 2006; Wang et al., 2009; Xing et al., 2011), thereby increasing viral induced cytokine and

bacteria infected inflammatory monocytes (Barbalat et al., 2009).

chemokine responses further. Stimulation of TLR2 or TLR3 also induces mucin expression by activating MAP kinases and inducing EGF receptor signaling (Chen et al., 2004; Kohri et al., 2002; Li et al., 1997; Zhu et al., 2009). MUC1, a transmembrane mucin is a negative regulator of TLRs and therefore may play an important role in limiting TLR- induced inflammatory responses (Ueno et al., 2008).

There are conflicting reports with regards to expression of TLRs and their role in innate immune responses in patients with COPD. Airway epithelial cells from patients with severe COPD showed decreased expression of TLR4, but not TLR2 (MacRedmond et al., 2007). In contrast, recently Pace et al observed increased neutrophils and decreased apoptosis of neutrophils in the bronchoalveolar lavage and increased expression of TLR4 in airway epithelium of COPD patients providing evidence that increased TLR4 may contribute to airway neutrophilia in COPD (Pace et al., 2011). Pace et al also demonstrated increased TLR4 expression and concurrent increased CXCL-8 in response to LPS challenge in cigarette smoke exposed airway epithelial cells(Pace et al., 2008), while other investigators showed decreased TLR4 expression which was associated with reduced CXCL-8 and hBD2 production (Kulkarni et al., 2010; MacRedmond et al., 2007). Our preliminary studies involving primary airway epithelial cells from COPD patients suggested heightened expression of CXCL-8 in responses to *P. aeruginosa* infection compared to normal airway epithelial cells (Ganesan and Sajjan, unpublished results). However, role of TLR in this context is yet to be established. Whether TLR4 expression is decreased or increased it has important implications in COPD airway inflammation and obstruction (Figure 2). The decreased expression of TLR4 may lead to decreased innate immune responses and increased persistence of infecting organism. On the other hand increased expression of TLR4 increases neutrophil recruitment and mucus production in response to bacterial or viral infection, thereby leading to increased airways inflammation and obstruction.

#### **3.2 RIG-I like receptors**

Another family of PRRs that play a role in innate defense mechanisms of airway epithelial cells is retinoic acid inducible (RIG)-I like receptors (RLR). This family of PRRs includes RIG-I, MDA-5 (melanoma differentiation associated protein 5) and LGP-2 (Laboratory of genetics and physiology 2). RLRs are the primary sensor molecules for detection of viral RNA in the cytoplasm (Meylan and Tschopp, 2006; Sun et al., 2006). Both RIG-I and MDA-5 contain a caspase recruitment domain (CARD) and a RNA helicase domain (Kang et al., 2002; Yoneyama et al., 2005; Yoneyama et al., 2004). On the other hand, LPG-2 has only RNA helicase domain but not CARD domain, which is required for recruiting adaptor protein MAVS (also known as VISA, Cardiff)(Yoneyama et al., 2005). Therefore recognition of viral RNA by RIG-I and MDA-5 leads to IFN or chemokine response, and LPG-2 suppresses this response (Yoneyama et al., 2005). RIG-I and MDA-5 recognize different RNA species. RIG-I recognizes single stranded (ss)RNA viruses, such as influenza virus, paramyxoviruses and deficiency in RIG-I increases the susceptibility of mice to RNA viruses (Kato et al., 2005). RIG-I specifically binds to the 5'-triphosphate moiety, the signature of which is exposed in the process of viral entry or replication. The host RNA which loses 5'triphosphate moiety during processing is therefore not recognized by RIG-I preventing cytokine and chemokine response due to self-recognition. RIG-I also recognizes short dsRNA (<1 kb) in 5'triphosphate-

Innate Immunity of Airway Epithelium and COPD 29

derivative, γ-D-glutamyl-mesodiaminopimelic acid from gram-negative bacteria (Chamaillard et al., 2003; Girardin et al., 2003a), whereas, NOD2 is considered as a general sensor of PGN through muramyl dipeptide (Girardin et al., 2003b). Upon recognizing PGN, both NOD1 and NOD2 activate NF-κB-mediated proinflammatory response via RIP-2 (Hasegawa et al., 2008). Both NOD1 and NOD2 are highly expressed in immune and inflammatory cells (Fritz et al., 2005; Kanneganti et al., 2007). These two NODs are also expressed in airway epithelium and are induced by bacterial stimuli (Bogefors et al., 2010; Mayer et al., 2007; Opitz et al., 2004; Travassos et al., 2005). NOD1 and NOD2 contribute to innate immune responses to different bacteria including *Pseudomonas aeruginosa*, *Chlamydia pneumonia*, *Haemophilus influenza* and *L. pneumophila* both *in vivo* and *in vitro* (Clarke et al.,

NOD2 not only recognizes bacterial peptidoglycan, but also viral ssRNA. NOD2 deficiency results in impaired type I IFN expression *in vitro* upon stimulation with viral ssRNA (Sabbah et al., 2009). This was dependent on NOD2 interaction with IPS-1 and activation of IRF3, but not on activation of RIP-2. NOD2 deficient mice were also found to be more susceptible to

Pyrin domain containing NLRs are normally called as NLRP. There are 14 members in this NLR subfamily. At least NLRP1-3 form multiprotein complex named "inflammasomes" which consists one or two NLRs, an adaptor molecule ASC (apoptosis-associated speck-like protein containing a CARD), and caspase-1(Martinon et al., 2002). Inflammasomes respond to several PAMPS or DAMPS and regulate caspase-1 mediated cell death called pyroptosis and production of IL-1β and IL-18 at post-transcriptional level. Therefore, unlike other cytokines, IL-1β production requires two signals. Signal I is often provided by TLRs which activates NFκB dependent pro-IL-1β, and signal II comes from inflammasomes, which mediate caspase 1 dependent cleavage of pro-IL-1β to its mature form. The activators of NLRP3 are microbial RNA, bacterial pore forming toxins, certain types of DNA and MDP (Kanneganti et al., 2006; Mariathasan et al., 2006; Martinon et al., 2004; Meixenberger et al., 2010; Muruve et al., 2008). Accordingly, NLRP3 null mice were shown to be susceptible to influenza virus, *Streptococcus pneumoniae* and *K. pneumonia* infection (Kanneganti, 2010; Allen et al., 2009; Ichinohe et al., 2010; Thomas et al., 2009). In addition NLRP3 is also activated by necrotic cells, uric acid metabolites, ATP, biglycan, hyaluronan that might be released after tissue injury (Babelova et al., 2009; Iyer et al., 2009; Mariathasan et al., 2006; Martinon et al., 2006; Yamasaki et al., 2009). In addition to NLRP, NLRC4 (NLR family CARD domain containing) and NAIP5 (NLR family, BIRdomain conaining) also form inflammasomes. While NAIP is expressed in both lung macrophages and epithelial cells, NLRC4 is expressed only in macrophages (Diez et al., 2000; Vinzing et al., 2008). NLRC4 inflammasome recognizes *L. pneumophila* and *P. aeruginosa* flagellin present in the host cytosol, independently of TLR5 (Franchi et al., 2006; Miao et al., 2006). NAIP controls intracellular replication of *L. pneumophila* depending on the

The widely expressed NLRX1 (NLR family member X1) is the only NLR receptor that is localized to mitochondria and it negatively regulates RIG-I and MDA-5 receptors. NLRX-1 mediates production of reactive oxygen species upon bacterial infection (Moore et al., 2008;

infection with respiratory syncytial virus and influenza virus than the wild-type mice.

2010; Frutuoso et al., 2010; Shimada et al., 2009; Zola et al., 2008).

recognition of flagellin (Vinzing et al., 2008).

Tattoli et al., 2008) and decreased dsRNA-stimulated IFN response.

independent manner and induces IFN responses (Kato et al., 2008). On the other hand, MDA-5 recognizes long dsRNA that is >1 kb. Since viruses from picornaviridea family including rhinovirus generate long dsRNA in infected cells, innate immune responses to these viruses depends on recognition of viral RNA by MDA-5 (Kato et al., 2006; Wang et al., 2009). Mice deficient in MDA-5 show increased inflammatory response, delayed IFN response and significantly increased viral load up to 48 h after rhinovirus infection (Wang et al., 2011) . Both RIG-I and MDA-5 uses a common adaptor protein called interferon beta promoter stimulator-1 (IPS-1, also known as MAVS, VISA, CARDIF)(Kawai et al., 2005; Meylan et al., 2005; Seth et al., 2005; Xu et al., 2005). IPS-1 has a CARD domain which is homologous to RIG-I and MDA-5 and has a transmembrane domain at its C-terminal end that spans the mitochondrial membrane (Seth et al., 2005). IPS-1 after binding to RIG-I or MDA-5 through CARD-CARD interaction, activates IRF3 and NF-κB via TBK1/IKKε and RIP-1/IKKα/IKKβ respectively. IPS-1 also interacts with receptor-interacting protein-1 (RIP-1), which is a death domain and is implicated in virus infection-induced IFN expression (Balachandran et al., 2004). However IPS-1 interaction with RIP-1 via the non-CARD region facilitates NF-κB activation, rather than IRF3 activation. Therefore IPS-1 regulates both IRF3 and NF-κB activation upon binding to RIG-I or MDA-5. IPS-1-deficient mice fail to activate IRF3 and NF-κB, with concomitant loss of type I IFN and inflammatory cytokine induction after viral infection and show increased persistence of virus (Kawai and Akira, 2008). Recently, cigarette smoke extract was demonstrated to inhibit RIG-I-stimulated innate immune responses to influenza infection in bronchial organ culture model (Wu et al., 2011). Exposure to cigarette smoke extract also interfered with STAT1 activation by IFN-γ, a type II interferon which stimulates expression of various antiviral proteins (Modestou et al., 2010). Further, cigarette smoke also attenuated the inhibitor effect of IFN-γ on RSV mRNA and protein expression. Eddleston et al demonstrated that exposure of airway epithelial cells to cigarette smoke extract suppressed mRNA induction of CXCL-10 and IFN-β by human rhinovirus and also viral dsRNA mimic polyinosinic:polycytidylic acid (poly I:C) (Eddleston et al., 2011). This was found to be due to decrease in activation of the IFN-STAT-1 and SAP-JNK pathways. Inhibition of antiviral responses, in particular IFN and CXCL-10 responses appear to be due to acute exposure to cigarette smoke that occurs *in vitro*, because the airway epithelial cells obtained from COPD patients showed antiviral responses to rhinovirus infection which was in fact significantly higher than the cells obtained from nonsmokers (Schneider et al., 2010). Similar to our observations, mice exposed to cigarette smoke and poly I:C or influenza virus showed increased IFN responses and this was attributed to pathogenesis of COPD (Kang et al., 2008).

#### **3.3 NOD-like receptors**

Nod-like receptors (NLR) are a family of proteins and sense microbial signatures in the cytosol. There are at least 22 identified NLRs in humans, although only few of them have been functionally characterized. All of them have a central nucleotide binding domain and C-terminal leucin-rich repeat domain, which possibly mediate ligand binding. In addition, they also contain different N-terminal effector domains such as CARD domain, pyrin domains or baculovirus inhibitor repeats and thus activate diverse downstream signaling pathways (Chen et al., 2009; Fritz et al., 2006). The most widely studied among the CARD containing NLRs are NOD1 and NOD2. NOD1 primarily recognizes peptidoglycan (PGN)

independent manner and induces IFN responses (Kato et al., 2008). On the other hand, MDA-5 recognizes long dsRNA that is >1 kb. Since viruses from picornaviridea family including rhinovirus generate long dsRNA in infected cells, innate immune responses to these viruses depends on recognition of viral RNA by MDA-5 (Kato et al., 2006; Wang et al., 2009). Mice deficient in MDA-5 show increased inflammatory response, delayed IFN response and significantly increased viral load up to 48 h after rhinovirus infection (Wang et al., 2011) . Both RIG-I and MDA-5 uses a common adaptor protein called interferon beta promoter stimulator-1 (IPS-1, also known as MAVS, VISA, CARDIF)(Kawai et al., 2005; Meylan et al., 2005; Seth et al., 2005; Xu et al., 2005). IPS-1 has a CARD domain which is homologous to RIG-I and MDA-5 and has a transmembrane domain at its C-terminal end that spans the mitochondrial membrane (Seth et al., 2005). IPS-1 after binding to RIG-I or MDA-5 through CARD-CARD interaction, activates IRF3 and NF-κB via TBK1/IKKε and RIP-1/IKKα/IKKβ respectively. IPS-1 also interacts with receptor-interacting protein-1 (RIP-1), which is a death domain and is implicated in virus infection-induced IFN expression (Balachandran et al., 2004). However IPS-1 interaction with RIP-1 via the non-CARD region facilitates NF-κB activation, rather than IRF3 activation. Therefore IPS-1 regulates both IRF3 and NF-κB activation upon binding to RIG-I or MDA-5. IPS-1-deficient mice fail to activate IRF3 and NF-κB, with concomitant loss of type I IFN and inflammatory cytokine induction after viral infection and show increased persistence of virus (Kawai and Akira, 2008). Recently, cigarette smoke extract was demonstrated to inhibit RIG-I-stimulated innate immune responses to influenza infection in bronchial organ culture model (Wu et al., 2011). Exposure to cigarette smoke extract also interfered with STAT1 activation by IFN-γ, a type II interferon which stimulates expression of various antiviral proteins (Modestou et al., 2010). Further, cigarette smoke also attenuated the inhibitor effect of IFN-γ on RSV mRNA and protein expression. Eddleston et al demonstrated that exposure of airway epithelial cells to cigarette smoke extract suppressed mRNA induction of CXCL-10 and IFN-β by human rhinovirus and also viral dsRNA mimic polyinosinic:polycytidylic acid (poly I:C) (Eddleston et al., 2011). This was found to be due to decrease in activation of the IFN-STAT-1 and SAP-JNK pathways. Inhibition of antiviral responses, in particular IFN and CXCL-10 responses appear to be due to acute exposure to cigarette smoke that occurs *in vitro*, because the airway epithelial cells obtained from COPD patients showed antiviral responses to rhinovirus infection which was in fact significantly higher than the cells obtained from nonsmokers (Schneider et al., 2010). Similar to our observations, mice exposed to cigarette smoke and poly I:C or influenza virus showed increased IFN responses and this was attributed to

Nod-like receptors (NLR) are a family of proteins and sense microbial signatures in the cytosol. There are at least 22 identified NLRs in humans, although only few of them have been functionally characterized. All of them have a central nucleotide binding domain and C-terminal leucin-rich repeat domain, which possibly mediate ligand binding. In addition, they also contain different N-terminal effector domains such as CARD domain, pyrin domains or baculovirus inhibitor repeats and thus activate diverse downstream signaling pathways (Chen et al., 2009; Fritz et al., 2006). The most widely studied among the CARD containing NLRs are NOD1 and NOD2. NOD1 primarily recognizes peptidoglycan (PGN)

pathogenesis of COPD (Kang et al., 2008).

**3.3 NOD-like receptors** 

derivative, γ-D-glutamyl-mesodiaminopimelic acid from gram-negative bacteria (Chamaillard et al., 2003; Girardin et al., 2003a), whereas, NOD2 is considered as a general sensor of PGN through muramyl dipeptide (Girardin et al., 2003b). Upon recognizing PGN, both NOD1 and NOD2 activate NF-κB-mediated proinflammatory response via RIP-2 (Hasegawa et al., 2008). Both NOD1 and NOD2 are highly expressed in immune and inflammatory cells (Fritz et al., 2005; Kanneganti et al., 2007). These two NODs are also expressed in airway epithelium and are induced by bacterial stimuli (Bogefors et al., 2010; Mayer et al., 2007; Opitz et al., 2004; Travassos et al., 2005). NOD1 and NOD2 contribute to innate immune responses to different bacteria including *Pseudomonas aeruginosa*, *Chlamydia pneumonia*, *Haemophilus influenza* and *L. pneumophila* both *in vivo* and *in vitro* (Clarke et al., 2010; Frutuoso et al., 2010; Shimada et al., 2009; Zola et al., 2008).

NOD2 not only recognizes bacterial peptidoglycan, but also viral ssRNA. NOD2 deficiency results in impaired type I IFN expression *in vitro* upon stimulation with viral ssRNA (Sabbah et al., 2009). This was dependent on NOD2 interaction with IPS-1 and activation of IRF3, but not on activation of RIP-2. NOD2 deficient mice were also found to be more susceptible to infection with respiratory syncytial virus and influenza virus than the wild-type mice.

Pyrin domain containing NLRs are normally called as NLRP. There are 14 members in this NLR subfamily. At least NLRP1-3 form multiprotein complex named "inflammasomes" which consists one or two NLRs, an adaptor molecule ASC (apoptosis-associated speck-like protein containing a CARD), and caspase-1(Martinon et al., 2002). Inflammasomes respond to several PAMPS or DAMPS and regulate caspase-1 mediated cell death called pyroptosis and production of IL-1β and IL-18 at post-transcriptional level. Therefore, unlike other cytokines, IL-1β production requires two signals. Signal I is often provided by TLRs which activates NFκB dependent pro-IL-1β, and signal II comes from inflammasomes, which mediate caspase 1 dependent cleavage of pro-IL-1β to its mature form. The activators of NLRP3 are microbial RNA, bacterial pore forming toxins, certain types of DNA and MDP (Kanneganti et al., 2006; Mariathasan et al., 2006; Martinon et al., 2004; Meixenberger et al., 2010; Muruve et al., 2008). Accordingly, NLRP3 null mice were shown to be susceptible to influenza virus, *Streptococcus pneumoniae* and *K. pneumonia* infection (Kanneganti, 2010; Allen et al., 2009; Ichinohe et al., 2010; Thomas et al., 2009). In addition NLRP3 is also activated by necrotic cells, uric acid metabolites, ATP, biglycan, hyaluronan that might be released after tissue injury (Babelova et al., 2009; Iyer et al., 2009; Mariathasan et al., 2006; Martinon et al., 2006; Yamasaki et al., 2009).

In addition to NLRP, NLRC4 (NLR family CARD domain containing) and NAIP5 (NLR family, BIRdomain conaining) also form inflammasomes. While NAIP is expressed in both lung macrophages and epithelial cells, NLRC4 is expressed only in macrophages (Diez et al., 2000; Vinzing et al., 2008). NLRC4 inflammasome recognizes *L. pneumophila* and *P. aeruginosa* flagellin present in the host cytosol, independently of TLR5 (Franchi et al., 2006; Miao et al., 2006). NAIP controls intracellular replication of *L. pneumophila* depending on the recognition of flagellin (Vinzing et al., 2008).

The widely expressed NLRX1 (NLR family member X1) is the only NLR receptor that is localized to mitochondria and it negatively regulates RIG-I and MDA-5 receptors. NLRX-1 mediates production of reactive oxygen species upon bacterial infection (Moore et al., 2008; Tattoli et al., 2008) and decreased dsRNA-stimulated IFN response.

Innate Immunity of Airway Epithelium and COPD 31

Fig. 3. COPD airway epithelial cells are impaired in clearing infecting bacteria. This leads to colonization of bacteria on the apical surface of airway epithelium. Subsequent rhinovirus infection disrupts barrier function and promotes traversal and interaction of bacteria with basolateral receptors leading to exaggerated chemokine response. At the same time COPD airway epithelial cells also show increased generation of reactive oxygen species and attenuated expression of antioxidant enzymes resulting in increased oxidative stress. This in turn suppresses interferon (antiviral) response stimulated by secondary rhinovirus infection. Together this may lead to persistence of bacteria and virus, and increased inflammation.

Impact of secondary viral or bacterial infection in patients colonized with bacteria is being increasingly recognized in recent years. For instance, despite chronic colonization with *P. aeruginosa*, cystic fibrosis patients show exacerbations periodically and some incidences are associated with acquiring secondary viral or bacterial infections (Ong et al., 1989; Ramsey et al., 1989; Wat et al., 2008). Similarly, in COPD patients who are chronically colonized with NTHi, exacerbations were associated with acquisition of new strain of NTHi, other species of bacteria or respiratory virus (Murphy, 2000; Murphy et al., 2008; Murphy et al., 2007; Papi et al., 2006; Sykes et al., 2007; Wilson, 2000). Recently, we showed that secondary bacterial infection in primary cystic fibrosis airway epithelial cells preinfected with *P. aeruginosa* increases C-X-C chemokine responses by increasing the load of planktonic bacteria which are more pro-inflammatory than their counterpart biofilm bacteria and also increased paracellular invasion of bacteria in differentiated airway epithelial cells (Chattoraj et al., 2011b). We also demonstrated that cystic fibrosis, but not normal airway epithelial cells infected with bacteria show suppressed type I IFN response to subsequent rhinovirus infection (Chattoraj et al., 2011a). This was due to increased oxidative stress in cystic fibrosis airway epithelial cells. Airway epithelial cells from COPD patients show increased oxidative stress similar to cystic fibrosis patients. Therefore we expect that bacterial preinfection may suppress innate immune responses to subsequent virus infection in COPD cells. Consistent with this notion, our preliminary studies indicate that infection with *P. aeruginosa* or NTHi infection increases oxidative stress further and decreases expression of antioxidant genes in COPD airway epithelial cells. In addition, we also observed suppression of IFN response in COPD airway epithelial cells infected with bacteria to subsequent rhinovirus infection (unpublished observations). Similar to our observations, LPS treatment was demonstrated

Rhinovirus

Increased Chemokines and reactive oxygen

species

Persistent bacteria

Decreased Interferons and antioxidant enzymes

Although, there is no evidence that NLRs play a role in innate immune responses to bacterial or viral infection in COPD so far, the emerging literature indicate inflammasome forming NLRs may contribute to COPD pathogenesis. Inhaled cigarette smoke, oxidative stress, necrotic cell death, hypoxia, hypercapnia may cause tissue injury and release of DAMPs (uric acid, ATP) and this in turn activates NLRP3 inflammasome (Wanderer, 2008). Consistent with this notion, uric acid concentration was increased in the bronchoalveolar lavage of COPD patients (Wanderer, 2008). COPD patients also had significantly increased amounts of IL-1β and this correlated with severity of the disease(Sapey et al., 2009). Mice exposed to cigarette smoke also showed increased IL-1β in their lungs (Doz et al., 2008) and finally mice overexpressing mature IL-1β in epithelial cells showed typical feature of COPD including emphysema, lung inflammation with increased neutrophils and macrophages and airway fibrosis (Lappalainen et al., 2005). ASC (inflammasome adaptor protein) null mice showed attenuated inflammation after exposing to elastase and less uric acid. Elastaseinduced inflammation was significantly reduced in wild-type mice treated with uricase or treated with IL-1R antagonist (Couillin et al., 2009). All these evidences suggest contribution of inflammasome forming NLRP3 to COPD pathogenesis.

#### **4. Innate immunity and co-infections**

Nontypeable *H. influenzae* (NTHi), *S. Pneumoniae* and *P. aeruginosa* are detectable in lower airways of appproximatley 25 to 50% of clinically stable COPD patients (Sethi and Murphy, 2008). Chronic colonization can alter the responses of airway epithelial cells and other innate and adaptive immune cells to subsequent viral or bacterial infections leading to increased severity of disease. Exacerbations due to concurrent or sequential infections was shown to be associated with increased severity of disease at least in one-quarter of COPD patient population (Papi et al., 2006; Sethi et al., 2006; Wilkinson et al., 2006). Risk of secondary bacterial infection following a viral infection dates back to 19th century, when cases of pneumonia correlated with influenza (flu) epidemic (McCullers, 2006). Influenza infection increases risk of secondary bacterial infection by increasing binding or invasion of bacterial pathogen to airway epithelial cells, desensitizing innate immune receptors such as TLRs, and causing immunosuppression by increasing glucocorticosteriod expression (Beadling and Slifka, 2004; Hament et al., 1999; Jamieson et al., 2010; McCullers, 2006; Seki et al., 2004; Sun and Metzger, 2008). Respiratory syncytial virus infection increased persistence of *P. aeruginosa* in mice and increased *P. aeruginosa* and NTHi binding to airway epithelial cells (de Vrankrijker et al., 2009; Jiang et al., 1999; Van Ewijk et al., 2007). Respiratory syncytial virus also increased persistence of NTHi by dysregulating the expression of β-defensin in chinchilla model of respiratory infection (McGillivary et al., 2009). Rhinovirus which causes common cold, in combination with *S. pnuemoniae* was associated with severe cases of community-acquired pneumonia in children (Honkinen et al., 2011). Various *in vitro* studies showed that rhinoviruses also increase bacterial binding to airway epithelial cells by increasing the expression of bacterial receptors on airway epithelial cells or by facilitating invasion of cells by bacteria (Ishizuka et al., 2003; Passariello et al., 2006). We demonstrated that rhinovirus infection also increases paracellular permeability and promote bacterial traversal across mucociliary- differentiated airway epithelium (Sajjan et al., 2008). Rhinovirus infection also decreases bacterial PAMPS-induced proinflammatory response by desensitizing TLRs (Oliver et al., 2008).

Although, there is no evidence that NLRs play a role in innate immune responses to bacterial or viral infection in COPD so far, the emerging literature indicate inflammasome forming NLRs may contribute to COPD pathogenesis. Inhaled cigarette smoke, oxidative stress, necrotic cell death, hypoxia, hypercapnia may cause tissue injury and release of DAMPs (uric acid, ATP) and this in turn activates NLRP3 inflammasome (Wanderer, 2008). Consistent with this notion, uric acid concentration was increased in the bronchoalveolar lavage of COPD patients (Wanderer, 2008). COPD patients also had significantly increased amounts of IL-1β and this correlated with severity of the disease(Sapey et al., 2009). Mice exposed to cigarette smoke also showed increased IL-1β in their lungs (Doz et al., 2008) and finally mice overexpressing mature IL-1β in epithelial cells showed typical feature of COPD including emphysema, lung inflammation with increased neutrophils and macrophages and airway fibrosis (Lappalainen et al., 2005). ASC (inflammasome adaptor protein) null mice showed attenuated inflammation after exposing to elastase and less uric acid. Elastaseinduced inflammation was significantly reduced in wild-type mice treated with uricase or treated with IL-1R antagonist (Couillin et al., 2009). All these evidences suggest contribution

Nontypeable *H. influenzae* (NTHi), *S. Pneumoniae* and *P. aeruginosa* are detectable in lower airways of appproximatley 25 to 50% of clinically stable COPD patients (Sethi and Murphy, 2008). Chronic colonization can alter the responses of airway epithelial cells and other innate and adaptive immune cells to subsequent viral or bacterial infections leading to increased severity of disease. Exacerbations due to concurrent or sequential infections was shown to be associated with increased severity of disease at least in one-quarter of COPD patient population (Papi et al., 2006; Sethi et al., 2006; Wilkinson et al., 2006). Risk of secondary bacterial infection following a viral infection dates back to 19th century, when cases of pneumonia correlated with influenza (flu) epidemic (McCullers, 2006). Influenza infection increases risk of secondary bacterial infection by increasing binding or invasion of bacterial pathogen to airway epithelial cells, desensitizing innate immune receptors such as TLRs, and causing immunosuppression by increasing glucocorticosteriod expression (Beadling and Slifka, 2004; Hament et al., 1999; Jamieson et al., 2010; McCullers, 2006; Seki et al., 2004; Sun and Metzger, 2008). Respiratory syncytial virus infection increased persistence of *P. aeruginosa* in mice and increased *P. aeruginosa* and NTHi binding to airway epithelial cells (de Vrankrijker et al., 2009; Jiang et al., 1999; Van Ewijk et al., 2007). Respiratory syncytial virus also increased persistence of NTHi by dysregulating the expression of β-defensin in chinchilla model of respiratory infection (McGillivary et al., 2009). Rhinovirus which causes common cold, in combination with *S. pnuemoniae* was associated with severe cases of community-acquired pneumonia in children (Honkinen et al., 2011). Various *in vitro* studies showed that rhinoviruses also increase bacterial binding to airway epithelial cells by increasing the expression of bacterial receptors on airway epithelial cells or by facilitating invasion of cells by bacteria (Ishizuka et al., 2003; Passariello et al., 2006). We demonstrated that rhinovirus infection also increases paracellular permeability and promote bacterial traversal across mucociliary- differentiated airway epithelium (Sajjan et al., 2008). Rhinovirus infection also decreases bacterial PAMPS-induced proinflammatory response by

of inflammasome forming NLRP3 to COPD pathogenesis.

**4. Innate immunity and co-infections** 

desensitizing TLRs (Oliver et al., 2008).

Fig. 3. COPD airway epithelial cells are impaired in clearing infecting bacteria. This leads to colonization of bacteria on the apical surface of airway epithelium. Subsequent rhinovirus infection disrupts barrier function and promotes traversal and interaction of bacteria with basolateral receptors leading to exaggerated chemokine response. At the same time COPD airway epithelial cells also show increased generation of reactive oxygen species and attenuated expression of antioxidant enzymes resulting in increased oxidative stress. This in turn suppresses interferon (antiviral) response stimulated by secondary rhinovirus infection. Together this may lead to persistence of bacteria and virus, and increased inflammation.

Impact of secondary viral or bacterial infection in patients colonized with bacteria is being increasingly recognized in recent years. For instance, despite chronic colonization with *P. aeruginosa*, cystic fibrosis patients show exacerbations periodically and some incidences are associated with acquiring secondary viral or bacterial infections (Ong et al., 1989; Ramsey et al., 1989; Wat et al., 2008). Similarly, in COPD patients who are chronically colonized with NTHi, exacerbations were associated with acquisition of new strain of NTHi, other species of bacteria or respiratory virus (Murphy, 2000; Murphy et al., 2008; Murphy et al., 2007; Papi et al., 2006; Sykes et al., 2007; Wilson, 2000). Recently, we showed that secondary bacterial infection in primary cystic fibrosis airway epithelial cells preinfected with *P. aeruginosa* increases C-X-C chemokine responses by increasing the load of planktonic bacteria which are more pro-inflammatory than their counterpart biofilm bacteria and also increased paracellular invasion of bacteria in differentiated airway epithelial cells (Chattoraj et al., 2011b). We also demonstrated that cystic fibrosis, but not normal airway epithelial cells infected with bacteria show suppressed type I IFN response to subsequent rhinovirus infection (Chattoraj et al., 2011a). This was due to increased oxidative stress in cystic fibrosis airway epithelial cells. Airway epithelial cells from COPD patients show increased oxidative stress similar to cystic fibrosis patients. Therefore we expect that bacterial preinfection may suppress innate immune responses to subsequent virus infection in COPD cells. Consistent with this notion, our preliminary studies indicate that infection with *P. aeruginosa* or NTHi infection increases oxidative stress further and decreases expression of antioxidant genes in COPD airway epithelial cells. In addition, we also observed suppression of IFN response in COPD airway epithelial cells infected with bacteria to subsequent rhinovirus infection (unpublished observations). Similar to our observations, LPS treatment was demonstrated

Innate Immunity of Airway Epithelium and COPD 33

This work was supported by NIH AT4793 and HL897720 to U.S. We thank Marisa Lynn for assisting with processing cell cultures for histology and Adam Comstock for his assistance

Adachi, O., Kawai, T., Takeda, K., Matsumoto, M., Tsutsui, H., Sakagami, M., Nakanishi, K.,

Adamo, R., Sokol, S., Soong, G., Gomez, M.I., and Prince, A. (2004). Pseudomonas

Akira, S., Takeda, K., and Kaisho, T. (2001). Toll-like receptors: critical proteins linking

Alexopoulou, L., Holt, A.C., Medzhitov, R., and Flavell, R.A. (2001). Recognition of double-

Aliprantis, A.O., Yang, R.B., Mark, M.R., Suggett, S., Devaux, B., Radolf, J.D., Klimpel, G.R.,

Allen, I.C., Scull, M.A., Moore, C.B., Holl, E.K., McElvania-TeKippe, E., Taxman, D.J.,

Araya, J., Cambier, S., Markovics, J.A., Wolters, P., Jablons, D., Hill, A., Finkbeiner, W.,

Araya, J., Cambier, S., Morris, A., Finkbeiner, W., and Nishimura, S.L. (2006). Integrin-

pulmonary epithelial-mesenchymal trophic unit. Am J Pathol *169*, 405-415. Babelova, A., Moreth, K., Tsalastra-Greul, W., Zeng-Brouwers, J., Eickelberg, O., Young,

Baginski, T.K., Dabbagh, K., Satjawatcharaphong, C., and Swinney, D.C. (2006). Cigarette

Balachandran, S., Thomas, E., and Barber, G.N. (2004). A FADD-dependent innate immune

Balda, M.S., and Matter, K. (2009). Tight junctions and the regulation of gene expression.

and lung cancer: new molecular insights. Respiration *81*, 265-284.

innate and acquired immunity. Nat Immunol *2*, 675-680.

lipoproteins through toll-like receptor-2. Science *285*, 736-739.

and IL-18-mediated function. Immunity *9*, 143-150.

and Akira, S. (1998). Targeted disruption of the MyD88 gene results in loss of IL-1-

aeruginosa flagella activate airway epithelial cells through asialoGM1 and toll-like receptor 2 as well as toll-like receptor 5. Am J Respir Cell Mol Biol *30*, 627-634. Adcock, I.M., Caramori, G., and Barnes, P.J. (2011). Chronic obstructive pulmonary disease

stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature *413*,

Godowski, P., and Zychlinsky, A. (1999). Cell activation and apoptosis by bacterial

Guthrie, E.H., Pickles, R.J., and Ting, J.P. (2009). The NLRP3 inflammasome mediates in vivo innate immunity to influenza A virus through recognition of viral

Jones, K., Broaddus, V.C., Sheppard, D.*, et al.* (2007). Squamous metaplasia amplifies pathologic epithelial-mesenchymal interactions in COPD patients. J Clin

mediated transforming growth factor-beta activation regulates homeostasis of the

M.F., Bruckner, P., Pfeilschifter, J., Schaefer, R.M., Grone, H.J.*, et al.* (2009). Biglycan, a danger signal that activates the NLRP3 inflammasome via toll-like and P2X

smoke synergistically enhances respiratory mucin induction by proinflammatory

**6. Acknowledgements** 

732-738.

RNA. Immunity *30*, 556-565.

receptors. J Biol Chem *284*, 24035-24048.

Biochim Biophys Acta *1788*, 761-767.

stimuli. Am J Respir Cell Mol Biol *35*, 165-174.

mechanism in mammalian cells. Nature *432*, 401-405.

Invest *117*, 3551-3562.

**7. References** 

in culturing primary epithelial cells.

to suppress IFN-β production in response to dsRNA in mice as well as in monocytes and macrophages (Piao et al., 2009; Sly et al., 2009). This was due to increased expression of SHIP, a MPA kinase phosphatase in LPS treated monocytes. In airway epithelial cells however, *P. aeruginosa* infection induced suppression of IFN response to rhinovirus infection was not due to increased expression of SHIP, but rather due to decreased Akt phosphorylation (Chattoraj et al 2011) which is required for maximal activation of IRF3 (Dong et al., 2008; Sarkar et al., 2004). Previously, we have shown that expression of IFN response to rhinovirus infection requires activation of IRF3 in airway epithelial cells (Wang et al., 2009). Based on these experimental evidences, it is possible that 30% of COPD patients who are chronically colonized with *NTHi* or *P. aeruginosa* in their lower airways may show suppressed antiviral responses and increased chemokine expression (Figure 3). This may lead to increased lung inflammation and progression of lung disease in COPD patients following exacerbation due to co-infections.

#### **5. Conclusion**

The airway epithelium contributes significantly to innate immune system in the lungs. It acts as a physical barrier that protects against inhaled substances and pathogens. Airway epithelial cells also express plethora of innate immune receptors which recognizes both PAMPS and DAMPS and stimulate appropriate responses to either clear the infecting organism and to repair of injured epithelium. However in COPD, chronic exposure to cigarette smoke or environmental hazards causes airway remodeling and also modulate innate immune responses of airway epithelial cells to infection (Figure 4). This results in impaired clearance of infecting organisms and aberrant cytokine and growth factor expression and increased lung inflammation leading to progression of lung disease.

Fig. 4. A schematic representation depecting the combined effects of cigarette smoke or other environmental hazards and bacterial infection on the progression of lung disease in COPD

### **6. Acknowledgements**

This work was supported by NIH AT4793 and HL897720 to U.S. We thank Marisa Lynn for assisting with processing cell cultures for histology and Adam Comstock for his assistance in culturing primary epithelial cells.

#### **7. References**

32 Emphysema

to suppress IFN-β production in response to dsRNA in mice as well as in monocytes and macrophages (Piao et al., 2009; Sly et al., 2009). This was due to increased expression of SHIP, a MPA kinase phosphatase in LPS treated monocytes. In airway epithelial cells however, *P. aeruginosa* infection induced suppression of IFN response to rhinovirus infection was not due to increased expression of SHIP, but rather due to decreased Akt phosphorylation (Chattoraj et al 2011) which is required for maximal activation of IRF3 (Dong et al., 2008; Sarkar et al., 2004). Previously, we have shown that expression of IFN response to rhinovirus infection requires activation of IRF3 in airway epithelial cells (Wang et al., 2009). Based on these experimental evidences, it is possible that 30% of COPD patients who are chronically colonized with *NTHi* or *P. aeruginosa* in their lower airways may show suppressed antiviral responses and increased chemokine expression (Figure 3). This may lead to increased lung inflammation and progression of lung disease in COPD patients

The airway epithelium contributes significantly to innate immune system in the lungs. It acts as a physical barrier that protects against inhaled substances and pathogens. Airway epithelial cells also express plethora of innate immune receptors which recognizes both PAMPS and DAMPS and stimulate appropriate responses to either clear the infecting organism and to repair of injured epithelium. However in COPD, chronic exposure to cigarette smoke or environmental hazards causes airway remodeling and also modulate innate immune responses of airway epithelial cells to infection (Figure 4). This results in impaired clearance of infecting organisms and aberrant cytokine and growth factor

expression and increased lung inflammation leading to progression of lung disease.

Normal airway epithelia

Increased mucus Altered expression of PRRs

Infection with bacteria /virus

Persistence of bacteria or virus Aberrant cytokine and growth factor expression

Fig. 4. A schematic representation depecting the combined effects of cigarette smoke or other environmental hazards and bacterial infection on the progression of lung disease in COPD

production Increased levels of pro-inflammatory factors

Repeated exposure to cigarette smoke or environmental hazards

> Progression of lung disease

Decreased barrier function Decreased antimicrobial factors

following exacerbation due to co-infections.

**5. Conclusion** 


Innate Immunity of Airway Epithelium and COPD 35

Chen, G., Shaw, M.H., Kim, Y.G., and Nunez, G. (2009). NOD-like receptors: role in innate

Chen, R., Lim, J.H., Jono, H., Gu, X.X., Kim, Y.S., Basbaum, C.B., Murphy, T.F., and Li, J.D.

Chen, Y.T., Gallup, M., Nikulina, K., Lazarev, S., Zlock, L., Finkbeiner, W., and McNamara,

Choi, W.I., Syrkina, O., Kwon, K.Y., Quinn, D.A., and Hales, C.A. (2010). JNK activation is

Clarke, T.B., Davis, K.M., Lysenko, E.S., Zhou, A.Y., Yu, Y., and Weiser, J.N. (2010).

Cohen, N.A., Zhang, S., Sharp, D.B., Tamashiro, E., Chen, B., Sorscher, E.J., and Woodworth,

Cosio, M., Ghezzo, H., Hogg, J.C., Corbin, R., Loveland, M., Dosman, J., and Macklem, P.T.

Couillin, I., Vasseur, V., Charron, S., Gasse, P., Tavernier, M., Guillet, J., Lagente, V., Fick, L.,

de Boer, W.I., van Schadewijk, A., Sont, J.K., Sharma, H.S., Stolk, J., Hiemstra, P.S., and van

de Vrankrijker, A.M., Wolfs, T.F., Ciofu, O., Hoiby, N., van der Ent, C.K., Poulsen, S.S., and

colonization of Pseudomonas aeruginosa in mice. J Med Virol *81*, 2096-2103. Diamond, G., Legarda, D., and Ryan, L.K. (2000). The innate immune response of the

Diebold, S.S., Kaisho, T., Hemmi, H., Akira, S., and Reis e Sousa, C. (2004). Innate antiviral

Diez, E., Yaraghi, Z., MacKenzie, A., and Gros, P. (2000). The neuronal apoptosis inhibitory

(2004). Nontypeable Haemophilus influenzae lipoprotein P6 induces MUC5AC mucin transcription via TLR2-TAK1-dependent p38 MAPK-AP1 and IKKbeta-IkappaBalpha-NF-kappaB signaling pathways. Biochem Biophys Res Commun *324*,

N. (2010). Cigarette smoke induces epidermal growth factor receptor-dependent redistribution of apical MUC1 and junctional beta-catenin in polarized human

responsible for mucus overproduction in smoke inhalation injury. Respir Res *11*, 172.

Recognition of peptidoglycan from the microbiota by Nod1 enhances systemic

B.A. (2009). Cigarette smoke condensate inhibits transepithelial chloride transport

(1978). The relations between structural changes in small airways and pulmonary-

Jacobs, M., Coelho, F.R.*, et al.* (2009). IL-1R1/MyD88 signaling is critical for elastase-induced lung inflammation and emphysema. J Immunol *183*, 8195-8202. Curran, D.R., and Cohn, L. (2010). Advances in mucous cell metaplasia: a plug for mucus as a therapeutic focus in chronic airway disease. Am J Respir Cell Mol Biol *42*, 268-275. Dauletbaev, N., Gropp, R., Frye, M., Loitsch, S., Wagner, T.O., and Bargon, J. (2002).

Expression of human beta defensin (HBD-1 and HBD-2) mRNA in nasal epithelia of adult cystic fibrosis patients, healthy individuals, and individuals with acute

Krieken, J.H. (1998). Transforming growth factor beta1 and recruitment of macrophages and mast cells in airways in chronic obstructive pulmonary disease.

Johansen, H.K. (2009). Respiratory syncytial virus infection facilitates acute

responses by means of TLR7-mediated recognition of single-stranded RNA. Science

protein (Naip) is expressed in macrophages and is modulated after phagocytosis

immunity and inflammatory disease. Annu Rev Pathol *4*, 365-398.

airway epithelial cells. Am J Pathol *177*, 1255-1264.

and ciliary beat frequency. Laryngoscope *119*, 2269-2274.

innate immunity. Nat Med *16*, 228-231.

function tests. N Engl J Med *298*, 1277-1281.

Am J Respir Crit Care Med *158*, 1951-1957.

respiratory epithelium. Immunol Rev *173*, 27-38.

cold. Respiration *69*, 46-51.

*303*, 1529-1531.

1087-1094.


Bals, R., and Hiemstra, P.S. (2004). Innate immunity in the lung: how epithelial cells fight

Bals, R., Wang, X., Zasloff, M., and Wilson, J.M. (1998). The peptide antibiotic LL-37/hCAP-

Barbieri, S.S., Ruggiero, L., Tremoli, E., and Weksler, B.B. (2008). Suppressing PTEN activity

Beadling, C., and Slifka, M.K. (2004). How do viral infections predispose patients to bacterial

Becker, M.N., Diamond, G., Verghese, M.W., and Randell, S.H. (2000). CD14-dependent

Beisswenger, C., Coyne, C.B., Shchepetov, M., and Weiser, J.N. (2007). Role of p38 MAP

Bhowmik, A., Chahal, K., Austin, G., and Chakravorty, I. (2009). Improving mucociliary clearance in chronic obstructive pulmonary disease. Respir Med *103*, 496-502. Bogefors, J., Rydberg, C., Uddman, R., Fransson, M., Mansson, A., Benson, M., Adner, M.,

Borchers, M.T., Carty, M.P., and Leikauf, G.D. (1999). Regulation of human airway mucins

Boucher, R.C., Johnson, J., Inoue, S., Hulbert, W., and Hogg, J.C. (1980). The effect of cigarette smoke on the permeability of guinea pig airways. Lab Invest *43*, 94-100. Casalino-Matsuda, S.M., Monzon, M.E., Day, A.J., and Forteza, R.M. (2009). Hyaluronan

Chamaillard, M., Hashimoto, M., Horie, Y., Masumoto, J., Qiu, S., Saab, L., Ogura, Y.,

Chattoraj, S.S., Ganesan, S., Faris, A., Comstock, A., Lee, W.M., and Sajjan, U.S. (2011a).

Chattoraj, S.S., Ganesan, S., Jones, A.M., Helm, J.M., Comstock, A.T., Bright-Thomas, R.,

of invasive bacterial pathogens. J Biol Chem *282*, 28700-28708.

treatment of allergic rhinitis? Allergy *65*, 1222-1226.

airway epithelium. Am J Respir Cell Mol Biol *40*, 277-285.

cystic fibrosis airway epithelial cells. Thorax *66*, 333-339.

activity at the airway surface. Proc Natl Acad Sci U S A *95*, 9541-9546. Barbalat, R., Lau, L., Locksley, R.M., and Barton, G.M. (2009). Toll-like receptor 2 on

18 is expressed in epithelia of the human lung where it has broad antimicrobial

inflammatory monocytes induces type I interferon in response to viral but not

by tobacco smoke plus interleukin-1beta modulates dissociation of VEcadherin/beta-catenin complexes in endothelium. Arterioscler Thromb Vasc Biol

lipopolysaccharide-induced beta-defensin-2 expression in human tracheobronchial

kinase and transforming growth factor-beta signaling in transepithelial migration

and Cardell, L.O. (2010). Nod1, Nod2 and Nalp3 receptors, new potential targets in

by acrolein and inflammatory mediators. The American journal of physiology *276*,

fragments/CD44 mediate oxidative stress-induced MUC5B up-regulation in

Kawasaki, A., Fukase, K., Kusumoto, S.*, et al.* (2003). An essential role for NOD1 in host recognition of bacterial peptidoglycan containing diaminopimelic acid. Nat

Pseudomonas aeruginosa suppresses interferon response to rhinovirus infection in Cystic fibrosis, but not in normal bronchial epithelial cells. Infect Immun 79, 4131-

LiPuma, J.J., Hershenson, M.B., and Sajjan, U.S. (2011b). Rhinovirus infection liberates planktonic bacteria from biofilm and increases chemokine responses in

against respiratory pathogens. Eur Respir J *23*, 327-333.

bacterial ligands. Nat Immunol *10*, 1200-1207.

infections? Curr Opin Infect Dis *17*, 185-191.

epithelium. J Biol Chem *275*, 29731-29736.

*28*, 732-738.

L549-555.

4145.

Immunol *4*, 702-707.


Innate Immunity of Airway Epithelium and COPD 37

Gern, J.E., French, D.A., Grindle, K.A., Brockman-Schneider, R.A., Konno, S., and Busse,

Girardin, S.E., Boneca, I.G., Carneiro, L.A., Antignac, A., Jehanno, M., Viala, J., Tedin, K.,

Goldman, M.J., Anderson, G.M., Stolzenberg, E.D., Kari, U.P., Zasloff, M., and Wilson, J.M.

Greene, C.M., Carroll, T.P., Smith, S.G., Taggart, C.C., Devaney, J., Griffin, S., O'Neill S, J.,

Guillot, L., Medjane, S., Le-Barillec, K., Balloy, V., Danel, C., Chignard, M., and Si-Tahar, M.

intracellular compartmentalization of TLR4. J Biol Chem *279*, 2712-2718. Hament, J.M., Kimpen, J.L., Fleer, A., and Wolfs, T.F. (1999). Respiratory viral infection

Hammad, H., and Lambrecht, B.N. (2011). Dendritic cells and airway epithelial cells at the interface between innate and adaptive immune responses. Allergy *66*, 579-587. Hartsock, A., and Nelson, W.J. (2008). Adherens and tight junctions: structure, function and connections to the actin cytoskeleton. Biochim Biophys Acta *1778*, 660-669. Hasegawa, M., Fujimoto, Y., Lucas, P.C., Nakano, H., Fukase, K., Nunez, G., and Inohara, N.

Haswell, L.E., Hewitt, K., Thorne, D., Richter, A., and Gaca, M.D. (2010). Cigarette smoke

bacterial flagellin is mediated by Toll-like receptor 5. Nature *410*, 1099-1103. Heil, F., Hemmi, H., Hochrein, H., Ampenberger, F., Kirschning, C., Akira, S., Lipford, G.,

Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo, H., Matsumoto, M., Hoshino,

Herr, C., Beisswenger, C., Hess, C., Kandler, K., Suttorp, N., Welte, T., Schroeder, J.M., and

potential model of goblet cell hyperplasia. Toxicol In Vitro *24*, 981-987. Hayashi, F., Smith, K.D., Ozinsky, A., Hawn, T.R., Yi, E.C., Goodlett, D.R., Eng, J.K., Akira,

RNA via toll-like receptor 7 and 8. Science *303*, 1526-1529.

by bronchial epithelial cells. Am J Respir Cell Mol Biol *28*, 731-737.

muramyl dipeptide (MDP) detection. J Biol Chem *278*, 8869-8872.

cystic fibrosis airway epithelial cells. J Immunol *174*, 1638-1646.

inactivated in cystic fibrosis. Cell *88*, 553-560.

39085-39093.

*26*, 189-195.

activation. EMBO J *27*, 373-383.

DNA. Nature *408*, 740-745.

Thorax *64*, 144-149.

production in lung cells requires oxygen radicals AP-1 and JNK. J Biol Chem *279*,

W.W. (2003). Double-stranded RNA induces the synthesis of specific chemokines

Taha, M.K., Labigne, A., Zahringer, U.*, et al.* (2003a). Nod1 detects a unique muropeptide from gram-negative bacterial peptidoglycan. Science *300*, 1584-1587. Girardin, S.E., Boneca, I.G., Viala, J., Chamaillard, M., Labigne, A., Thomas, G., Philpott, D.J.,

and Sansonetti, P.J. (2003b). Nod2 is a general sensor of peptidoglycan through

(1997). Human beta-defensin-1 is a salt-sensitive antibiotic in lung that is

and McElvaney, N.G. (2005). TLR-induced inflammation in cystic fibrosis and non-

(2004). Response of human pulmonary epithelial cells to lipopolysaccharide involves Toll-like receptor 4 (TLR4)-dependent signaling pathways: evidence for an

predisposing for bacterial disease: a concise review. FEMS Immunol Med Microbiol

(2008). A critical role of RICK/RIP2 polyubiquitination in Nod-induced NF-kappaB

total particulate matter increases mucous secreting cell numbers in vitro: a

S., Underhill, D.M., and Aderem, A. (2001). The innate immune response to

Wagner, H., and Bauer, S. (2004). Species-specific recognition of single-stranded

K., Wagner, H., Takeda, K.*, et al.* (2000). A Toll-like receptor recognizes bacterial

Vogelmeier, C. (2009). Suppression of pulmonary innate host defence in smokers.

and during intracellular infection with Legionella pneumophila. J Immunol *164*, 1470-1477.


Dohrman, A., Miyata, S., Gallup, M., Li, J.D., Chapelin, C., Coste, A., Escudier, E., Nadel, J.,

Doz, E., Noulin, N., Boichot, E., Guenon, I., Fick, L., Le Bert, M., Lagente, V., Ryffel, B.,

Duits, L.A., Nibbering, P.H., van Strijen, E., Vos, J.B., Mannesse-Lazeroms, S.P., van

Eddleston, J., Lee, R.U., Doerner, A.M., Herschbach, J., and Zuraw, B.L. (2011). Cigarette

Ellison, R.T., 3rd, and Giehl, T.J. (1991). Killing of gram-negative bacteria by lactoferrin and

Franchi, L., Amer, A., Body-Malapel, M., Kanneganti, T.D., Ozoren, N., Jagirdar, R., Inohara,

Fritz, J.H., Ferrero, R.L., Philpott, D.J., and Girardin, S.E. (2006). Nod-like proteins in

Fritz, J.H., Girardin, S.E., Fitting, C., Werts, C., Mengin-Lecreulx, D., Caroff, M., Cavaillon,

Frutuoso, M.S., Hori, J.I., Pereira, M.S., Junior, D.S., Sonego, F., Kobayashi, K.S., Flavell,

Gangl, K., Reininger, R., Bernhard, D., Campana, R., Pree, I., Reisinger, J., Kneidinger, M.,

Ganz, T. (2003). Defensins: antimicrobial peptides of innate immunity. Nat Rev Immunol *3*,

Gensch, E., Gallup, M., Sucher, A., Li, D., Gebremichael, A., Lemjabbar, H., Mengistab, A.,

allergen penetration across respiratory epithelium. Allergy *64*, 398-405. Ganz, T. (2002). Antimicrobial polypeptides in host defense of the respiratory tract. J Clin

immunity, inflammation and disease. Nat Immunol *7*, 1250-1257.

1470-1477.

*45*, 3025-3035.

Immunol *180*, 1169-1178.

Immunol Med Microbiol *38*, 59-64.

Respir Cell Mol Biol *44*, 118-126.

lysozyme. J Clin Invest *88*, 1080-1091.

macrophages. Nat Immunol *7*, 576-582.

Invest *109*, 693-697.

710-720.

activating agonists. Eur J Immunol *35*, 2459-2470.

with Legionella pneumophila. Microbes Infect *12*, 819-827.

and during intracellular infection with Legionella pneumophila. J Immunol *164*,

and Basbaum, C. (1998). Mucin gene (MUC 2 and MUC 5AC) upregulation by Gram-positive and Gram-negative bacteria. Biochim Biophys Acta *1406*, 251-259. Dong, L.W., Kong, X.N., Yan, H.X., Yu, L.X., Chen, L., Yang, W., Liu, Q., Huang, D.D., Wu,

M.C., and Wang, H.Y. (2008). Signal regulatory protein alpha negatively regulates both TLR3 and cytoplasmic pathways in type I interferon induction. Mol Immunol

Schnyder, B., Quesniaux, V.F.*, et al.* (2008). Cigarette smoke-induced pulmonary inflammation is TLR4/MyD88 and IL-1R1/MyD88 signaling dependent. J

Sterkenburg, M.A., and Hiemstra, P.S. (2003). Rhinovirus increases human betadefensin-2 and -3 mRNA expression in cultured bronchial epithelial cells. FEMS

smoke decreases innate responses of epithelial cells to rhinovirus infection. Am J

N., Vandenabeele, P., Bertin, J., Coyle, A.*, et al.* (2006). Cytosolic flagellin requires Ipaf for activation of caspase-1 and interleukin 1beta in salmonella-infected

J.M., Philpott, D.J., and Adib-Conquy, M. (2005). Synergistic stimulation of human monocytes and dendritic cells by Toll-like receptor 4 and NOD1- and NOD2-

R.A., Cunha, F.Q., and Zamboni, D.S. (2010). The pattern recognition receptors Nod1 and Nod2 account for neutrophil recruitment to the lungs of mice infected

Kundi, M., Dolznig, H., Thurnher, D.*, et al.* (2009). Cigarette smoke facilitates

Dasari, V., Hotchkiss, J., Harkema, J.*, et al.* (2004). Tobacco smoke control of mucin

production in lung cells requires oxygen radicals AP-1 and JNK. J Biol Chem *279*, 39085-39093.


Innate Immunity of Airway Epithelium and COPD 39

John, G., Yildirim, A.O., Rubin, B.K., Gruenert, D.C., and Henke, M.O. (2010). TLR-4-

Kagan, J.C., Su, T., Horng, T., Chow, A., Akira, S., and Medzhitov, R. (2008). TRAM couples

Kang, D.C., Gopalkrishnan, R.V., Wu, Q., Jankowsky, E., Pyle, A.M., and Fisher, P.B. (2002).

Kang, M.J., Lee, C.G., Lee, J.Y., Dela Cruz, C.S., Chen, Z.J., Enelow, R., and Elias, J.A. (2008).

Kanneganti, T.D., Lamkanfi, M., and Nunez, G. (2007). Intracellular NOD-like receptors in

Kanneganti, T.D., Ozoren, N., Body-Malapel, M., Amer, A., Park, J.H., Franchi, L., Whitfield,

Kato, A., and Schleimer, R.P. (2007). Beyond inflammation: airway epithelial cells are at the interface of innate and adaptive immunity. Curr Opin Immunol *19*, 711-720. Kato, H., Sato, S., Yoneyama, M., Yamamoto, M., Uematsu, S., Matsui, K., Tsujimura, T.,

Kato, H., Takeuchi, O., Mikamo-Satoh, E., Hirai, R., Kawai, T., Matsushita, K., Hiiragi, A.,

Kato, H., Takeuchi, O., Sato, S., Yoneyama, M., Yamamoto, M., Matsui, K., Uematsu, S.,

Kawai, T., Adachi, O., Ogawa, T., Takeda, K., and Akira, S. (1999). Unresponsiveness of

Kawai, T., and Akira, S. (2008). Toll-like receptor and RIG-I-like receptor signaling. Ann N Y

Kawai, T., and Akira, S. (2009). The roles of TLRs, RLRs and NLRs in pathogen recognition.

Kawai, T., Takahashi, K., Sato, S., Coban, C., Kumar, H., Kato, H., Ishii, K.J., Takeuchi, O.,

Khan, E.M., Lanir, R., Danielson, A.R., and Goldkorn, T. (2008). Epidermal growth factor

and Akira, S. (2005). IPS-1, an adaptor triggering RIG-I- and Mda5-mediated type I

receptor exposed to cigarette smoke is aberrantly activated and undergoes

Cell Mol Biol *42*, 424-431.

Natl Acad Sci U S A *99*, 637-642.

host defense and disease. Immunity *27*, 549-559.

RIG-I in antiviral response. Immunity *23*, 19-28.

interferon induction. Nat Immunol *6*, 981-988.

perinuclear trafficking. FASEB J *22*, 910-917.

differentiation-associated gene 5. J Exp Med *205*, 1601-1610.

MyD88-deficient mice to endotoxin. Immunity *11*, 115-122.

helicases in the recognition of RNA viruses. Nature *441*, 101-105.

Rev Immunol *10*, 688-698.

*9*, 361-368.

*440*, 233-236.

Acad Sci *1143*, 1-20.

Int Immunol *21*, 317-337.

mediated innate immunity is reduced in cystic fibrosis airway cells. Am J Respir

endocytosis of Toll-like receptor 4 to the induction of interferon-beta. Nat Immunol

mda-5: An interferon-inducible putative RNA helicase with double-stranded RNAdependent ATPase activity and melanoma growth-suppressive properties. Proc

Cigarette smoke selectively enhances viral PAMP- and virus-induced pulmonary innate immune and remodeling responses in mice. J Clin Invest *118*, 2771-2784. Kanneganti, T.D. (2010). Central roles of NLRs and inflammasomes in viral infection. Nat

J., Barchet, W., Colonna, M., Vandenabeele, P.*, et al.* (2006). Bacterial RNA and small antiviral compounds activate caspase-1 through cryopyrin/Nalp3. Nature

Takeda, K., Fujita, T., Takeuchi, O.*, et al.* (2005). Cell type-specific involvement of

Dermody, T.S., Fujita, T., and Akira, S. (2008). Length-dependent recognition of double-stranded ribonucleic acids by retinoic acid-inducible gene-I and melanoma

Jung, A., Kawai, T., Ishii, K.J.*, et al.* (2006). Differential roles of MDA5 and RIG-I


Hoebe, K., Du, X., Georgel, P., Janssen, E., Tabeta, K., Kim, S.O., Goode, J., Lin, P., Mann, N.,

Hogg, J.C., and Timens, W. (2009). The pathology of chronic obstructive pulmonary disease.

Honkinen, M., Lahti, E., Osterback, R., Ruuskanen, O., and Waris, M. (2011). Viruses and

Horng, T., Barton, G.M., Flavell, R.A., and Medzhitov, R. (2002). The adaptor molecule TIRAP provides signalling specificity for Toll-like receptors. Nature *420*, 329-333. Ibrahim, H.R., Aoki, T., and Pellegrini, A. (2002). Strategies for new antimicrobial proteins and peptides: lysozyme and aprotinin as model molecules. Curr Pharm Des *8*, 671-693. Ichinohe, T., Pang, I.K., and Iwasaki, A. (2010). Influenza virus activates inflammasomes via

Innes, A.L., Woodruff, P.G., Ferrando, R.E., Donnelly, S., Dolganov, G.M., Lazarus, S.C., and

Ishizuka, S., Yamaya, M., Suzuki, T., Takahashi, H., Ida, S., Sasaki, T., Inoue, D., Sekizawa,

Iyer, S.S., Pulskens, W.P., Sadler, J.J., Butter, L.M., Teske, G.J., Ulland, T.K., Eisenbarth, S.C.,

Jacquot, J., Tournier, J.M., and Puchelle, E. (1985). In vitro evidence that human airway

Jamieson, A.M., Yu, S., Annicelli, C.H., and Medzhitov, R. (2010). Influenza virus-induced

Jansen, H.M., Sachs, A.P., and van Alphen, L. (1995). Predisposing conditions to bacterial

Jeong, E., and Lee, J.Y. (2011). Intrinsic and extrinsic regulation of innate immune receptors.

Jia, H.P., Kline, J.N., Penisten, A., Apicella, M.A., Gioannini, T.L., Weiss, J., and McCray,

low expression of MD-2. Am J Physiol Lung Cell Mol Physiol *287*, L428-437. Jiang, Z., Nagata, N., Molina, E., Bakaletz, L.O., Hawkins, H., and Patel, J.A. (1999). Fimbria-

Fahy, J.V. (2006). Epithelial mucin stores are increased in the large airways of

K., Nishimura, H., and Sasaki, H. (2003). Effects of rhinovirus infection on the adherence of Streptococcus pneumoniae to cultured human airway epithelial cells.

Florquin, S., Flavell, R.A., Leemans, J.C.*, et al.* (2009). Necrotic cells trigger a sterile inflammatory response through the Nlrp3 inflammasome. Proc Natl Acad Sci U S

lysozyme is cleaved and inactivated by Pseudomonas aeruginosa elastase and not

glucocorticoids compromise innate host defense against a secondary bacterial

infections in chronic obstructive pulmonary disease. Am J Respir Crit Care Med

P.B., Jr. (2004). Endotoxin responsiveness of human airway epithelia is limited by

mediated enhanced attachment of nontypeable Haemophilus influenzae to respiratory syncytial virus-infected respiratory epithelial cells. Infect Immun *67*,

independent TIR signalling. Nature *424*, 743-748.

Annu Rev Pathol *4*, 435-459.

J Infect Dis *188*, 1928-1939.

A *106*, 20388-20393.

*151*, 2073-2080.

187-192.

Yonsei Med J *52*, 379-392.

Microbiol Infect.

Hoffmann, J.A. (2003). The immune response of Drosophila. Nature *426*, 33-38.

its intracellular M2 ion channel. Nat Immunol *11*, 404-410.

smokers with airflow obstruction. Chest *130*, 1102-1108.

by human leukocyte elastase. Infect Immun *47*, 555-560.

infection. Cell Host Microbe *7*, 103-114.

Mudd, S.*, et al.* (2003). Identification of Lps2 as a key transducer of MyD88-

bacteria in sputum samples of children with community-acquired pneumonia. Clin


Innate Immunity of Airway Epithelium and COPD 41

Livraghi, A., and Randell, S.H. (2007). Cystic fibrosis and other respiratory diseases of

MacRedmond, R.E., Greene, C.M., Dorscheid, D.R., McElvaney, N.G., and O'Neill, S.J.

Mariathasan, S., Weiss, D.S., Newton, K., McBride, J., O'Rourke, K., Roose-Girma, M., Lee,

Martinon, F., Agostini, L., Meylan, E., and Tschopp, J. (2004). Identification of bacterial

Martinon, F., Burns, K., and Tschopp, J. (2002). The inflammasome: a molecular platform

Martinon, F., Petrilli, V., Mayor, A., Tardivel, A., and Tschopp, J. (2006). Gout-associated uric acid crystals activate the NALP3 inflammasome. Nature *440*, 237-241. Masui, T., Lechner, J.F., Yoakum, G.H., Willey, J.C., and Harris, C.C. (1986a). Growth and

Masui, T., Wakefield, L.M., Lechner, J.F., LaVeck, M.A., Sporn, M.B., and Harris, C.C.

Matrosovich, M., and Klenk, H.D. (2003). Natural and synthetic sialic acid-containing inhibitors of influenza virus receptor binding. Rev Med Virol *13*, 85-97. Mayer, A.K., Muehmer, M., Mages, J., Gueinzius, K., Hess, C., Heeg, K., Bals, R., Lang, R.,

McCray, P.B., Jr., and Bentley, L. (1997). Human airway epithelia express a beta-defensin.

McCullers, J.A. (2006). Insights into the interaction between influenza virus and

McElvaney, N.G., Nakamura, H., Birrer, P., Hebert, C.A., Wong, W.L., Alphonso, M., Baker,

McGillivary, G., Mason, K.M., Jurcisek, J.A., Peeples, M.E., and Bakaletz, L.O. (2009).

Mebratu, Y.A., Schwalm, K., Smith, K.R., Schuyler, M., and Tesfaigzi, Y. (2011). Cigarette

J.B., Catalano, M.A., and Crystal, R.G. (1992). Modulation of airway inflammation in cystic fibrosis. In vivo suppression of interleukin-8 levels on the respiratory epithelial surface by aerosolization of recombinant secretory leukoprotease

Respiratory syncytial virus-induced dysregulation of expression of a mucosal betadefensin augments colonization of the upper airway by non-typeable Haemophilus

smoke suppresses Bik to cause epithelial cell hyperplasia and mucous cell

ligands in human bronchial epithelial cells. J Immunol *178*, 3134-3142. Mazieres, J., He, B., You, L., Xu, Z., and Jablons, D.M. (2005). Wnt signaling in lung cancer.

inflammasome in response to toxins and ATP. Nature *440*, 228-232.

(2007). Epithelial expression of TLR4 is modulated in COPD and by steroids,

W.P., Weinrauch, Y., Monack, D.M., and Dixit, V.M. (2006). Cryopyrin activates the

muramyl dipeptide as activator of the NALP3/cryopyrin inflammasome. Curr Biol

triggering activation of inflammatory caspases and processing of proIL-beta. Mol

differentiation of normal and transformed human bronchial epithelial cells. J Cell

(1986b). Type beta transforming growth factor is the primary differentiationinducing serum factor for normal human bronchial epithelial cells. Proc Natl Acad

and Dalpke, A.H. (2007). Differential recognition of TLR-dependent microbial

impaired mucus clearance. Toxicol Pathol *35*, 116-129.

salmeterol and cigarette smoke. Respir Res *8*, 84.

*14*, 1929-1934.

Cell *10*, 417-426.

Physiol Suppl *4*, 73-81.

Sci U S A *83*, 2438-2442.

Cancer Lett *222*, 1-10.

Am J Respir Cell Mol Biol *16*, 343-349.

inhibitor. J Clin Invest *90*, 1296-1301.

influenzae. Cell Microbiol *11*, 1399-1408.

metaplasia. Am J Respir Crit Care Med *183*, 1531-1538.

pneumococcus. Clin Microbiol Rev *19*, 571-582.


Knowles, M.R., and Boucher, R.C. (2002). Mucus clearance as a primary innate defense

Koch, S., and Nusrat, A. (2009). Dynamic regulation of epithelial cell fate and barrier function by intercellular junctions. Ann N Y Acad Sci *1165*, 220-227. Kohri, K., Ueki, I.F., Shim, J.J., Burgel, P.R., Oh, Y.M., Tam, D.C., Dao-Pick, T., and Nadel,

Komori, M., Inoue, H., Matsumoto, K., Koto, H., Fukuyama, S., Aizawa, H., and Hara, N.

Kota, S., Sabbah, A., Chang, T.H., Harnack, R., Xiang, Y., Meng, X., and Bose, S. (2008). Role

Kulkarni, R., Rampersaud, R., Aguilar, J.L., Randis, T.M., Kreindler, J.L., and Ratner, A.J.

Kurt-Jones, E.A., Popova, L., Kwinn, L., Haynes, L.M., Jones, L.P., Tripp, R.A., Walsh, E.E.,

Lachowicz-Scroggins, M.E., Boushey, H.A., Finkbeiner, W.E., and Widdicombe, J.H. (2010).

Lappalainen, U., Whitsett, J.A., Wert, S.E., Tichelaar, J.W., and Bry, K. (2005). Interleukin-

Laube, D.M., Yim, S., Ryan, L.K., Kisich, K.O., and Diamond, G. (2006). Antimicrobial

Lemjabbar, H., Li, D., Gallup, M., Sidhu, S., Drori, E., and Basbaum, C. (2003). Tobacco

Li, J.D., Dohrman, A.F., Gallup, M., Miyata, S., Gum, J.R., Kim, Y.S., Nadel, J.A., Prince, A.,

Liu, P., Jamaluddin, M., Li, K., Garofalo, R.P., Casola, A., and Brasier, A.R. (2007). Retinoic

peptides in the airway. Curr Top Microbiol Immunol *306*, 153-182.

converting enzyme and amphiregulin. J Biol Chem *278*, 26202-26207. Li, C., Zienkiewicz, J., and Hawiger, J. (2005). Interactive sites in the MyD88 Toll/interleukin

adult murine lung. Am J Respir Cell Mol Biol *32*, 311-318.

J.A. (2002). Pseudomonas aeruginosa induces MUC5AC production via epidermal

(2001). PAF mediates cigarette smoke-induced goblet cell metaplasia in guinea pig

of human beta-defensin-2 during tumor necrosis factor-alpha/NF-kappaBmediated innate antiviral response against human respiratory syncytial virus. J Biol

(2010). Cigarette smoke inhibits airway epithelial cell innate immune responses to

Freeman, M.W., Golenbock, D.T., Anderson, L.J.*, et al.* (2000). Pattern recognition receptors TLR4 and CD14 mediate response to respiratory syncytial virus. Nat

Interleukin-13-induced mucous metaplasia increases susceptibility of human airway epithelium to rhinovirus infection. Am J Respir Cell Mol Biol *43*, 652-661. Landry, R.M., An, D., Hupp, J.T., Singh, P.K., and Parsek, M.R. (2006). Mucin-Pseudomonas

aeruginosa interactions promote biofilm formation and antibiotic resistance. Mol

1beta causes pulmonary inflammation, emphysema, and airway remodeling in the

smoke-induced lung cell proliferation mediated by tumor necrosis factor alpha-

(IL) 1 receptor domain responsible for coupling to the IL1beta signaling pathway. J

and Basbaum, C.B. (1997). Transcriptional activation of mucin by Pseudomonas aeruginosa lipopolysaccharide in the pathogenesis of cystic fibrosis lung disease.

acid-inducible gene I mediates early antiviral response and Toll-like receptor 3 expression in respiratory syncytial virus-infected airway epithelial cells. J Virol *81*,

mechanism for mammalian airways. J Clin Invest *109*, 571-577.

airways. Am J Physiol Lung Cell Mol Physiol *280*, L436-441.

growth factor receptor. Eur Respir J *20*, 1263-1270.

Chem *283*, 22417-22429.

Immunol *1*, 398-401.

Microbiol *59*, 142-151.

Biol Chem *280*, 26152-26159.

1401-1411.

Proc Natl Acad Sci U S A *94*, 967-972.

bacteria. Infect Immun *78*, 2146-2152.


Innate Immunity of Airway Epithelium and COPD 43

Oliver, B.G., Lim, S., Wark, P., Laza-Stanca, V., King, N., Black, J.L., Burgess, J.K., Roth, M.,

Ong, E.L., Ellis, M.E., Webb, A.K., Neal, K.R., Dodd, M., Caul, E.O., and Burgess, S. (1989).

Opitz, B., Puschel, A., Schmeck, B., Hocke, A.C., Rosseau, S., Hammerschmidt, S.,

Pace, E., Ferraro, M., Siena, L., Melis, M., Montalbano, A.M., Johnson, M., Bonsignore, M.R.,

Pace, E., Giarratano, A., Ferraro, M., Bruno, A., Siena, L., Mangione, S., Johnson, M., and

Papi, A., Bellettato, C.M., Braccioni, F., Romagnoli, M., Casolari, P., Caramori, G., Fabbri,

Parameswaran, G.I., Sethi, S., and Murphy, T.F. (2011). Effects of Bacterial Infection on

Parameswaran, G.I., Wrona, C.T., Murphy, T.F., and Sethi, S. (2009). Moraxella catarrhalis

Passariello, C., Schippa, S., Conti, C., Russo, P., Poggiali, F., Garaci, E., and Palamara, A.T.

Piao, W., Song, C., Chen, H., Diaz, M.A., Wahl, L.M., Fitzgerald, K.A., Li, L., and Medvedev,

Plotkowski, M.C., Bajolet-Laudinat, O., and Puchelle, E. (1993). Cellular and molecular mechanisms of bacterial adhesion to respiratory mucosa. Eur Respir J *6*, 903-916. Pohl, C., Hermanns, M.I., Uboldi, C., Bock, M., Fuchs, S., Dei-Anang, J., Mayer, E., Kehe, K.,

fully permissive cultured pneumocytes. Microbes Infect *8*, 758-766.

obstructive pulmonary disease. BMC Infect Dis *9*, 178.

epithelial cells. J Immunol *173*, 1219-1223.

bacterial products in human alveolar macrophages. Thorax *63*, 519-525. Olivera, D.S., Boggs, S.E., Beenhouwer, C., Aden, J., and Knall, C. (2007). Cellular

permeability changes in vitro. Inhal Toxicol *19*, 13-22.

viruses and atypical microorganisms. Thorax *44*, 739-742.

Streptococcus pneumoniae. J Biol Chem *279*, 36426-36432.

epithelial cells. Immunology *124*, 401-411.

Hum Immunol *72*, 54-62.

*173*, 1114-1121.

Disease. Chest.

and Johnston, S.L. (2008). Rhinovirus exposure impairs immune responses to

mechanisms of mainstream cigarette smoke-induced lung epithelial tight junction

Infective respiratory exacerbations in young adults with cystic fibrosis: role of

Schumann, R.R., Suttorp, N., and Hippenstiel, S. (2004). Nucleotide-binding oligomerization domain proteins are innate immune receptors for internalized

Bonsignore, G., and Gjomarkaj, M. (2008). Cigarette smoke increases Toll-like receptor 4 and modifies lipopolysaccharide-mediated responses in airway

Gjomarkaj, M. (2011). TLR4 upregulation underpins airway neutrophilia in smokers with chronic obstructive pulmonary disease and acute respiratory failure.

L.M., and Johnston, S.L. (2006). Infections and airway inflammation in chronic obstructive pulmonary disease severe exacerbations. Am J Respir Crit Care Med

Airway Antimicrobial Peptides and Proteins in Chronic Obstructive Pulmonary

acquisition, airway inflammation and protease-antiprotease balance in chronic

(2006). Rhinoviruses promote internalisation of Staphylococcus aureus into non-

A.E. (2009). Endotoxin tolerance dysregulates MyD88- and Toll/IL-1R domaincontaining adapter inducing IFN-beta-dependent pathways and increases expression of negative regulators of TLR signaling. J Leukoc Biol *86*, 863-875. Platz, J., Beisswenger, C., Dalpke, A., Koczulla, R., Pinkenburg, O., Vogelmeier, C., and Bals,

R. (2004). Microbial DNA induces a host defense reaction of human respiratory

Kummer, W., and Kirkpatrick, C.J. (2009). Barrier functions and paracellular

Medzhitov, R. (2001). Toll-like receptors and innate immunity. Nat Rev Immunol *1*, 135-145.


Medzhitov, R. (2001). Toll-like receptors and innate immunity. Nat Rev Immunol *1*, 135-145. Medzhitov, R., and Janeway, C.A., Jr. (1997). Innate immunity: impact on the adaptive

Mehta, H., Nazzal, K., and Sadikot, R.T. (2008). Cigarette smoking and innate immunity.

Meixenberger, K., Pache, F., Eitel, J., Schmeck, B., Hippenstiel, S., Slevogt, H., N'Guessan, P.,

Meylan, E., Curran, J., Hofmann, K., Moradpour, D., Binder, M., Bartenschlager, R., and

Meylan, E., and Tschopp, J. (2006). Toll-like receptors and RNA helicases: two parallel ways

Miao, E.A., Alpuche-Aranda, C.M., Dors, M., Clark, A.E., Bader, M.W., Miller, S.I., and

Modestou, M.A., Manzel, L.J., El-Mahdy, S., and Look, D.C. (2010). Inhibition of IFN-

Moore, C.B., Bergstralh, D.T., Duncan, J.A., Lei, Y., Morrison, T.E., Zimmermann, A.G.,

Mukaida, N., Mahe, Y., and Matsushima, K. (1990). Cooperative interaction of nuclear

Murphy, T.F. (2000). Haemophilus influenzae in chronic bronchitis. Semin Respir Infect *15*,

Murphy, T.F., Brauer, A.L., Eschberger, K., Lobbins, P., Grove, L., Cai, X., and Sethi, S.

Murphy, T.F., Brauer, A.L., Sethi, S., Kilian, M., Cai, X., and Lesse, A.J. (2007). Haemophilus

Muruve, D.A., Petrilli, V., Zaiss, A.K., White, L.R., Clark, S.A., Ross, P.J., Parks, R.J., and

DNA and triggers an innate immune response. Nature *452*, 103-107. O'Donnell, R.A., Richter, A., Ward, J., Angco, G., Mehta, A., Rousseau, K., Swallow, D.M.,

regulator of mitochondrial antiviral immunity. Nature *451*, 573-577. Moskwa, P., Lorentzen, D., Excoffon, K.J., Zabner, J., McCray, P.B., Jr., Nauseef, W.M.,

in cystic fibrosis. Am J Respir Crit Care Med *175*, 174-183.

Witzenrath, M., Netea, M.G., Chakraborty, T.*, et al.* (2010). Listeria monocytogenesinfected human peripheral blood mononuclear cells produce IL-1beta, depending

Tschopp, J. (2005). Cardif is an adaptor protein in the RIG-I antiviral pathway and

Aderem, A. (2006). Cytoplasmic flagellin activates caspase-1 and secretion of

gamma-dependent antiviral airway epithelial defense by cigarette smoke. Respir

Accavitti-Loper, M.A., Madden, V.J., Sun, L., Ye, Z.*, et al.* (2008). NLRX1 is a

Dupuy, C., and Banfi, B. (2007). A novel host defense system of airways is defective

factor-kappa B- and cis-regulatory enhancer binding protein-like factor binding elements in activating the interleukin-8 gene by pro-inflammatory cytokines. J Biol

(2008). Pseudomonas aeruginosa in chronic obstructive pulmonary disease. Am J

haemolyticus: a human respiratory tract commensal to be distinguished from

Tschopp, J. (2008). The inflammasome recognizes cytosolic microbial and host

Holgate, S.T., Djukanovic, R., Davies, D.E.*, et al.* (2004). Expression of ErbB receptors and mucins in the airways of long term current smokers. Thorax *59*, 1032-1040.

immune response. Curr Opin Immunol *9*, 4-9.

on listeriolysin O and NLRP3. J Immunol *184*, 922-930.

is targeted by hepatitis C virus. Nature *437*, 1167-1172.

to trigger antiviral responses. Mol Cell *22*, 561-569.

interleukin 1beta via Ipaf. Nat Immunol *7*, 569-575.

Inflamm Res *57*, 497-503.

Res *11*, 64.

41-51.

Chem *265*, 21128-21133.

Respir Crit Care Med *177*, 853-860.

Haemophilus influenzae. J Infect Dis *195*, 81-89.


Innate Immunity of Airway Epithelium and COPD 45

Schneider, D., Ganesan, S., Comstock, A.T., Meldrum, C.A., Mahidhara, R., Goldsmith,

obstructive pulmonary disease. Am J Respir Crit Care Med *182*, 332-340. Schwandner, R., Dziarski, R., Wesche, H., Rothe, M., and Kirschning, C.J. (1999).

Seki, M., Higashiyama, Y., Tomono, K., Yanagihara, K., Ohno, H., Kaneko, Y., Izumikawa,

Serikov, V.B., Leutenegger, C., Krutilina, R., Kropotov, A., Pleskach, N., Suh, J.H., and

V and increases airway epithelial permeability. Inhal Toxicol *18*, 79-92. Seth, R.B., Sun, L., Ea, C.K., and Chen, Z.J. (2005). Identification and characterization of

Sethi, S. (2000). Bacterial infection and the pathogenesis of COPD. Chest *117*, 286S-291S. Sethi, S., Maloney, J., Grove, L., Wrona, C., and Berenson, C.S. (2006). Airway inflammation

obstructive pulmonary disease. N Engl J Med *359*, 2355-2365.

Sethi, S., and Murphy, T.F. (2008). Infection in the pathogenesis and course of chronic

Sha, Q., Truong-Tran, A.Q., Plitt, J.R., Beck, L.A., and Schleimer, R.P. (2004). Activation of

Shao, M.X., Nakanaga, T., and Nadel, J.A. (2004). Cigarette smoke induces MUC5AC mucin

Shimada, K., Chen, S., Dempsey, P.W., Sorrentino, R., Alsabeh, R., Slepenkin, A.V., Peterson,

Shin, K., Fogg, V.C., and Margolis, B. (2006). Tight junctions and cell polarity. Annu Rev Cell

Simet, S.M., Sisson, J.H., Pavlik, J.A., Devasure, J.M., Boyer, C., Liu, X., Kawasaki, S., Sharp,

Singh, P.K., Jia, H.P., Wiles, K., Hesselberth, J., Liu, L., Conway, B.A., Greenberg, E.P.,

human airway epithelia. Proc Natl Acad Sci U S A *95*, 14961-14966.

receptor 2. J Biol Chem *274*, 17406-17409.

IRF 3. Cell *122*, 669-682.

358-364.

J Respir Crit Care Med *173*, 991-998.

Cell Mol Life Sci *68*, 877-892.

Dev Biol *22*, 207-235.

infection. PLoS Pathog *5*, e1000379.

Pseudomonas aeruginosa. Clin Exp Immunol *137*, 35-40.

A.M., Curtis, J.L., Martinez, F.J., Hershenson, M.B., and Sajjan, U. (2010). Increased cytokine response of rhinovirus-infected airway epithelial cells in chronic

Peptidoglycan- and lipoteichoic acid-induced cell activation is mediated by toll-like

K., Miyazaki, Y., Hirakata, Y., Mizuta, Y.*, et al.* (2004). Acute infection with influenza virus enhances susceptibility to fatal pneumonia following Streptococcus pneumoniae infection in mice with chronic pulmonary colonization with

Tomilin, N.V. (2006). Cigarette smoke extract inhibits expression of peroxiredoxin

MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and

and bronchial bacterial colonization in chronic obstructive pulmonary disease. Am

airway epithelial cells by toll-like receptor agonists. Am J Respir Cell Mol Biol *31*,

overproduction via tumor necrosis factor-alpha-converting enzyme in human airway epithelial (NCI-H292) cells. American journal of physiology *287*, L420-427. Shaykhiev, R., Otaki, F., Bonsu, P., Dang, D.T., Teater, M., Strulovici-Barel, Y., Salit, J.,

Harvey, B.G., and Crystal, R.G. (2011). Cigarette smoking reprograms apical junctional complex molecular architecture in the human airway epithelium in vivo.

E., Doherty, T.M., Underhill, D., Crother, T.R.*, et al.* (2009). The NOD/RIP2 pathway is essential for host defenses against Chlamydophila pneumoniae lung

J.G., Rennard, S.I., and Wyatt, T.A. (2010). Long-term cigarette smoke exposure in a mouse model of ciliated epithelial cell function. Am J Respir Cell Mol Biol *43*, 635-640.

Valore, E.V., Welsh, M.J., Ganz, T.*, et al.* (1998). Production of beta-defensins by

integrity in human cell culture models of the proximal respiratory unit. Eur J Pharm Biopharm *72*, 339-349.


Poltorak, A., He, X., Smirnova, I., Liu, M.Y., Van Huffel, C., Du, X., Birdwell, D., Alejos, E.,

Rose, M.C., Nickola, T.J., and Voynow, J.A. (2001). Airway mucus obstruction: mucin

Rose, M.C., and Voynow, J.A. (2006). Respiratory tract mucin genes and mucin

Ryan, P.A., Pancholi, V., and Fischetti, V.A. (2001). Group A streptococci bind to mucin and

Sabbah, A., Chang, T.H., Harnack, R., Frohlich, V., Tominaga, K., Dube, P.H., Xiang, Y., and

Sajjan, S.U., and Forstner, J.F. (1992). Identification of the mucin-binding adhesin of

Sajjan, U., Ganesan, S., Comstock, A.T., Shim, J., Wang, Q., Nagarkar, D.R., Zhao, Y.,

Sajjan, U., Wang, Q., Zhao, Y., Gruenert, D.C., and Hershenson, M.B. (2008). Rhinovirus

Sajjan, U.S., Corey, M., Karmali, M.A., and Forstner, J.F. (1992). Binding of Pseudomonas

Sajjan, U.S., Jia, Y., Newcomb, D.C., Bentley, J.K., Lukacs, N.W., LiPuma, J.J., and

Sarkar, S.N., Peters, K.L., Elco, C.P., Sakamoto, S., Pal, S., and Sen, G.C. (2004). Novel roles of

Savitski, A.N., Mesaros, C., Blair, I.A., Cohen, N.A., and Kreindler, J.L. (2009). Secondhand

Schneeberger, E.E., and Lynch, R.D. (2004). The tight junction: a multifunctional complex.

glycoproteins in health and disease. Physiol Rev *86*, 245-278.

C57BL/10ScCr mice: mutations in Tlr4 gene. Science *282*, 2085-2088. Ramsey, B.W., Gore, E.J., Smith, A.L., Cooney, M.K., Redding, G.J., and Foy, H. (1989). The

Pharm Biopharm *72*, 339-349.

*143*, 662-668.

7402-7412.

1434-1440.

Mol Biol *25*, 533-537.

Immunol *10*, 1073-1080.

Physiol *297*, L931-944.

Care Med *178*, 1271-1281.

with cystic fibrosis. J Clin Invest *89*, 648-656.

Nat Struct Mol Biol *11*, 1060-1067.

Am J Physiol Cell Physiol *286*, C1213-1228.

cells. Respir Res *10*, 120.

antagonists in stable COPD. J Clin Immunol *29*, 508-516.

integrity in human cell culture models of the proximal respiratory unit. Eur J

Silva, M., Galanos, C.*, et al.* (1998). Defective LPS signaling in C3H/HeJ and

effect of respiratory viral infections on patients with cystic fibrosis. Am J Dis Child

glycoproteins, MUC gene regulation and goblet cell hyperplasia. Am J Respir Cell

human pharyngeal cells through sialic acid-containing receptors. Infect Immun *69*,

Bose, S. (2009). Activation of innate immune antiviral responses by Nod2. Nat

Pseudomonas cepacia isolated from patients with cystic fibrosis. Infect Immun *60*,

Goldsmith, A.M., Sonstein, J., Linn, M.J.*, et al.* (2009). Elastase- and LPS-exposed mice display altered responses to rhinovirus infection. Am J Physiol Lung Cell Mol

disrupts the barrier function of polarized airway epithelial cells. Am J Respir Crit

cepacia to normal human intestinal mucin and respiratory mucin from patients

Hershenson, M.B. (2006). H. influenzae potentiates airway epithelial cell responses to rhinovirus by increasing ICAM-1 and TLR3 expression. FASEB J *20*, 2121-2123. Sapey, E., Ahmad, A., Bayley, D., Newbold, P., Snell, N., Rugman, P., and Stockley, R.A.

(2009). Imbalances between interleukin-1 and tumor necrosis factor agonists and

TLR3 tyrosine phosphorylation and PI3 kinase in double-stranded RNA signaling.

smoke inhibits both Cl- and K+ conductances in normal human bronchial epithelial


Innate Immunity of Airway Epithelium and COPD 47

Tsoumakidou, M., Bouloukaki, I., Thimaki, K., Tzanakis, N., and Siafakas, N.M. (2010).

Ueno, K., Koga, T., Kato, K., Golenbock, D.T., Gendler, S.J., Kai, H., and Kim, K.C. (2008).

Urban, T.A., Griffith, A., Torok, A.M., Smolkin, M.E., Burns, J.L., and Goldberg, J.B. (2004).

van der Strate, B.W., Beljaars, L., Molema, G., Harmsen, M.C., and Meijer, D.K. (2001).

Van Ewijk, B.E., Wolfs, T.F., Aerts, P.C., Van Kessel, K.P., Fleer, A., Kimpen, J.L., and Van

Vinzing, M., Eitel, J., Lippmann, J., Hocke, A.C., Zahlten, J., Slevogt, H., N'Guessan P, D.,

Wanderer, A.A. (2008). Interleukin-1beta targeted therapy in severe persistent asthma (SPA)

Wang, A., Yokosaki, Y., Ferrando, R., Balmes, J., and Sheppard, D. (1996). Differential

Wang, Q., Miller, D.J., Bowman, E.R., Nagarkar, D.R., Schneider, D., Zhao, Y., Linn, M.J.,

Wang, Q., Nagarkar, D.R., Bowman, E.R., Schneider, D., Gosangi, B., Lei, J., Zhao, Y.,

Wat, D., Gelder, C., Hibbitts, S., Cafferty, F., Bowler, I., Pierrepoint, M., Evans, R., and Doull, I. (2008). The role of respiratory viruses in cystic fibrosis. J Cyst Fibros *7*, 320-328. Wilkinson, T.M., Hurst, J.R., Perera, W.R., Wilks, M., Donaldson, G.C., and Wedzicha, J.A.

Wilson, R. (2000). Evidence of bacterial infection in acute exacerbations of chronic bronchitis.

inflammation and hyperresponsiveness. PLoS Pathog *7*, e1002070.

infection in exacerbations of COPD. Chest *129*, 317-324.

Antiviral activities of lactoferrin. Antiviral Res *52*, 225-239.

fibrosis and normal epithelial cells. Pediatr Res *61*, 398-403.

pulmonary fibrosis. Exp Lung Res *36*, 373-380.

Mol Biol *38*, 263-268.

Infect Immun *72*, 5126-5134.

AAV5. J Biol Chem *277*, 23709-23713.

responses. J Immunol *183*, 6989-6997.

Semin Respir Infect *15*, 208-215.

Assoc J *10*, 837-842.

Biol *15*, 664-672.

Innate immunity proteins in chronic obstructive pulmonary disease and idiopathic

MUC1 mucin is a negative regulator of toll-like receptor signaling. Am J Respir Cell

Contribution of Burkholderia cenocepacia flagella to infectivity and inflammation.

der Ent, C.K. (2007). RSV mediates Pseudomonas aeruginosa binding to cystic

Gunther, S., Schmeck, B., Hippenstiel, S.*, et al.* (2008). NAIP and Ipaf control Legionella pneumophila replication in human cells. J Immunol *180*, 6808-6815. Voynow, J.A., Gendler, S.J., and Rose, M.C. (2006). Regulation of mucin genes in chronic inflammatory airway diseases. Am J Respir Cell Mol Biol *34*, 661-665. Walters, R.W., Pilewski, J.M., Chiorini, J.A., and Zabner, J. (2002). Secreted and

transmembrane mucins inhibit gene transfer with AAV4 more efficiently than

and chronic obstructive pulmonary disease (COPD): proposed similarities between biphasic pathobiology of SPA/COPD and ischemia-reperfusion injury. Isr Med

regulation of airway epithelial integrins by growth factors. Am J Respir Cell Mol

Goldsmith, A.M., Bentley, J.K., Sajjan, U.S.*, et al.* (2011). MDA5 and TLR3 initiate pro-inflammatory signaling pathways leading to rhinovirus-induced airways

McHenry, C.L., Burgens, R.V., Miller, D.J.*, et al.* (2009). Role of double-stranded RNA pattern recognition receptors in rhinovirus-induced airway epithelial cell

(2006). Effect of interactions between lower airway bacterial and rhinoviral


Sly, L.M., Hamilton, M.J., Kuroda, E., Ho, V.W., Antignano, F.L., Omeis, S.L., van Netten-

Soong, G., Reddy, B., Sokol, S., Adamo, R., and Prince, A. (2004). TLR2 is mobilized into an

Sun, K., and Metzger, D.W. (2008). Inhibition of pulmonary antibacterial defense by interferon-gamma during recovery from influenza infection. Nat Med *14*, 558-564. Sun, Q., Sun, L., Liu, H.H., Chen, X., Seth, R.B., Forman, J., and Chen, Z.J. (2006). The specific

Sykes, A., Mallia, P., and Johnston, S.L. (2007). Diagnosis of pathogens in exacerbations of chronic obstructive pulmonary disease. Proc Am Thorac Soc *4*, 642-646. Taggart, C.C., Lowe, G.J., Greene, C.M., Mulgrew, A.T., O'Neill, S.J., Levine, R.L., and

Takeuchi, O., Kawai, T., Muhlradt, P.F., Morr, M., Radolf, J.D., Zychlinsky, A., Takeda, K.,

Takeuchi, O., Sato, S., Horiuchi, T., Hoshino, K., Takeda, K., Dong, Z., Modlin, R.L., and

Tamashiro, E., Xiong, G., Anselmo-Lima, W.T., Kreindler, J.L., Palmer, J.N., and Cohen, N.A.

Tattoli, I., Carneiro, L.A., Jehanno, M., Magalhaes, J.G., Shu, Y., Philpott, D.J., Arnoult, D.,

Taylor, D.C., Cripps, A.W., and Clancy, R.L. (1995). A possible role for lysozyme in

Thomas, P.G., Dash, P., Aldridge, J.R., Jr., Ellebedy, A.H., Reynolds, C., Funk, A.J., Martin,

Thornton, D.J., Rousseau, K., and McGuckin, M.A. (2008). Structure and function of the polymeric mucins in airways mucus. Annu Rev Physiol *70*, 459-486. Tian, D., Zhu, M., Li, J., Ma, Y., and Wu, R. (2009). Cigarette smoke extract induces

Travassos, L.H., Carneiro, L.A., Girardin, S.E., Boneca, I.G., Lemos, R., Bozza, M.T.,

leucoprotease inhibitor. J Biol Chem *276*, 33345-33352.

response to microbial lipoproteins. J Immunol *169*, 10-14.

2954.

633-642.

Invest *113*, 1482-1489.

Int Immunol *13*, 933-940.

Rhinol Allergy *23*, 117-122.

36714-36718.

production. EMBO Rep *9*, 293-300.

regulation of caspase-1. Immunity *30*, 566-575.

alveolar epithelial cell line. Toxicol Lett *187*, 58-62.

Thomas, C.J., Wong, D., Brugger, H.K., Williams, O.*, et al.* (2009). SHIP prevents lipopolysaccharide from triggering an antiviral response in mice. Blood *113*, 2945-

apical lipid raft receptor complex to signal infection in airway epithelial cells. J Clin

and essential role of MAVS in antiviral innate immune responses. Immunity *24*,

McElvaney, N.G. (2001). Cathepsin B, L, and S cleave and inactivate secretory

and Akira, S. (2001). Discrimination of bacterial lipoproteins by Toll-like receptor 6.

Akira, S. (2002). Cutting edge: role of Toll-like receptor 1 in mediating immune

(2009). Cigarette smoke exposure impairs respiratory epithelial ciliogenesis. Am J

and Girardin, S.E. (2008). NLRX1 is a mitochondrial NOD-like receptor that amplifies NF-kappaB and JNK pathways by inducing reactive oxygen species

determining acute exacerbation in chronic bronchitis. Clin Exp Immunol *102*, 406-416.

W.J., Lamkanfi, M., Webby, R.J., Boyd, K.L.*, et al.* (2009). The intracellular sensor NLRP3 mediates key innate and healing responses to influenza A virus via the

activation of beta-catenin/TCF signaling through inhibiting GSK3beta in human

Domingues, R.C., Coyle, A.J., Bertin, J., Philpott, D.J.*, et al.* (2005). Nod1 participates in the innate immune response to Pseudomonas aeruginosa. J Biol Chem *280*,


**3** 

 *Cambridge United Kingdom* 

Sam Alam and Ravi Mahadeva

**The Role of Alpha–1 Antitrypsin in Emphysema** 

Alpha-1 antitrypsin (AT) is a member of the serine proteinase inhibitor (SERPIN) superfamily. It is an acute phase protein produced constitutively, primarily by hepatocytes, and is secreted in to the plasma from where it diffuses into the lung. AT is the most abundant proteinase inhibitor within the lung whose main physiological role is to regulate neutrophil elastase (NE) liberated from activated neutrophils (Brantly et al., 1988a; Lomas

The importance of AT in pulmonary biology was demonstrated by the association between severe plasma deficiency and pulmonary emphysema (Laurell and Eriksson., 1963). These findings in conjunction with Gross et al., 1965 formed the basis of the proteinaseantiproteinase hypothesis for the development of emphysema and other lung diseases. It was subsequently identified that the Z variant is the commonest cause of severe AT deficiency. It results in aggregation of the protein in the hepatocyte (with a predisposition to liver disease) resulting in a secretory defect and deficiency. Initially it was presumed that NE and AT were the most important proteinase and anti-proteinase respectively within the lung, but it is now appreciated that several proteinases and inhibitors exist within the lung and other mechanisms are important e.g. apoptosis, ageing, oxidants. Nevertheless, no other PI and proteinase have been so clearly linked with pulmonary emphysema, thus emphasizing the important role of AT in lung biology. Despite this long association, epidemiological studies suggest that AT deficiency is under-recognized or misdiagnosed

The AT protein is an extremely polymorphic molecule; there are over 100 variants of AT resulting from mutations in the *SERPINA1* gene. They are named by the letter of the alphabet according to the migration of the glycosylated form of the protein on isoelectric focusing (IEF). The wildtype protein is therefore termed M-AT as it is associated with normal level of serum AT and it has a medium rate of migration on IEF. Variants that migrate faster than M-AT are classified as A to L or slower than M-AT are classified as N to Z (A being the fastest and Z the slowest) (Brantly et al., 1988a; 1991; Cox et al., 1980; Fagerhol and Laurell, 1967). On the basis of their plasma level and function, the majority of

(Bull World Health Organ, 1997; ATS-ERS statement, 2003).

**1.1 Nomenclature and detection of mutants** 

individuals are M homozygotes (M1-5 subtypes).

**1. Introduction** 

and Mahadeva, 2002).

*Department of Medicine, Addenbrooke's Hospital, University of Cambridge* 


## **The Role of Alpha–1 Antitrypsin in Emphysema**

Sam Alam and Ravi Mahadeva

*Department of Medicine, Addenbrooke's Hospital, University of Cambridge Cambridge United Kingdom* 

#### **1. Introduction**

48 Emphysema

Wu, W., Patel, K.B., Booth, J.L., Zhang, W., and Metcalf, J.P. (2011). Cigarette smoke extract

Xing, Z., Harper, R., Anunciacion, J., Yang, Z., Gao, W., Qu, B., Guan, Y., and Cardona, C.J.

human tracheobronchial epithelial cells. Am J Respir Cell Mol Biol *44*, 24-33. Xu, L.G., Wang, Y.Y., Han, K.J., Li, L.Y., Zhai, Z., and Shu, H.B. (2005). VISA is an adapter protein required for virus-triggered IFN-beta signaling. Mol Cell *19*, 727-740. Xu, W., and Kimelman, D. (2007). Mechanistic insights from structural studies of beta-

Yamasaki, K., Muto, J., Taylor, K.R., Cogen, A.L., Audish, D., Bertin, J., Grant, E.P., Coyle,

trigger of inflammation in response to injury. J Biol Chem *284*, 12762-12771. Yoneyama, M., Kikuchi, M., Matsumoto, K., Imaizumi, T., Miyagishi, M., Taira, K., Foy, E.,

Yoneyama, M., Kikuchi, M., Natsukawa, T., Shinobu, N., Imaizumi, T., Miyagishi, M., Taira,

Yu, H., Li, Q., Kolosov, V.P., Perelman, J.M., and Zhou, X. (2011b). Regulation of cigarette

Zheng, S., De, B.P., Choudhary, S., Comhair, S.A., Goggans, T., Slee, R., Williams, B.R.,

Zheng, S., Xu, W., Bose, S., Banerjee, A.K., Haque, S.J., and Erzurum, S.C. (2004). Impaired

Zhu, L., Lee, P.K., Lee, W.M., Zhao, Y., Yu, D., and Chen, Y. (2009). Rhinovirus-induced

Zola, T.A., Lysenko, E.S., and Weiser, J.N. (2008). Mucosal clearance of capsule-expressing

airway epithelial cells. Basic Clin Pharmacol Toxicol *109*, 63-72.

flagellin by Toll-like receptor 5. Infect Immun *73*, 7151-7160.

Physiol Lung Cell Mol Physiol *287*, L374-381.

Am J Respir Cell Mol Biol *40*, 610-619.

signaling. J Immunol *181*, 7909-7916.

human lung. Am J Physiol Lung Cell Mol Physiol *300*, L821-830.

catenin and its binding partners. J Cell Sci *120*, 3337-3344.

Immunol *175*, 2851-2858.

619-630.

suppresses the RIG-I-initiated innate immune response to influenza virus in the

(2011). Host immune and apoptotic responses to avian influenza virus H9N2 in

A.J., Misaghi, A., Hoffman, H.M.*, et al.* (2009). NLRP3/cryopyrin is necessary for interleukin-1beta (IL-1beta) release in response to hyaluronan, an endogenous

Loo, Y.M., Gale, M., Jr., Akira, S.*, et al.* (2005). Shared and unique functions of the DExD/H-box helicases RIG-I, MDA5, and LGP2 in antiviral innate immunity. J

K., Akira, S., and Fujita, T. (2004). The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat Immunol *5*, 730-737. Yu, H., Li, Q., Kolosov, V.P., Perelman, J.M., and Zhou, X. (2011a). Regulation of cigarette

smoke-induced mucin expression by neuregulin1beta/ErbB3 signalling in human

smoke-mediated mucin expression by hypoxia-inducible factor-1alpha via epidermal growth factor receptor-mediated signaling pathways. J Appl Toxicol. Zhang, Z., Louboutin, J.P., Weiner, D.J., Goldberg, J.B., and Wilson, J.M. (2005). Human

airway epithelial cells sense Pseudomonas aeruginosa infection via recognition of

Pilewski, J., Haque, S.J., and Erzurum, S.C. (2003). Impaired innate host defense causes susceptibility to respiratory virus infections in cystic fibrosis. Immunity *18*,

nitric oxide synthase-2 signaling pathway in cystic fibrosis airway epithelium. Am J

major airway mucin production involves a novel TLR3-EGFR-dependent pathway.

bacteria requires both TLR and nucleotide-binding oligomerization domain 1

Alpha-1 antitrypsin (AT) is a member of the serine proteinase inhibitor (SERPIN) superfamily. It is an acute phase protein produced constitutively, primarily by hepatocytes, and is secreted in to the plasma from where it diffuses into the lung. AT is the most abundant proteinase inhibitor within the lung whose main physiological role is to regulate neutrophil elastase (NE) liberated from activated neutrophils (Brantly et al., 1988a; Lomas and Mahadeva, 2002).

The importance of AT in pulmonary biology was demonstrated by the association between severe plasma deficiency and pulmonary emphysema (Laurell and Eriksson., 1963). These findings in conjunction with Gross et al., 1965 formed the basis of the proteinaseantiproteinase hypothesis for the development of emphysema and other lung diseases. It was subsequently identified that the Z variant is the commonest cause of severe AT deficiency. It results in aggregation of the protein in the hepatocyte (with a predisposition to liver disease) resulting in a secretory defect and deficiency. Initially it was presumed that NE and AT were the most important proteinase and anti-proteinase respectively within the lung, but it is now appreciated that several proteinases and inhibitors exist within the lung and other mechanisms are important e.g. apoptosis, ageing, oxidants. Nevertheless, no other PI and proteinase have been so clearly linked with pulmonary emphysema, thus emphasizing the important role of AT in lung biology. Despite this long association, epidemiological studies suggest that AT deficiency is under-recognized or misdiagnosed (Bull World Health Organ, 1997; ATS-ERS statement, 2003).

#### **1.1 Nomenclature and detection of mutants**

The AT protein is an extremely polymorphic molecule; there are over 100 variants of AT resulting from mutations in the *SERPINA1* gene. They are named by the letter of the alphabet according to the migration of the glycosylated form of the protein on isoelectric focusing (IEF). The wildtype protein is therefore termed M-AT as it is associated with normal level of serum AT and it has a medium rate of migration on IEF. Variants that migrate faster than M-AT are classified as A to L or slower than M-AT are classified as N to Z (A being the fastest and Z the slowest) (Brantly et al., 1988a; 1991; Cox et al., 1980; Fagerhol and Laurell, 1967). On the basis of their plasma level and function, the majority of individuals are M homozygotes (M1-5 subtypes).

The Role of Alpha–1 Antitrypsin in Emphysema 51

cells; reduced inhibitory activity; deficiency

and plasma; deficiency

polymerization in liver cells; deficiency

deficiency

activity; deficiency

deficiency

abnormal intracellular degradation; polymerization in liver cells

decreased inhibitory activity; mild deficiency

biosynthesis; loss of conserved Gly; disturbed packing; degraded in liver cells; decreased inhibitory activity; deficiency

inhibitory activity

(G67E) aberrant posttranslational

**disease** 

Emphysema Liver disease

Emphysema Liver disease

Emphysema Liver disease

bleeding

**References** 

Oakeshott et al., 1985; Carell, 1990; Ogushi et al., 1987

Seyama et al., 1991

Cox and Billingsley, 1989;Roberts et al., 1984

1981

al., 1988; Holmes et al., 1990 (a)

1990 (b)

Owen et al., 1983

Elliott et al., 1996a; Schindler, 1984; Engh et al., 1989

al., 1999; Graham et al., 1989; Baur and Bencze, 1987

1990

Okayama et al., 1991; Hayes et al., 1992

Emphysema Kramps et al.,

Emphysema Takahashi et

Emphysema Holmes et al.,

Emphysema Mahadeva et

Emphysema Curiel et al.,

 **Variants Mutations Mechanism of deficiency Clinical** 

Z (E342K) polymerization in liver

Siiyama (F53S) polymerization in liver cells

Mheerlen (P369L) intracellular degradation;

P (D256V) intracellular degradation;

S (E264V) incorrect splicing of mRNA;

I (R39C) polymerization; slightly

F (R223C) polymerization; decreased

Table 1. Alpha-1-antitrypsin variants associated with plasma deficiency

Pittsburgh (M358R) altered substrate specificity Serous

Mprocida (L41P) decreased inhibitory

deleted)

Mmalton (52Phe

Mmineral Springs

**Severe** 

**Mild** 

#### **1.2 Genotyping**

Currently, diagnosis of AT deficiency is based on the measurement of AT levels in the serum and/or phenotyping by IEF of the serum within a narrow pH range on the polyacrylamide gel. The latter has been standard practice for many years, but is time consuming, difficult to interpret and limited to a few reference laboratories. Genotyping by real-time PCR and Restriction Fragment-Length Polymorphism PCR (RFLP-PCR) are highly effective, relatively inexpensive and reliable, and as a consequence are now commonly performed. Direct sequencing of coding exons of the gene can also be used as an adjunct in selected cases to clarify genotyping (Zorzetto et al., 2008; Miravitlles et al., 2010).

#### **1.3 Alpha-1-antitrypsin gene expression**

The AT gene is located on chromosome 14q31-32.1, and is co-dominantly expressed (Schroeder et al., 1985; Bull World Health Organ, 1997; Brantly et al., 1988). The gene is 12 kb in length and contains seven exons (Ia, b c and II–V) and six introns. Exon I contains the 3' untranslated promoter sequences: Ia and Ib contains the promoter sequence for macrophage-specific, and Ic for hepatocyte-specific transcription, respectively. The coding regions (Exons II-V) are 1434 base-pairs (bp) in length and the reactive centre is within Exon V (Long et al., 1984). Aside from the promoter elements, there are other regulatory sequences including an enhancer element in the 5' and 3' flanking sequences of exonic regions of the AT gene. A polymorphism in the 3' flanking region is associated with susceptibility to COPD (Kalsheker et al., 1987; Morgan et al., 1992). A map of single nucleotide polymorphisms (SNPs) in the 5' and 3' flanking regions showed that among the 15 SNPs, five SNPs increased the risk of COPD by 6- to 50-fold (Chappell et al., 2006). These polymorphisms within regulatory sequences are associated with normal basal plasma levels, but can result in reduced levels of AT transcription in response to stimulation *in vitro*, which is postulated to relate to the susceptibility to COPD (Henry et al., 2001; Chappell et al., 2006). However, this has not been proven *in vivo* (Mahadeva et al., 1998; de Faria et al., 2005; Courtney et al., 2006, Brennan, 2007).

#### **1.4 Variants associated with alpha-1 antitrypsin deficiency**

The commonest cause of severe deficiency in Caucasians is Z-AT (Glu342Lys). Four percent of North Europeans carry this variant, and amongst them 1/2000 are PiZ homozygotes (Fagerhol, 1974). The frequencies of PiZ are 1/2700 in USA and 1/5000 in UK (Cook, 1975; de Serres, 2002; Brantly et al., 1988a). The distribution of the genetic types (PI alleles) of AT has been investigated in many populations. Some variants are only common in specific populations; the Z mutant is rare in Asian and African populations, whereas the S (364 Glu-Val) variant is more frequent in the Mediterranean area. The plasma level of the principal AT phenotypes are MM (20-39 µmol/L), MS (19-35 µmol/L), SS (14-20 µmol/L), MZ (13-23 µmol/L), SZ (9-15 µmol/L), ZZ (2-8 µmol/L). About 20 variants are associated with lower but detectable AT in plasma (Table 1). The dysfunctional Pittsburgh (M358R) variant converts AT from an elastase inhibitor to a thrombin inhibitor due to mutation in the active site. The Null (QO) variants occur as a result of insertion or deletion of nucleotides. They are associated with only trace amounts (less than 1%) of AT in plasma and associated with increased risk for emphysema (Bull Health World Organ, 1997; ATS-ERS, 2003).

Currently, diagnosis of AT deficiency is based on the measurement of AT levels in the serum and/or phenotyping by IEF of the serum within a narrow pH range on the polyacrylamide gel. The latter has been standard practice for many years, but is time consuming, difficult to interpret and limited to a few reference laboratories. Genotyping by real-time PCR and Restriction Fragment-Length Polymorphism PCR (RFLP-PCR) are highly effective, relatively inexpensive and reliable, and as a consequence are now commonly performed. Direct sequencing of coding exons of the gene can also be used as an adjunct in

The AT gene is located on chromosome 14q31-32.1, and is co-dominantly expressed (Schroeder et al., 1985; Bull World Health Organ, 1997; Brantly et al., 1988). The gene is 12 kb in length and contains seven exons (Ia, b c and II–V) and six introns. Exon I contains the 3' untranslated promoter sequences: Ia and Ib contains the promoter sequence for macrophage-specific, and Ic for hepatocyte-specific transcription, respectively. The coding regions (Exons II-V) are 1434 base-pairs (bp) in length and the reactive centre is within Exon V (Long et al., 1984). Aside from the promoter elements, there are other regulatory sequences including an enhancer element in the 5' and 3' flanking sequences of exonic regions of the AT gene. A polymorphism in the 3' flanking region is associated with susceptibility to COPD (Kalsheker et al., 1987; Morgan et al., 1992). A map of single nucleotide polymorphisms (SNPs) in the 5' and 3' flanking regions showed that among the 15 SNPs, five SNPs increased the risk of COPD by 6- to 50-fold (Chappell et al., 2006). These polymorphisms within regulatory sequences are associated with normal basal plasma levels, but can result in reduced levels of AT transcription in response to stimulation *in vitro*, which is postulated to relate to the susceptibility to COPD (Henry et al., 2001; Chappell et al., 2006). However, this has not been proven *in vivo* (Mahadeva et al., 1998; de Faria et al., 2005;

The commonest cause of severe deficiency in Caucasians is Z-AT (Glu342Lys). Four percent of North Europeans carry this variant, and amongst them 1/2000 are PiZ homozygotes (Fagerhol, 1974). The frequencies of PiZ are 1/2700 in USA and 1/5000 in UK (Cook, 1975; de Serres, 2002; Brantly et al., 1988a). The distribution of the genetic types (PI alleles) of AT has been investigated in many populations. Some variants are only common in specific populations; the Z mutant is rare in Asian and African populations, whereas the S (364 Glu-Val) variant is more frequent in the Mediterranean area. The plasma level of the principal AT phenotypes are MM (20-39 µmol/L), MS (19-35 µmol/L), SS (14-20 µmol/L), MZ (13-23 µmol/L), SZ (9-15 µmol/L), ZZ (2-8 µmol/L). About 20 variants are associated with lower but detectable AT in plasma (Table 1). The dysfunctional Pittsburgh (M358R) variant converts AT from an elastase inhibitor to a thrombin inhibitor due to mutation in the active site. The Null (QO) variants occur as a result of insertion or deletion of nucleotides. They are associated with only trace amounts (less than 1%) of AT in plasma and associated with

increased risk for emphysema (Bull Health World Organ, 1997; ATS-ERS, 2003).

selected cases to clarify genotyping (Zorzetto et al., 2008; Miravitlles et al., 2010).

**1.2 Genotyping** 

**1.3 Alpha-1-antitrypsin gene expression** 

Courtney et al., 2006, Brennan, 2007).

**1.4 Variants associated with alpha-1 antitrypsin deficiency** 


Table 1. Alpha-1-antitrypsin variants associated with plasma deficiency

The Role of Alpha–1 Antitrypsin in Emphysema 53

1988a; Cichy et al., 1997). Daily production of AT is 34 mg/kg. The large amount of AT in the circulation and lung is primarily present to control the activity of elastase in the lung. NE is the main substrate for AT, accordingly it inhibits 90% of the NE in circulation and interstitium of lung. AT also inhibits the serine proteinases cathepsin G and proteinase 3.

In addition to acting as an antiproteinase, AT plays important role in modulating inflammation. It may inhibit immune responses, and fibroblast-proliferation and fibroblast procollagen production thereby contributing to repair and matrix production (Dabbagh et al., 2001), and have antibacterial activities (Hadzic et al., 2006), and also blocks the cytotoxic and stimulatory activity of defensins (Hiemstra et al., 1998). AT also has direct and indirect anti-apoptotic properties by inhibiting caspase-3 or NE mediated apoptosis, respectively (Petrache et al., 2006). AT is also involved in calcium-induced activation mechanisms; AT inactivates calpain I (μ-calpain), induces a rapid cell polarization and random migration of neutrophils The role of AT in neutrophil regulation was further supported by its ability to transiently increase calcium from intracellular stores, which is linked to neutrophil polarization. AT modulated increase in intracellular lipids, activation of the Rho GTPases, Rac1 and Cdc42, and extracellular signal-regulated kinase (ERK1/2) all these kinases are indeed found to be activated or phosphoryated in polarized neutrophils with significant mobility (Al-Omari et al., 2011). Furthermore, a recent study demonstrated that AT can control immune complex–mediated neutrophil chemotaxis by inhibiting ADAM-17 (TACE) activity and preventing the release of glycosylphosphatidylinositol-linked (GPI-linked) membrane protein, FcγRIIIb, from the cell (Bergin et al., 2010). The same study also demonstrated *in vivo,* that AT is a potent inhibitor of neutrophil chemotaxis in Z-AT individuals compared with M-AT individuals correlating with increased chemotactic responses of both CXCR1 and immune complex receptor (FcγRIIIb) (Bergin et al., 2010).

The process of inhibition is initiated by the specific binding of the proteinase to the RCL of AT to form a non-covalent Michaelis complex and is one of "suicide substrate inhibition" (Gettins, 2002). The inhibitory mechanism of AT relies upon cleavage of the methionineserine P1-P1' by NE (1:1 AT-elastase complex). The protease is then swung 70 Å (1 Å = 0.1 nm) from the upper pole to the lower pole of the protein in association with the insertion of the reactive loop as an extra strand into β-sheet A. The complex inactivates the protease by distortion of the catalytic triad at the active site (Huntington et al., 2000; Wilczynska et al., 1997; Stratikos and Gettins, 1997). The stable complex is subsequently recognized and cleared by the liver. The complexes are short lived (a few hours) in the circulation compared with the native AT (5-6 days), and the low-density lipoprotein receptor related protein (LRP) on liver cells appears to be the principal receptor for clearance of the AT-proteinase

Molecular mobility and the P1 methionine is essential for elastase inhibitory behaviour, but is also its Achilles heel making the molecule vulnerable to the effects of critically situated

**1.7 Other biological effects of alpha-antitrypsin** 

**1.8 Mechanism of proteinase inhibition** 

complexes (Kounnas et al., 1996).

**1.9 Mutations and their effect on conformation of AT** 

#### **1.5 Molecular structure of α1-antitrypsin**

AT consists of three *β*-sheets, eight α-helices, and a reactive centre loop (RCL), which contains the residues that directly interact with the proteinase substrate (Fig. 1). β-Sheet A is composed of five strands spreading along the long axis of the protein: the first strand has 5-6 residues and the other strands have 12-15 residues. In the native conformation, the 17 amino acid RCL locates at an external position in relation to the body of the molecule between the C-terminus of β-sheet A3 and the N-terminus of β-sheet C1. The N-terminal side of the reactive loop including M358 (P1) is directly related to the recognition and binding of the substrate (Fig. 1) (Song et al., 1995; Silverman et al., 2001; Elliott et al., 1996a; Ryu et al., 1996; Kim et al., 2001)

Pathways of polymerization of α1-antitrypsin. The structure of α1-antitrypsin is centred on β-sheet A and the mobile RCL. Polymer formation results from the Z-AT (E342K at P17; Z) or other mutations in the shutter domain, which open β-sheet A to favour partial loop insertion and the formation of an unstable intermediate (M\*). The patent β-sheet A can accept either the loop of another molecule, to form a dimer (D), which then extends into polymers (P), or else its own loop, to form a latent conformation (L). The individual molecules of AT within the polymer are shown in different shades of grey. Reproduced from Lomas and Mahadeva, 2002.

Fig. 1. Mechanism of Z α1-antitrypsin polymerization

#### **1.6 Physiology and function of α1-antitrypsin**

AT is a 394 amino acid (52 KDa) glycoprotein produced primarily by hepatocytes. Other cells produce the protein to a lesser extent in peripheral blood monocytes, alveolar macrophages and bronchial epithelial cells and gastrointestinal mucosa. (Brantly et al.,

AT consists of three *β*-sheets, eight α-helices, and a reactive centre loop (RCL), which contains the residues that directly interact with the proteinase substrate (Fig. 1). β-Sheet A is composed of five strands spreading along the long axis of the protein: the first strand has 5-6 residues and the other strands have 12-15 residues. In the native conformation, the 17 amino acid RCL locates at an external position in relation to the body of the molecule between the C-terminus of β-sheet A3 and the N-terminus of β-sheet C1. The N-terminal side of the reactive loop including M358 (P1) is directly related to the recognition and binding of the substrate (Fig. 1) (Song et al., 1995; Silverman et al., 2001; Elliott et al., 1996a; Ryu et al., 1996;

Pathways of polymerization of α1-antitrypsin. The structure of α1-antitrypsin is centred on β-sheet A and the mobile RCL. Polymer formation results from the Z-AT (E342K at P17; Z) or other mutations in the shutter domain, which open β-sheet A to favour partial loop insertion and the formation of an unstable intermediate (M\*). The patent β-sheet A can accept either the loop of another molecule, to form a dimer (D), which then extends into polymers (P), or else its own loop, to form a latent conformation (L). The individual molecules of AT within the polymer are shown in different shades of grey.

AT is a 394 amino acid (52 KDa) glycoprotein produced primarily by hepatocytes. Other cells produce the protein to a lesser extent in peripheral blood monocytes, alveolar macrophages and bronchial epithelial cells and gastrointestinal mucosa. (Brantly et al.,

**1.5 Molecular structure of α1-antitrypsin** 

Reproduced from Lomas and Mahadeva, 2002.

Fig. 1. Mechanism of Z α1-antitrypsin polymerization

**1.6 Physiology and function of α1-antitrypsin** 

Kim et al., 2001)

1988a; Cichy et al., 1997). Daily production of AT is 34 mg/kg. The large amount of AT in the circulation and lung is primarily present to control the activity of elastase in the lung. NE is the main substrate for AT, accordingly it inhibits 90% of the NE in circulation and interstitium of lung. AT also inhibits the serine proteinases cathepsin G and proteinase 3.

#### **1.7 Other biological effects of alpha-antitrypsin**

In addition to acting as an antiproteinase, AT plays important role in modulating inflammation. It may inhibit immune responses, and fibroblast-proliferation and fibroblast procollagen production thereby contributing to repair and matrix production (Dabbagh et al., 2001), and have antibacterial activities (Hadzic et al., 2006), and also blocks the cytotoxic and stimulatory activity of defensins (Hiemstra et al., 1998). AT also has direct and indirect anti-apoptotic properties by inhibiting caspase-3 or NE mediated apoptosis, respectively (Petrache et al., 2006). AT is also involved in calcium-induced activation mechanisms; AT inactivates calpain I (μ-calpain), induces a rapid cell polarization and random migration of neutrophils The role of AT in neutrophil regulation was further supported by its ability to transiently increase calcium from intracellular stores, which is linked to neutrophil polarization. AT modulated increase in intracellular lipids, activation of the Rho GTPases, Rac1 and Cdc42, and extracellular signal-regulated kinase (ERK1/2) all these kinases are indeed found to be activated or phosphoryated in polarized neutrophils with significant mobility (Al-Omari et al., 2011). Furthermore, a recent study demonstrated that AT can control immune complex–mediated neutrophil chemotaxis by inhibiting ADAM-17 (TACE) activity and preventing the release of glycosylphosphatidylinositol-linked (GPI-linked) membrane protein, FcγRIIIb, from the cell (Bergin et al., 2010). The same study also demonstrated *in vivo,* that AT is a potent inhibitor of neutrophil chemotaxis in Z-AT individuals compared with M-AT individuals correlating with increased chemotactic responses of both CXCR1 and immune complex receptor (FcγRIIIb) (Bergin et al., 2010).

#### **1.8 Mechanism of proteinase inhibition**

The process of inhibition is initiated by the specific binding of the proteinase to the RCL of AT to form a non-covalent Michaelis complex and is one of "suicide substrate inhibition" (Gettins, 2002). The inhibitory mechanism of AT relies upon cleavage of the methionineserine P1-P1' by NE (1:1 AT-elastase complex). The protease is then swung 70 Å (1 Å = 0.1 nm) from the upper pole to the lower pole of the protein in association with the insertion of the reactive loop as an extra strand into β-sheet A. The complex inactivates the protease by distortion of the catalytic triad at the active site (Huntington et al., 2000; Wilczynska et al., 1997; Stratikos and Gettins, 1997). The stable complex is subsequently recognized and cleared by the liver. The complexes are short lived (a few hours) in the circulation compared with the native AT (5-6 days), and the low-density lipoprotein receptor related protein (LRP) on liver cells appears to be the principal receptor for clearance of the AT-proteinase complexes (Kounnas et al., 1996).

#### **1.9 Mutations and their effect on conformation of AT**

Molecular mobility and the P1 methionine is essential for elastase inhibitory behaviour, but is also its Achilles heel making the molecule vulnerable to the effects of critically situated

The Role of Alpha–1 Antitrypsin in Emphysema 55

emphysematous alveolar walls (Mahadeva et al., 2005). Polymers of Z-AT are chemotactic for neutrophils (Parmar et al., 2002; Mulgrew et al., 2004). Polymers of Z-AT are also ineffective anti-inflammatory molecules or inhibitors of NE, (Alam et al., 2011; Bergin et al., 2010; Al-Omari et al., 2011). Recent findings show that ER accumulation of Z-AT polymers is associated with up-regulation of PKR-like ER kinase (PERK), regulator of G-protein signaling (RGS) 16, and calnexin, and NF-B activation and secretion of inflammatory mediators; IL-6 and IL-8 in keeping with activation of the ER overload response (EOR) linked to excess inflammatory

The normal plasma concentration of AT is about 30 μM, providing 24 μM in lung interstitium, which is thought to be critical in inhibiting elastase. It has been calculated that a concentration of 11 μM of plasma AT is the threshold for providing sufficient AT in the lung (Wewers et al., 1987; Stockely, 2003). Hence, although some phenotypes of AT are associated with abnormally low concentrations of AT in the plasma; PiMS: 80% of normal, PiSS: 60%; PiMZ: 57.5%, only PiSZ, 40%, and PiZ:10-15% and Null variants have been linked to the development of lung disease (Brantly et al., 1988a;b). AT deficiency appears to be underdiagnosed in some populations (Bull World Health Organ, 1997; de Serres, 2003) with only a small proportion of those predicted according to allele frequencies to have AT deficiency to have been identified: 4.5% in UK, 6% in Sweden, and 5% in USA (Tobin et al., 1983; Larsson,

Cigarette smoking is the most important independent risk factor for the development of emphysema in the Western world. A landmark study (Fletcher et al., 1977) showed that 15 to 25% of smokers with normal AT develop clinically significant COPD, and that the rate of FEV1 (forced expiratory volume in 1 second) decline was around 50 ml/year in smokers compared with 25 ml/year in non-smokers. Among the AT-deficient population, the decline of FEV1 is 70 ml/year in current smokers compared with 41 ml/year in ex-smokers (Piitulainen and Eriksson, 1999). Smokers with severe deficiency of AT develop symptoms of emphysema 10-15 years earlier than those non-smoking individuals and have a higher

Severe AT deficiency usually due to Z-AT accounts for about 2% of cases of emphysema (Morse, 1978), and has also been linked to asthma and bronchiectasis (Parr et al., 2007; Eden et al., 1997; King et al., 1996; Bleumink and Klokke, 1985). Individuals, who have never smoked, rarely develop symptoms before the age of 50. Twenty-40% of patients have chronic bronchitis and bronchiectasis, and about half have exacerbations (Needham and Stockley, 2004). Most PiZ non-index cases have normal or slightly abnormal lung function in the absence of symptoms (Tobin et al., 1983). The development of lung disease is intimately related to cigarette smoking. However, the severity of lung disease can show some variability: lung function is well maintained in some AT-deficient smokers, while can be impaired in non-smokers (Brantly et al., 1988b; Janus et al., 1985). It is also

activity of the Z-AT cell (Alam et al., Unpublished observation).

**3. Mechanisms of disease and pathology 3.1 Alpha-1-antitrypsin associated diseases** 

**3.2 Z α1-antitrypsin associated lung disease** 

mortality (Buist et al., 1983; Janus et al., 1985).

1978; Silverman et al., 1989).

point mutations and oxidation (Stein and Carrell, 1995). AT molecules can undergo conformational transitions, which not only inactivate is antiproteinase function, but also confers it with other biological properties.

### **2 Conformations and their effect on structure and function of α1-antirypsin**

#### **2.1 Conformations of α1-antirypsin**

*In vivo* AT can exist in different conformational forms; native, oxidized, polymerized, oxidized-polymers, RCL cleaved and latent, and the AT-elastase complex. The conformational changes can be the result of inflammation, such as the cleavage by nontarget proteinases and oxidation by reactive oxygen species (ROS). Mutaations e.g Z, S predispose it to polymerization. Whilst these conformations result in a loss of proteinase inhibitory activity, they can have biological effects such as inflammatory cell activation and chemotaxis, cytokine release or apoptosis (Janciauskiene, 2001).

#### **2.2 Oxidized AT**

Oxidation of AT is a sulphoxide modification of the methionine residues of AT (Johnson and Travis, 1979; Beatty et al., 1980; Taggart et al., 2000). Methionine can be attacked by various oxidants, such as peroxide, hydroxyl radicals, hypochloride, chloramines and peroxynitrite (Vogt, 1995; Rahman and MacNee, 1996), which are mainly produced by activated inflammatory cells. Oxidized AT (Ox-AT) *in vivo* has been confirmed by the finding that the inactive AT purified from the inflammatory synovial fluid contains methionine sulphoxide residues, and that 41% of total AT in the fluid is inactive, oxidized and/or cleaved (Zhang et al., 1993). Smoking is a major external source of oxidants (Rahman and MacNee, 1996; Church and Pryor, 1985; Schaberg et al., 1992). In smoking-related emphysema, 5-10% of total AT is in the oxidized state (Wong and Travis, 1980). Oxidation of the P1 methionine (M358) significantly reduces the activity of AT against NE to 1/2000 of the normal (Johnson and Travis, 1979). Recent data demonstrates that oxidation of Z-AT promotes polymerization of Z-AT thus increasing the risk of emphysema of Z-AT deficient patients (Alam et al., 2011). Ox-AT has also been shown to stimulate release of MCP-1 and IL-8 from lung epithelial cells (Li et al., 2009) and stimulate monocyte activation, inducing an elevation in MCP-1, IL-6, TNF-α expression and NADPH oxidase activity (Moraga and Janciauskiene., 2004)

#### **2.3 Polymerized form**

The Z-variant accumulates in the hepatocyte involving a process of loop-sheet polymerization whereby the RCL of one molecule inserts into β-sheet A of a second and so on to form chains of Z-AT polymers (Lomas et al., 1992; Mahadeva et al., 1999). M-AT has not been found to polymerize *in vivo* (Mahadeva et al., 2005). Polymerization can occur in other variants of AT, such as Siiyama, Mmalton, I, and S (Mahadeva et al., 1999; Janciauskiene et al., 2004; Elliott et al., 199b). I α1-antitrypsin and S-AT polymerize slower than Z-AT but faster than M-AT, and hence are associated with less severe plasma deficiency (Dafforn et al.,1999).

The occurrence of Z-AT polymerization *in vivo* has been confirmed by the finding of AT polymers in lungs (Elliot et al., 1998b; Mulgrew et al., 2004). Polymers of Z-AT are found in

point mutations and oxidation (Stein and Carrell, 1995). AT molecules can undergo conformational transitions, which not only inactivate is antiproteinase function, but also

**2 Conformations and their effect on structure and function of α1-antirypsin** 

*In vivo* AT can exist in different conformational forms; native, oxidized, polymerized, oxidized-polymers, RCL cleaved and latent, and the AT-elastase complex. The conformational changes can be the result of inflammation, such as the cleavage by nontarget proteinases and oxidation by reactive oxygen species (ROS). Mutaations e.g Z, S predispose it to polymerization. Whilst these conformations result in a loss of proteinase inhibitory activity, they can have biological effects such as inflammatory cell activation and

Oxidation of AT is a sulphoxide modification of the methionine residues of AT (Johnson and Travis, 1979; Beatty et al., 1980; Taggart et al., 2000). Methionine can be attacked by various oxidants, such as peroxide, hydroxyl radicals, hypochloride, chloramines and peroxynitrite (Vogt, 1995; Rahman and MacNee, 1996), which are mainly produced by activated inflammatory cells. Oxidized AT (Ox-AT) *in vivo* has been confirmed by the finding that the inactive AT purified from the inflammatory synovial fluid contains methionine sulphoxide residues, and that 41% of total AT in the fluid is inactive, oxidized and/or cleaved (Zhang et al., 1993). Smoking is a major external source of oxidants (Rahman and MacNee, 1996; Church and Pryor, 1985; Schaberg et al., 1992). In smoking-related emphysema, 5-10% of total AT is in the oxidized state (Wong and Travis, 1980). Oxidation of the P1 methionine (M358) significantly reduces the activity of AT against NE to 1/2000 of the normal (Johnson and Travis, 1979). Recent data demonstrates that oxidation of Z-AT promotes polymerization of Z-AT thus increasing the risk of emphysema of Z-AT deficient patients (Alam et al., 2011). Ox-AT has also been shown to stimulate release of MCP-1 and IL-8 from lung epithelial cells (Li et al., 2009) and stimulate monocyte activation, inducing an elevation in MCP-1, IL-6, TNF-α

The Z-variant accumulates in the hepatocyte involving a process of loop-sheet polymerization whereby the RCL of one molecule inserts into β-sheet A of a second and so on to form chains of Z-AT polymers (Lomas et al., 1992; Mahadeva et al., 1999). M-AT has not been found to polymerize *in vivo* (Mahadeva et al., 2005). Polymerization can occur in other variants of AT, such as Siiyama, Mmalton, I, and S (Mahadeva et al., 1999; Janciauskiene et al., 2004; Elliott et al., 199b). I α1-antitrypsin and S-AT polymerize slower than Z-AT but faster than M-AT, and hence are associated with less severe plasma

The occurrence of Z-AT polymerization *in vivo* has been confirmed by the finding of AT polymers in lungs (Elliot et al., 1998b; Mulgrew et al., 2004). Polymers of Z-AT are found in

confers it with other biological properties.

chemotaxis, cytokine release or apoptosis (Janciauskiene, 2001).

expression and NADPH oxidase activity (Moraga and Janciauskiene., 2004)

**2.1 Conformations of α1-antirypsin** 

**2.2 Oxidized AT** 

**2.3 Polymerized form** 

deficiency (Dafforn et al.,1999).

emphysematous alveolar walls (Mahadeva et al., 2005). Polymers of Z-AT are chemotactic for neutrophils (Parmar et al., 2002; Mulgrew et al., 2004). Polymers of Z-AT are also ineffective anti-inflammatory molecules or inhibitors of NE, (Alam et al., 2011; Bergin et al., 2010; Al-Omari et al., 2011). Recent findings show that ER accumulation of Z-AT polymers is associated with up-regulation of PKR-like ER kinase (PERK), regulator of G-protein signaling (RGS) 16, and calnexin, and NF-B activation and secretion of inflammatory mediators; IL-6 and IL-8 in keeping with activation of the ER overload response (EOR) linked to excess inflammatory activity of the Z-AT cell (Alam et al., Unpublished observation).

#### **3. Mechanisms of disease and pathology**

#### **3.1 Alpha-1-antitrypsin associated diseases**

The normal plasma concentration of AT is about 30 μM, providing 24 μM in lung interstitium, which is thought to be critical in inhibiting elastase. It has been calculated that a concentration of 11 μM of plasma AT is the threshold for providing sufficient AT in the lung (Wewers et al., 1987; Stockely, 2003). Hence, although some phenotypes of AT are associated with abnormally low concentrations of AT in the plasma; PiMS: 80% of normal, PiSS: 60%; PiMZ: 57.5%, only PiSZ, 40%, and PiZ:10-15% and Null variants have been linked to the development of lung disease (Brantly et al., 1988a;b). AT deficiency appears to be underdiagnosed in some populations (Bull World Health Organ, 1997; de Serres, 2003) with only a small proportion of those predicted according to allele frequencies to have AT deficiency to have been identified: 4.5% in UK, 6% in Sweden, and 5% in USA (Tobin et al., 1983; Larsson, 1978; Silverman et al., 1989).

#### **3.2 Z α1-antitrypsin associated lung disease**

Cigarette smoking is the most important independent risk factor for the development of emphysema in the Western world. A landmark study (Fletcher et al., 1977) showed that 15 to 25% of smokers with normal AT develop clinically significant COPD, and that the rate of FEV1 (forced expiratory volume in 1 second) decline was around 50 ml/year in smokers compared with 25 ml/year in non-smokers. Among the AT-deficient population, the decline of FEV1 is 70 ml/year in current smokers compared with 41 ml/year in ex-smokers (Piitulainen and Eriksson, 1999). Smokers with severe deficiency of AT develop symptoms of emphysema 10-15 years earlier than those non-smoking individuals and have a higher mortality (Buist et al., 1983; Janus et al., 1985).

Severe AT deficiency usually due to Z-AT accounts for about 2% of cases of emphysema (Morse, 1978), and has also been linked to asthma and bronchiectasis (Parr et al., 2007; Eden et al., 1997; King et al., 1996; Bleumink and Klokke, 1985). Individuals, who have never smoked, rarely develop symptoms before the age of 50. Twenty-40% of patients have chronic bronchitis and bronchiectasis, and about half have exacerbations (Needham and Stockley, 2004). Most PiZ non-index cases have normal or slightly abnormal lung function in the absence of symptoms (Tobin et al., 1983). The development of lung disease is intimately related to cigarette smoking. However, the severity of lung disease can show some variability: lung function is well maintained in some AT-deficient smokers, while can be impaired in non-smokers (Brantly et al., 1988b; Janus et al., 1985). It is also

The Role of Alpha–1 Antitrypsin in Emphysema 57

individuals tends to have more polymorpholeucocytes compared with M-AT emphysema

The main mechanism contributing to the development of emphysema in individuals with Z-AT is the imbalance of AT-elastase, in favour of elastase caused by severe AT deficiency. It is now well established that the conformational changes originating from this mutation predispose Z-AT molecules to irreversible polymerization, with consequent accumulation within the ER of hepatocytes (Lomas et al., 1992; Mahadeva et al., 2002). As a consequence, only approximately 15% of the molecules produced reach the circulation. In addition, in the presence of cigarette smoking a major portion of these secreted proteins has been shown to be either oxidized monomeric AT or in its polymeric form (Alam et al., 2011), which are inactive as proteinase inhibitors. Z-AT also has a reduced activity against elastase (Oakeshhott et al., 1985; Lomas et al., 2003). The inactivation of AT as in M-AT related emphysema can also occur by cleavage by non-target proteinases. The end result of these

Polymeric conformation of Z-AT has also been found in Z-AT emphysematous lungs in association with neutrophils (Mahadeva et al., 2005). Polymers of Z-AT are also thought to contribute the inflammation and lung damage in emphysema. Polymers of Z-AT are thought to be produced locally within the lung, however, a recent study reported finding of polymers of Z-AT not only in the lung, but also in the serum of transgenic mice expressing human Z-AT that had been exposed to cigarette smoke (Alam et al., 2011) (Section 3.4). Formation of Z-AT polymers may be accelerated by local inflammation e.g. bacterial infection. The polymers are themselves chemotactic for human neutrophils *in vitro* and *in vivo* and are co-localized with neutrophils in the alveoli of individuals with Z-AT-related emphysema (Elliott et al., 1998a; Parmar et al., 2002; Mulgrew et al., 2004). The transition of native Z-AT to polymers inactivates its anti-proteinase and anti-inflammatory function, and also converts it to a pro-inflammatory stimulus and may explain the excess numbers of neutrophils in bronchoalveolar lavage fluid (BALF) and lung tissue from Z-AT homozygotes (Morrison et al., 1987; Mahadeva et al., 2005) and in transgenic Z-AT mice (Alam et al., 2011). The presence of polymers may also contribute to the progression of PiZ

Z-AT related emphysema is potentiated by cigarette smoking, characteristically occurring in the third to fourth decade compared with fifth to sixth decade in non-smokers (Luisetti and Seersholme, 2004; Evald et al., 1990). The mechanism of accelerated decline in smokers with Z-AT is in part due to the independent effects of cigarette smoke, but also due to oxidation of Z-AT which promotes polymerization (production of oxidized polymers) of Z-AT (Fig. 2) (Alam et al., 2011). Polymers are inactive as an anti-elastase, and are not only unable to perform their normal anti-inflammatory role, but are also chemotactic for neutrophils (Alam et al., 2011; Mulgrew et al., 2004; Morrison et al., 1987; Parmar et al., 2002; Bergin et al., 2010; Al-Omari et al., 2011). The acceleration of COPD by cigarette smoke in Z-AT individuals exemplifies the critical importance of gene-environmental interactions to the development of COPD. This provides a molecular explanation for the striking association of premature

processes is a further reduction in the quantity of functional AT.

lung disease after smoking cessation.

**3.5 Cigarette smoking and emphysema** 

emphysema in ZZ homozygotes who smoke.

(Morrison et al., 1987).

postulated that host factors, such as individual bronchodilator reversibility, baseline lung function, sex, age, and other unidentified genetic factors as well as other environmental factor such as dust-exposure and recurrent respiratory infections may influence the clinical phenotype (Needham and Stockley, 2004). A recent familial study estimated heritability for FEV1/forced vital capacity (FVC) in 378 ZZ homozygotes from 167 families identified cigarette smoking as the genetic modifier in the pathogenesis and severity of COPD (DeMeo et al., 2009).

#### **3.3 MZ α1-antitrypsin associated lung disease**

The serum levels of AT in MZ heterozygotes is lower than in MM homozygotes (Section 1.4), but whether MZ individuals have an increased risk of COPD remains controversial. Increased COPD risk in this group may have public health implications because there are about 117 million of MZ and MS phenotypes worldwide (de Serres 2002; Brantly et al., 1991). Many studies have addressed the risk of lung function reduction and increased risk of COPD in MZ heterozygotes, but the results have not been consistent. A meta-analysis demonstrated increased risk of COPD in MZ compared to MM, but there was no difference in mean FEV1 between MZ and MM individuals when combining the results from population-based studies (Hersh et al., 2004), which is in agreement with a cohort of MZ heterozygotes analyzed from the Danish Alpha-1-Antitrypsin Deficiency Registry (Seersholm et al., 2000) and a longitudinal study of the general population in Arizona (Silva et al., 2003). Many of the previous studies have been limited by small sample sizes, varying phenotype definitions, or failure to adjust for smoking. However, recent studies investigated two large, well characterized populations of current and ex-smokers, a casecontrol study and a multicenter family-based study using quantitative CT scan measurements of emphysema and airway disease, established an association of reduced FEV1/FVC in MZ compared to MM (Sandhaus et al., 2008; Sørheim et al., 2010). This suggests that at least some MZ heterozygotes are more susceptible to the development of COPD. Interestingly, MZ with a low smoking history (<20 pack-year) had more severe emphysema on chest CT scan. It remains to be established whether all MZ individuals have an increased risk or whether a subset is more susceptible because of other genetic or environmental factors.

#### **3.4 Mechanism of Z α1-antitrypsin-related emphysema**

The majority of emphysema occurs in cigarette smokers who have normal AT concentrations and smoking can injure the lungs by many mechanisms, such as, A. increasing the oxidant burden (Alam et al., 2011; Church and Pryor, 1985; Carp and Janoff, 1978); B. direct stimulation of neutrophils and macrophages to produce proteinases (Bracke et al., 2005; Hautamaki et al., 1997); C. inactivation of AT and other proteinase inhibitors by oxidation (Alam et al., 2011; Wong and Travis, 1980); D. interfering the repairing process by repeated damage (Janoff et al., 1983). These mechanisms can all occur in Z-AT individuals. However, there are some noteable differences in emphysema due to Z-AT compared to those with normal AT. Firstly, the emphysema has a predilection (although not exclusively) at least initially to affect the lower lobes in Z-AT emphysema compared with the upper lobes in M-AT emphysema. Secondly, the emphysema in Z-AT

postulated that host factors, such as individual bronchodilator reversibility, baseline lung function, sex, age, and other unidentified genetic factors as well as other environmental factor such as dust-exposure and recurrent respiratory infections may influence the clinical phenotype (Needham and Stockley, 2004). A recent familial study estimated heritability for FEV1/forced vital capacity (FVC) in 378 ZZ homozygotes from 167 families identified cigarette smoking as the genetic modifier in the pathogenesis and severity of

The serum levels of AT in MZ heterozygotes is lower than in MM homozygotes (Section 1.4), but whether MZ individuals have an increased risk of COPD remains controversial. Increased COPD risk in this group may have public health implications because there are about 117 million of MZ and MS phenotypes worldwide (de Serres 2002; Brantly et al., 1991). Many studies have addressed the risk of lung function reduction and increased risk of COPD in MZ heterozygotes, but the results have not been consistent. A meta-analysis demonstrated increased risk of COPD in MZ compared to MM, but there was no difference in mean FEV1 between MZ and MM individuals when combining the results from population-based studies (Hersh et al., 2004), which is in agreement with a cohort of MZ heterozygotes analyzed from the Danish Alpha-1-Antitrypsin Deficiency Registry (Seersholm et al., 2000) and a longitudinal study of the general population in Arizona (Silva et al., 2003). Many of the previous studies have been limited by small sample sizes, varying phenotype definitions, or failure to adjust for smoking. However, recent studies investigated two large, well characterized populations of current and ex-smokers, a casecontrol study and a multicenter family-based study using quantitative CT scan measurements of emphysema and airway disease, established an association of reduced FEV1/FVC in MZ compared to MM (Sandhaus et al., 2008; Sørheim et al., 2010). This suggests that at least some MZ heterozygotes are more susceptible to the development of COPD. Interestingly, MZ with a low smoking history (<20 pack-year) had more severe emphysema on chest CT scan. It remains to be established whether all MZ individuals have an increased risk or whether a subset is more susceptible because of other genetic or

The majority of emphysema occurs in cigarette smokers who have normal AT concentrations and smoking can injure the lungs by many mechanisms, such as, A. increasing the oxidant burden (Alam et al., 2011; Church and Pryor, 1985; Carp and Janoff, 1978); B. direct stimulation of neutrophils and macrophages to produce proteinases (Bracke et al., 2005; Hautamaki et al., 1997); C. inactivation of AT and other proteinase inhibitors by oxidation (Alam et al., 2011; Wong and Travis, 1980); D. interfering the repairing process by repeated damage (Janoff et al., 1983). These mechanisms can all occur in Z-AT individuals. However, there are some noteable differences in emphysema due to Z-AT compared to those with normal AT. Firstly, the emphysema has a predilection (although not exclusively) at least initially to affect the lower lobes in Z-AT emphysema compared with the upper lobes in M-AT emphysema. Secondly, the emphysema in Z-AT

COPD (DeMeo et al., 2009).

environmental factors.

**3.4 Mechanism of Z α1-antitrypsin-related emphysema** 

**3.3 MZ α1-antitrypsin associated lung disease** 

individuals tends to have more polymorpholeucocytes compared with M-AT emphysema (Morrison et al., 1987).

The main mechanism contributing to the development of emphysema in individuals with Z-AT is the imbalance of AT-elastase, in favour of elastase caused by severe AT deficiency. It is now well established that the conformational changes originating from this mutation predispose Z-AT molecules to irreversible polymerization, with consequent accumulation within the ER of hepatocytes (Lomas et al., 1992; Mahadeva et al., 2002). As a consequence, only approximately 15% of the molecules produced reach the circulation. In addition, in the presence of cigarette smoking a major portion of these secreted proteins has been shown to be either oxidized monomeric AT or in its polymeric form (Alam et al., 2011), which are inactive as proteinase inhibitors. Z-AT also has a reduced activity against elastase (Oakeshhott et al., 1985; Lomas et al., 2003). The inactivation of AT as in M-AT related emphysema can also occur by cleavage by non-target proteinases. The end result of these processes is a further reduction in the quantity of functional AT.

Polymeric conformation of Z-AT has also been found in Z-AT emphysematous lungs in association with neutrophils (Mahadeva et al., 2005). Polymers of Z-AT are also thought to contribute the inflammation and lung damage in emphysema. Polymers of Z-AT are thought to be produced locally within the lung, however, a recent study reported finding of polymers of Z-AT not only in the lung, but also in the serum of transgenic mice expressing human Z-AT that had been exposed to cigarette smoke (Alam et al., 2011) (Section 3.4). Formation of Z-AT polymers may be accelerated by local inflammation e.g. bacterial infection. The polymers are themselves chemotactic for human neutrophils *in vitro* and *in vivo* and are co-localized with neutrophils in the alveoli of individuals with Z-AT-related emphysema (Elliott et al., 1998a; Parmar et al., 2002; Mulgrew et al., 2004). The transition of native Z-AT to polymers inactivates its anti-proteinase and anti-inflammatory function, and also converts it to a pro-inflammatory stimulus and may explain the excess numbers of neutrophils in bronchoalveolar lavage fluid (BALF) and lung tissue from Z-AT homozygotes (Morrison et al., 1987; Mahadeva et al., 2005) and in transgenic Z-AT mice (Alam et al., 2011). The presence of polymers may also contribute to the progression of PiZ lung disease after smoking cessation.

#### **3.5 Cigarette smoking and emphysema**

Z-AT related emphysema is potentiated by cigarette smoking, characteristically occurring in the third to fourth decade compared with fifth to sixth decade in non-smokers (Luisetti and Seersholme, 2004; Evald et al., 1990). The mechanism of accelerated decline in smokers with Z-AT is in part due to the independent effects of cigarette smoke, but also due to oxidation of Z-AT which promotes polymerization (production of oxidized polymers) of Z-AT (Fig. 2) (Alam et al., 2011). Polymers are inactive as an anti-elastase, and are not only unable to perform their normal anti-inflammatory role, but are also chemotactic for neutrophils (Alam et al., 2011; Mulgrew et al., 2004; Morrison et al., 1987; Parmar et al., 2002; Bergin et al., 2010; Al-Omari et al., 2011). The acceleration of COPD by cigarette smoke in Z-AT individuals exemplifies the critical importance of gene-environmental interactions to the development of COPD. This provides a molecular explanation for the striking association of premature emphysema in ZZ homozygotes who smoke.

The Role of Alpha–1 Antitrypsin in Emphysema 59

year (Gildea et al., 2003). There is no definitive evidence to suggest superiority (specific functional inhibitor activity) of any one of the formulation comparing them to Prolastin. Prolastin was the first approved human purified plasma AT, which is usually administrated intravenously at 60 mg/kg weekly. This dose increased the serum AT level and remained above the putative protective threshold level of 11µM/L after 3 weeks of infusion. However only 2% of the infused purified plasma AT drug reaches to the lung and therefore administration *via* aerosol has also been assessed (Wencker et al., 1998; Hubbard et al., 1989; Smith et al., 1989). Some positive effects of augmentation therapy have been observed in those with moderately impaired lung function (FEV1 30-65%) (Alpha-1-antitrypsin-Deficiency-Study-Group, 1998; Abusriwil and Stockley, 2006), and some studies have also demonstrated that the treatment reduces airway LTB4, which plays important role in exacerbations (Stockley et al., 2002). However, the therapeutic effect of augmentation therapy is debated due to the lack of a randomized controlled clinical trial (Burrows, 1983; Wewers and Gadek, 1987). There are however problems with conducting such studies: in particular the large numbers of patients with this rare disease required for placebocontrolled and randomized clinical trials; the length of follow-up required to assess efficacy

and the limited supply and cost of the treatment (Abusriwil and Stockley, 2006).

preparation (viral inactivation)

Solvent detergent and nanofiltration

detergent and nanofiltration

Table 2. Preparations of purified human plasma antitrypsin are available world wide Modrykamien and Stoller, 2009; Stockley et al., 2010; Louie et al., 2005; Barker et al., 1997.

Minimum specific functional inhibitor activity per mg total protein

Pasteurization ≥ 0.7 mg US

≥ 0.55 mg US

≥ 0.7 mg Argentina, Brazil,

Chile, Mexico, Spain,

Pasteurization ≥ 0.35 mg Argentina, Austria,

Countries approved for use

Bahamas, Barbados, Belgium, Bermuda, Canada, Denmark, Finland, Germany, Greece, Guam, Ireland, Italy, Netherlands, Norway, Oman, Poland, Portugal, Puerto Rico, Qatar, Spain, Sweden, Switzerland, US

Drug Manufacturer Method of

Biotherapeutics, Research Traingle Park, NC

King of Prussia, PA

Baxter, Deerfield, IL

Trypsone® Grifols, SA. Solvent

Prolastin® Talecris

ZemairaTM CSL Behring,

AralastTM (which was initially called Respitin)

Proposed model for the pathogenesis of emphysema in patients with Z-AT deficiency. Cigarette smoke induces oxidation of Z-AT, which accelerates Z-AT polymerization. Plasma deficiency and reduced inhibitory activity of Z-AT would be exacerbated by the oxidation and polymerization of AT within the lungs, thereby further reducing the antiproteinase screen. Conversion from a monomer to a polymer results in a loss of anti-inflammatory effect. Z-AT polymers also act as a pro-inflammatory stimulus to attract and activate neutrophils, resulting in further increase in neutrophils and liberation of NE thereby imbalance of AT-elastase in the favour of NE leading to tissue damage and subsequently causing emphysema. Adapted from Alam et al., 2011.

Fig. 2. Schematic diagram depicting the role of conformations of α1-antitrypsin and the interaction with cigarette smoke in the development of emphysema.

#### **4. Prognosis and review of current treatments**

#### **4.1 Treatment of Z α1-antitrypsin-associated lung disease**

The major goals in the management of patients with Z-AT related emphysema are to prevent lung disease, and to reduce progression of the disease. Smoking cessation and standard management for COPD with normal AT levels is of crucial importance once the diagnosis has been made. Repeated respiratory infections can lead to permanent lung injury in patients with Z-AT deficiency. Thus, reducing exacerbation rate is also essential.

Purified plasma AT (half-life of 4.5 day) and recombinant AT (half-life of a few hours) are both commercially available. Currently four different preparations of purified plasma AT are available worldwide; Prolastin®, ZemairaTM, AralastTM and Trypsone® and have been approved for use by the regulatory agencies in several countries (Table 2). The former three preparations are available in the United States at an estimated cost of \$60,000 to \$150,000 per

Proposed model for the pathogenesis of emphysema in patients with Z-AT deficiency. Cigarette smoke induces oxidation of Z-AT, which accelerates Z-AT polymerization. Plasma deficiency and reduced inhibitory activity of Z-AT would be exacerbated by the oxidation and polymerization of AT within the lungs, thereby further reducing the antiproteinase screen. Conversion from a monomer to a polymer results in a loss of anti-inflammatory effect. Z-AT polymers also act as a pro-inflammatory stimulus to attract and activate neutrophils, resulting in further increase in neutrophils and liberation of NE thereby imbalance of AT-elastase in the favour of NE leading to tissue damage and subsequently causing

Fig. 2. Schematic diagram depicting the role of conformations of α1-antitrypsin and the

The major goals in the management of patients with Z-AT related emphysema are to prevent lung disease, and to reduce progression of the disease. Smoking cessation and standard management for COPD with normal AT levels is of crucial importance once the diagnosis has been made. Repeated respiratory infections can lead to permanent lung injury

Purified plasma AT (half-life of 4.5 day) and recombinant AT (half-life of a few hours) are both commercially available. Currently four different preparations of purified plasma AT are available worldwide; Prolastin®, ZemairaTM, AralastTM and Trypsone® and have been approved for use by the regulatory agencies in several countries (Table 2). The former three preparations are available in the United States at an estimated cost of \$60,000 to \$150,000 per

in patients with Z-AT deficiency. Thus, reducing exacerbation rate is also essential.

interaction with cigarette smoke in the development of emphysema.

**4. Prognosis and review of current treatments** 

**4.1 Treatment of Z α1-antitrypsin-associated lung disease** 

emphysema. Adapted from Alam et al., 2011.

year (Gildea et al., 2003). There is no definitive evidence to suggest superiority (specific functional inhibitor activity) of any one of the formulation comparing them to Prolastin. Prolastin was the first approved human purified plasma AT, which is usually administrated intravenously at 60 mg/kg weekly. This dose increased the serum AT level and remained above the putative protective threshold level of 11µM/L after 3 weeks of infusion. However only 2% of the infused purified plasma AT drug reaches to the lung and therefore administration *via* aerosol has also been assessed (Wencker et al., 1998; Hubbard et al., 1989; Smith et al., 1989). Some positive effects of augmentation therapy have been observed in those with moderately impaired lung function (FEV1 30-65%) (Alpha-1-antitrypsin-Deficiency-Study-Group, 1998; Abusriwil and Stockley, 2006), and some studies have also demonstrated that the treatment reduces airway LTB4, which plays important role in exacerbations (Stockley et al., 2002). However, the therapeutic effect of augmentation therapy is debated due to the lack of a randomized controlled clinical trial (Burrows, 1983; Wewers and Gadek, 1987). There are however problems with conducting such studies: in particular the large numbers of patients with this rare disease required for placebocontrolled and randomized clinical trials; the length of follow-up required to assess efficacy and the limited supply and cost of the treatment (Abusriwil and Stockley, 2006).


Table 2. Preparations of purified human plasma antitrypsin are available world wide Modrykamien and Stoller, 2009; Stockley et al., 2010; Louie et al., 2005; Barker et al., 1997.

The Role of Alpha–1 Antitrypsin in Emphysema 61

improvement and better quality of life are clear benefits deriving from lung transplantation, while a survival advantage has not yet been proven (Marulli and Rea, 2008). Studies have shown advantage of single versus double lung transplantation for COPD or AT deficiency. However, a common cause of death PiZ post transplantation was due to pulmonary infection and bronchiolitis obliterans syndrome (BOS), and sepsis in the presence of excess NE (Meyer et al., 2001; Tanash et al., 2011; de Perrot et al., 2004). A recent study analyzed a total of 83 PiZZ patients with severe emphysema who underwent lung transplantation between 1990 and June 2010 compared to 70 age, gender, smoking history and lung function matched controls (Tanash et al., 2011). Of 83 transplanted patients, 62 (75%) underwent single-lung transplantation. During follow-up, 37 (45%) deaths occurred in transplanted patients and 45 (64%) in the non transplanted patients. In the transplanted patients, the estimated median survival time was 11 years (95% confidence interval [CI] 9 to 14 years), compared with 8 years (95% CI 4 to 6 years) for the non transplanted patients (p = 0.006) (Tanash et al., 2011). Constant annual death rates due to BOS and other complications result in a 50% 5-year survival (Patterson and Cooper, 1995). In addition, Mal and colleagues (Mal et al., 2004) have shown an association between cigarette smoking induced NE activity and recurrence of pulmonary emphysema in the transplanted lung of a 49 year old PiZ patient 11 years after receiving single lung transplant. Therefore, lung transplantation should only

A major distinction between pathogenesis of lung and liver disease in Z-AT deficiency is loss of function and gain of function, respectively. In liver disease it relates to the intracellular accumulation of misfolded and unsecreted AT from hepatocytes rather than unopposed elastolysis in the lung due to lack of AT. Therefore, augmentation therapy does not confer protect against and not indicated for liver disease relating to severe AT deficiency. Other strategies have been assessed for treatment of Z-AT related liver disease including targeting a lateral hydrophobic cavity to prevent polymerization, and enhancing clearance of Z-AT aggregates by drugs promoting autophagy (Zhou et al., 2004; Burrows et al., 2000; Mallya et al., 2007; Hedvegi et al., 2010; Devlin et al., 2001; Kaushal et al., 2010). These methods reduce intracellular aggregation of Z-AT but do not increase the secretion Z-AT. Use of short synthetic peptides targeting β-sheet A may show therapeutic potential for Z-AT related liver and lung disease (Figure 3) (Chang et al., 2006; 2009; Alam et al.,

Future therapies for α1-antitrypsin deficiency include gene therapy. Supplementing AT by gene delivery is an alternative way to increase the local AT in lung. Preclinical studies have shown that the Adeno-associated viral vector is capable in increasing the AT concentration to over 11μM in the lung. The safety and efficiency of this approach is under evaluation (Flotte, 2002; Flotte et al., 2004; Stecenko and Brigham, 2003; Flotte and

be offered to selected candidates.

Unpublished observation).

**5.1 Gene therapy** 

Mueller, 2011).

**5. Novel treatments in development** 

**4.4 Treatment of Z α1-antitrypsin-associated liver disease** 

A recent study analyzed results from two randomized, double-blind, placebo-controlled trials to date; a 2-center Danish-Dutch study (n = 54) and the 3-center EXAcerbations and CT scan as Lung Endpoints (EXACTLE) study (n = 65) (Stockley et al., 2010). The study investigated the efficacy of IV AT augmentation therapy on emphysema progression using CT densitometry over an average mean follow-up of about 2.5 years. The study confirmed that IV augmentation therapy significantly reduces the decline in lung density. Decline from baseline to last CT scan was -4.082 g/L versus -6.379 g/L for placebo, with the treatment difference of 2.297 (95% CI, 0.669 to 3.926; p=0.006), the corresponding annual declines were -1.73 and -2.74 g/L/yr, respectively) and may therefore reduce the future risk of mortality in patients with AT deficiency related emphysema, in favour of IV AT augmentation therapy.

There is no evidence that IV augmentation therapy with purified plasma AT preparations is effective in MZ genotypes. MZ patients are at risk for accelerated airflow obstruction/lung disease as mentioned above and that augmentation therapy in MZ patients can be associated with side effects (Stoller et al., 2003/2009). The Medical and Scientific Advisory Committee of the Alpha-1 Foundation (Sandhaus et al., 2008) concluded that augmentation therapy for MZ phenotypes should be avoided.

#### **4.2 Alpha-1 antitrypsin deficiency and Lung volume reduction surgery**

Recently lung volume reduction surgery (LVRS) has been proposed as a treatment for severe emphysema. Over the years studies reported both in favour and against LVRS in AT deficient patients (Dauriat et al., 2006; Tutic et al., 2004). Because LVRS offers only shortterm benefits for most AT deficient patients LVRS should not be recommended in these patients pending additional studies (ATS–ERS, 2003). This data is further supported by landmark studies from the National Emphysema Treatment Trial (NETT) (Fishman et al 2003; Stoller et al 2007) that included 1218 randomized subjects and 10 who were randomized had severe Z-AT deficiency and underwent LVRS. Deficient individuals had a shorter duration in FEV1 rise, smaller increase in exercise capacity at 6 months, and higher mortality (20% vs. 0% compared with medical treatment) after 2 years. Although these conclusions are inherently limited by the small number of patients analyzed, LVRS cannot clearly be recommended for this population based on the above data (Stoller et al 2007). In addition, most patients with Z-AT deficiency have lower lobe predominant emphysema, which showed the least surgical benefit in NETT (leading to worse outcomes in good exercise capacity patients) (Stoller et al 2007). Although LVRS has small functional gains and a shorter-lasting effect in AT deficient patients than in patients with normal AT emphysema, it could potentially serve as a bridging procedure that postpones the need for lung transplantation (Dauriat et al., 2006; Tutic et al., 2004). Emerging techniques for bronchoscopic lung volume reduction are covered in another chapter.

#### **4.3 Alpha-1 antitrypsin deficiency and Lung transplantation**

End stage pulmonary emphysema is the most common indication for lung transplantation worldwide. Lung transplantation is considered in patients with declining lung function or symptomatic patients with a poor quality of life after receiving all conservative treatment options including smoking cessation and rehabilitation programmes. A functional

A recent study analyzed results from two randomized, double-blind, placebo-controlled trials to date; a 2-center Danish-Dutch study (n = 54) and the 3-center EXAcerbations and CT scan as Lung Endpoints (EXACTLE) study (n = 65) (Stockley et al., 2010). The study investigated the efficacy of IV AT augmentation therapy on emphysema progression using CT densitometry over an average mean follow-up of about 2.5 years. The study confirmed that IV augmentation therapy significantly reduces the decline in lung density. Decline from baseline to last CT scan was -4.082 g/L versus -6.379 g/L for placebo, with the treatment difference of 2.297 (95% CI, 0.669 to 3.926; p=0.006), the corresponding annual declines were -1.73 and -2.74 g/L/yr, respectively) and may therefore reduce the future risk of mortality in patients with AT deficiency related emphysema, in favour of IV AT augmentation therapy. There is no evidence that IV augmentation therapy with purified plasma AT preparations is effective in MZ genotypes. MZ patients are at risk for accelerated airflow obstruction/lung disease as mentioned above and that augmentation therapy in MZ patients can be associated with side effects (Stoller et al., 2003/2009). The Medical and Scientific Advisory Committee of the Alpha-1 Foundation (Sandhaus et al., 2008) concluded that augmentation

therapy for MZ phenotypes should be avoided.

**4.2 Alpha-1 antitrypsin deficiency and Lung volume reduction surgery** 

bronchoscopic lung volume reduction are covered in another chapter.

**4.3 Alpha-1 antitrypsin deficiency and Lung transplantation** 

Recently lung volume reduction surgery (LVRS) has been proposed as a treatment for severe emphysema. Over the years studies reported both in favour and against LVRS in AT deficient patients (Dauriat et al., 2006; Tutic et al., 2004). Because LVRS offers only shortterm benefits for most AT deficient patients LVRS should not be recommended in these patients pending additional studies (ATS–ERS, 2003). This data is further supported by landmark studies from the National Emphysema Treatment Trial (NETT) (Fishman et al 2003; Stoller et al 2007) that included 1218 randomized subjects and 10 who were randomized had severe Z-AT deficiency and underwent LVRS. Deficient individuals had a shorter duration in FEV1 rise, smaller increase in exercise capacity at 6 months, and higher mortality (20% vs. 0% compared with medical treatment) after 2 years. Although these conclusions are inherently limited by the small number of patients analyzed, LVRS cannot clearly be recommended for this population based on the above data (Stoller et al 2007). In addition, most patients with Z-AT deficiency have lower lobe predominant emphysema, which showed the least surgical benefit in NETT (leading to worse outcomes in good exercise capacity patients) (Stoller et al 2007). Although LVRS has small functional gains and a shorter-lasting effect in AT deficient patients than in patients with normal AT emphysema, it could potentially serve as a bridging procedure that postpones the need for lung transplantation (Dauriat et al., 2006; Tutic et al., 2004). Emerging techniques for

End stage pulmonary emphysema is the most common indication for lung transplantation worldwide. Lung transplantation is considered in patients with declining lung function or symptomatic patients with a poor quality of life after receiving all conservative treatment options including smoking cessation and rehabilitation programmes. A functional improvement and better quality of life are clear benefits deriving from lung transplantation, while a survival advantage has not yet been proven (Marulli and Rea, 2008). Studies have shown advantage of single versus double lung transplantation for COPD or AT deficiency. However, a common cause of death PiZ post transplantation was due to pulmonary infection and bronchiolitis obliterans syndrome (BOS), and sepsis in the presence of excess NE (Meyer et al., 2001; Tanash et al., 2011; de Perrot et al., 2004). A recent study analyzed a total of 83 PiZZ patients with severe emphysema who underwent lung transplantation between 1990 and June 2010 compared to 70 age, gender, smoking history and lung function matched controls (Tanash et al., 2011). Of 83 transplanted patients, 62 (75%) underwent single-lung transplantation. During follow-up, 37 (45%) deaths occurred in transplanted patients and 45 (64%) in the non transplanted patients. In the transplanted patients, the estimated median survival time was 11 years (95% confidence interval [CI] 9 to 14 years), compared with 8 years (95% CI 4 to 6 years) for the non transplanted patients (p = 0.006) (Tanash et al., 2011). Constant annual death rates due to BOS and other complications result in a 50% 5-year survival (Patterson and Cooper, 1995). In addition, Mal and colleagues (Mal et al., 2004) have shown an association between cigarette smoking induced NE activity and recurrence of pulmonary emphysema in the transplanted lung of a 49 year old PiZ patient 11 years after receiving single lung transplant. Therefore, lung transplantation should only be offered to selected candidates.

#### **4.4 Treatment of Z α1-antitrypsin-associated liver disease**

A major distinction between pathogenesis of lung and liver disease in Z-AT deficiency is loss of function and gain of function, respectively. In liver disease it relates to the intracellular accumulation of misfolded and unsecreted AT from hepatocytes rather than unopposed elastolysis in the lung due to lack of AT. Therefore, augmentation therapy does not confer protect against and not indicated for liver disease relating to severe AT deficiency. Other strategies have been assessed for treatment of Z-AT related liver disease including targeting a lateral hydrophobic cavity to prevent polymerization, and enhancing clearance of Z-AT aggregates by drugs promoting autophagy (Zhou et al., 2004; Burrows et al., 2000; Mallya et al., 2007; Hedvegi et al., 2010; Devlin et al., 2001; Kaushal et al., 2010). These methods reduce intracellular aggregation of Z-AT but do not increase the secretion Z-AT. Use of short synthetic peptides targeting β-sheet A may show therapeutic potential for Z-AT related liver and lung disease (Figure 3) (Chang et al., 2006; 2009; Alam et al., Unpublished observation).

#### **5. Novel treatments in development**

#### **5.1 Gene therapy**

Future therapies for α1-antitrypsin deficiency include gene therapy. Supplementing AT by gene delivery is an alternative way to increase the local AT in lung. Preclinical studies have shown that the Adeno-associated viral vector is capable in increasing the AT concentration to over 11μM in the lung. The safety and efficiency of this approach is under evaluation (Flotte, 2002; Flotte et al., 2004; Stecenko and Brigham, 2003; Flotte and Mueller, 2011).

The Role of Alpha–1 Antitrypsin in Emphysema 63

polymerizes within the liver and this accounts for its severe plasma deficiency, and α1 antitrypsin polymers may have a role in the progression of emphysema, but this requires further investigation. Recent and ongoing studies should clarify the role of augmentation therapy and lung volume reduction in subgroups of PiZZ homozygotes, and the understanding of polymer formation has raised the exciting prospect of developing new

Alam, S., Li, Z., Janciauskiene, S. & Mahadeva, R. (2011) *Am J Respir Cell Mol Biol* 45(2):261-

ATS-ERS; American Thoracic Society/European Respiratory Society statement (2003) *Am J* 

Bergin, D. A., Reeves, E. P., Meleady, P., Henry, M., McElvaney, O. J., Carroll, T. P.,

Bracke, K., Cataldo, D., Maes, T., Gueders, M., Noel, A., Foidart, J. M., Brusselle, G. &

Brantly, M. L., Paul, L. D., Miller, B. H., Falk, R. T., Wu, M. & Crystal, R. G. (1988b) *Am Rev* 

Brantly, M. L., Wittes, J. T., Vogelmeier, C. F., Hubbard, R. C., Fells, G. A. & Crystal, R. G.

Buist, A. S., Burrows, B., Eriksson, S., Mittman, C., & Wu, M. (1983) *Am Rev Resp Dis* 127(2),

Chang, Y. P., Mahadeva, R., Chang, W. S., Shukla, A., Dafforn, T. R., & Chu, Y. H. (2006)*Am* 

Chang, Y. P., Mahadeva, R., Chang, W. S., Lin, S. C. & Chu, Y. H. (2009) *J Cell Mol Med*

Burrows, J. A., Willis, L. K. & Perlmutter, D. H. (2000) *PNAS USA* 97(4), 1796-1801

Condron, C., Chotirmall, S. H., Clynes, M., O'Neill, S. J. & McElvaney, N. G. (2010) *J* 

Alam, S., Wang, J., Janciauskiene, S. & Mahadeva R, (2011) (Unpublished observation). Al-Omari, M., Korenbaum, E., Ballmaier, M., Lehmann, U., Jonigk, D., Manstein, D. J., Welte, T., Mahadeva, R. & Janciauskiene, S. (2011) *Mol Med.* [Epub ahead of print] Alpha-1-antitrypsin-Deficiency-Registry-Study-Group. (1998) *Am J Respir Crit Care Med* 

Barker, A.F., Iwata-Morgan, I., Oveson, L. & Roussel R. (1997) *Chest* 112(4):872-4.

therapeutic strategies for the liver and lung disease associated with Z α1-antitrypsin.

Abusriwil, H. & Stockley, R. A. (2006) *Cur Opin Pul Med* 12(2), 125-131

*Respir Crit Care Med* 168(7):818-900.

Beatty, K., Bieth, J. & Travis J. (1980) *J Biol Chem* 255: 3931–3934.

Bleumink, E. & Klokke, A. H. (1985) *Arch Dermatol Res* 1985;277(4):328-9.

Pauwels, R. A. (2005) *Int Arch Allerg and Imm* 138(2), 169-179 Brantly, M., Nukiwa, T. & Crystal, R. G. (1988a) *Am J Med* 84(6A), 13-31

Baur, X. & Bencze, K. (1987) *Respir* 51(3), 188-195

*Clin Invest* 120(12):4236-5420.

*Respir Dis* 138(2), 327-336

(1991) *Chest* 100(3):703-708. Brennan, S. (2007) *Eur Respir J* 29(2), 229-230 *Bull World Health Organ* (1997) 75(5):397–415.

Carell, R. W. (1990) *Lung* 168 Suppl, 530-534

13(8B):2304-2316.

Burrows, B. (1983) *Am Rev Respir Dis* 127(2), S42-43

*J Respir Cell Mol Biol* 35(5), 540-548

Carp, H. & Janoff, A. (1978) *Am Rev Resp Dis* 118(3), 617-621

**7. References** 

269.

158(1)

S43-45

**B.** Polymer inhibition by targeting β-sheet A

A. Z mutation (E342K) perturbs the structure of AT to allow opening of β-sheet A, which then accepts the RCL of another molecule to form a dimer (*left*) that can extend to form chains of polymers as depicted in Fig. 1. 12-mer peptide can anneal to β-sheet A thereby preventing polymer formation (*right*). B. Z mutation allows partial insertion of the RCL. This opens the lower part of β-sheet A thereby favouring polymerization (*left*). Understanding the configuration of the reactive loop and interacting with β-sheet A prompted the hypothesis that a 6-mer with homology to P7-2 of the RCL would specifically bind to Z-AT and so prevent polymerization and explained why the 12-mer peptide preferentially bound to M-AT (*right*). Reproduced and adapted from Lomas and Mahadeva, 2002; Mahadeva et al., 2002; Chang et al., 2009.

Fig. 3. Representation Z α1-antitrypsin polymerization and the design of a selective inhibitor.

#### **6. Summary**

Alpha-1-antitrypsin is the most important proteinase inhibitor in maintaining the proteinase/antiproteinase balance within the lung. The recognition of the association between plasma deficiency of α1-antitrypsin and emphysema over 40 years ago has led to the proteinase-antiproteinase hypothesis of lung disease which remains central to understanding lung biology. In the last 20 years there has been significant progress in our understanding of α1-antitrypsin. Alpha-1 antitrypsin may modulate other biological processes such as apoptosis and inflammatory cell recruitment. Z α1-antitrypsin polymerizes within the liver and this accounts for its severe plasma deficiency, and α1 antitrypsin polymers may have a role in the progression of emphysema, but this requires further investigation. Recent and ongoing studies should clarify the role of augmentation therapy and lung volume reduction in subgroups of PiZZ homozygotes, and the understanding of polymer formation has raised the exciting prospect of developing new therapeutic strategies for the liver and lung disease associated with Z α1-antitrypsin.

#### **7. References**

62 Emphysema

+12 mer peptide

**A.** Polymer inhibition by 12 mer peptide

**B.** Polymer inhibition by targeting β-sheet A

Mahadeva et al., 2002; Chang et al., 2009.

inhibitor.

**6. Summary** 

Peptide

A. Z mutation (E342K) perturbs the structure of AT to allow opening of β-sheet A, which then accepts the RCL of another molecule to form a dimer (*left*) that can extend to form chains of polymers as depicted in Fig. 1. 12-mer peptide can anneal to β-sheet A thereby preventing polymer formation (*right*). B. Z mutation allows partial insertion of the RCL. This opens the lower part of β-sheet A thereby favouring polymerization (*left*). Understanding the configuration of the reactive loop and interacting with β-sheet A prompted the hypothesis that a 6-mer with homology to P7-2 of the RCL would specifically bind to Z-AT and so prevent polymerization and explained why the 12-mer peptide preferentially bound to M-AT (*right*). Reproduced and adapted from Lomas and Mahadeva, 2002;

Fig. 3. Representation Z α1-antitrypsin polymerization and the design of a selective

Alpha-1-antitrypsin is the most important proteinase inhibitor in maintaining the proteinase/antiproteinase balance within the lung. The recognition of the association between plasma deficiency of α1-antitrypsin and emphysema over 40 years ago has led to the proteinase-antiproteinase hypothesis of lung disease which remains central to understanding lung biology. In the last 20 years there has been significant progress in our understanding of α1-antitrypsin. Alpha-1 antitrypsin may modulate other biological processes such as apoptosis and inflammatory cell recruitment. Z α1-antitrypsin Abusriwil, H. & Stockley, R. A. (2006) *Cur Opin Pul Med* 12(2), 125-131


The Role of Alpha–1 Antitrypsin in Emphysema 65

Flotte, T. R., Brantly, M. L., Spencer, L. T., Byrne, B. J., Spencer, C. T., Baker, D. J. &

Gildea, T. R., Shermock, K. M., Singer, M. E. & Stoller, J. K. (2003) *Am J Respir Crit Care Med*

Graham, A., Kalsheker, N. A., Newton, C. R., Bamforth, F. J., Powell, S. J. & Markham, A. F.

Hautamaki, R. D., Kobayashi, D. K., Senior, R. M. & Shapiro, S. D. (1997) *Sci (New York, N.Y*

Hadzic, R., Nita, I., Tassidis, H., Riesbeck, K., Wingren, A. G. & Janciauskiene, S. (2006)

Henry, M. T., Cave, S., Rendall, J., O'Connor, C. M., Morgan, K., FitzGerald, M. X. &

Hersh, C. P., Dahl, M., Ly, N. P., Berkey, C. S,. Nordestgaard, B. G. & Silverman, E. K. (2004)

Hidvegi, T., Schmidt, B. Z., Hale, P. & Perlmutter, D. H. (2005) *J Biol Chem* 280(47), 39002-

Hidvegi, T., Ewing, M., Hale, P., Dippold, C., Beckett, C., Kemp, C., Maurice, N., Mukherjee,

Hodges, J. R., Millward-Sadler, G. H., Barbatis, C. & Wright, R. (1981) *N Engl J Med* 304(10),

Janciauskiene, S., Eriksson, S., Callea, F., Mallya, M., Zhou, A., Seyama, K., Hata, S. & Lomas

Kalsheker, N. A., Hodgson, I. J., Watkins, G. L., White, J. P., Morrison, H. M. & Stockley, R.

King, M. A., Stone, J. A., Diaz, P. T., Mueller, C. F., Becker, W. J. & Gadek JE. (1996) *Radiol*

Kounnas, M. Z., Church, F. C., Argraves, W. S. & Strickland, D. K. (1996) *J Biol Chem* 271(11),

Kaushal, S., Annamali, M., Blomenkamp, K., Rudnick, D., Halloran, D., Brunt, E.M., &

Kramps, J. A., Brouwers, J. W., Maesen, F. & Dijkman, J. H. (1981) *Hum Genet* 59(2), 104-107

Holmes, M. D., Brantly, M. L. & Crystal, R. G. (1990a) *Am Rev Respir Dis* 142(5), 1185-1192 Holmes, M. D., Brantly, M. L., Fells, G. A. & Crystal, R. G. (1990b) *BBRC* 170(3), 1013-1020 Hubbard, R. C., McElvaney, N. G., Sellers, S. E., Healy, J. T., Czerski, D. B. & Crystal, R. G.

Huntington, J. A., Read, R. J. & Carrell, R. W. (2000) *Nature* 407(6806), 923-926

Janoff, A., Carp, H., Laurent, P. & Raju, L. (1983) *Am Rev Respir Dis* 127(2), S31-38

Janus, E. D., Phillips, N. T. & Carrell, R. W. (1985) *Lancet* 1(8421), 152-154

A. (1987) *Br Med J (Clin Res Ed)* 294(6586), 1511-1514 Kim, S., Woo, J., Seo, E. J., Yu, M. & Ryu, S. (2001) *J Mol Biol* 306(1), 109-119

Teckman, J. H. (2010) *Exp Biol Med* 235(6):700-709.

A., Goldbach, C., Watkins, S., Michalopoulos, G., Perlmutter, D. H. (2010) *Sci* 

Humphries, M. (2004) *Hum Gen Therp* 15(1), 93-128 Flotte, T. R. & Mueller, C. (2011) *Hum Mol Genet* 20(R1):R87-92.

Hayes, K., Graham, A., & Kalsheker, N. (1992) *Biochem Soc Trans* 20(2), 182S

Kalsheker, N. (2001) *Eur J Hum Genet* 9(4), 273-278

Hiemstra, P., Wetering, S. van, & Stolk, J. (1998) *Eur Respir J* 12

(1989) *J Clin Invest* 84(4), 1349-1354

D. A. (2004) *Hepatol* 40(5):1203-10.

Johnson, D. & Travis, J. (1979) *J Biol Chem* 254(10):4022-6.

Janciauskiene, S. (2001) *Biochim Biophys Acta* 1535(3):221-35.

Gettins, P. G. (2002) *Chem Rev* 102(12):4751-4804.

(1989) *Hum Genet* 84(1), 55-58

167:1387–1392

277(5334), 2002-2004

*Immunol Lett* 102:141-147.

*Thorax* 59(10): 843-849.

329(5988):229-232.

39015

557-560

199(1):137-41.

6523-6529


Chappell, S., Daly, L., Morgan, K., Guetta Baranes, T., Roca, J., Rabinovich, R., Millar, A.,

Monti, S., O'Connor, C. M. & Kalsheker, N. (2006) *Hum Mut* 27(1), 103-109

Courtney, J. M., Plant, B. J., Morgan, K., Rendall, J., Gallagher, C., Ennis, M., Kalsheker, N.,

Curiel, D. T., Vogelmeier, C., Hubbard, R. C., Stier, L. E. & Crystal, R. G. (1990) *Mole Cell Biol*

Dabbagh, K., Laurent, G. J., Shock, A., Leoni, P., Papakrivopoulou, J., and Chambers, R. C.

Dafforn, T. R., Mahadeva, R., Elliott, P. R., Sivasothy, P., and Lomas, D. A. (1999) *J Biol Chem*

Dauriat, G., Mal, H., Jebrak, G., Brugière, O., Castier, Y., Camuset, J., Marceau, A., Taillé, C., Lesèche, G. & Fournier, M. (2006) *Int J Chron Obstruct Pulmon Dis* 1(2):201-6. de Faria, E. J., de Faria, I. C., Alvarez, A. E., Ribeiro, J. D., Ribeiro, A. F. & Bertuzzo, C. S.

DeMeo, D. L., Campbell, E. J., Brantly, M. L., Barker, A. F., Eden, E,. McElvaney, N. G.,

de Perrot, M., Chaparro, C., McRae, K., Waddell, T. K., Hadjiliadis, D., Singer, L. G., Pierre,

Devlin, G. L., Parfrey, H., Tew, D. J., Lomas, D.A. & Bottomley, S.P. (2001) *Am J Respir Cell* 

Elliott, P. R., Lomas, D. A., Carrell, R. W., and Abrahams, J. P. (1996a) *Nat Struct Biol* 3(8),

Evald, T., Dirksen, A., Keittelmann, S., Viskum, K. & Kok-Jensen, A. (1990) *Lung* 168 Suppl,

Fishman, A., Martinez, F., Naunheim, K., Piantadosi, S., Wise, R., Ries, A., Weinmann, G. &

Fletcher, A. P., Alkjaersig, N. K., O'Brien, J. R., & Tulevski, V. (1977) *J Lab Clin Med* 89(6),

Wood, D. E; National Emphysema Treatment Trial Research Group. (2003) *N Engl J* 

Elliott PR, Stein PE, Bilton D, Carrell RW, Lomas DA. (1996b) *Nat Struct Biol* 3(11):910-911. Engh, R., Lobermann, H., Schneider, M., Wiegand, G., Huber, R. & Laurell, C. B. (1989) *Prot* 

Elliott, P. R., Abrahams, J. P. & Lomas, D. A. (1998a) *J Mol Biol* 275(3), 419-425 Elliott, P. R., Bilton, D. & Lomas, D. A. (1998b) *Am J Resp Cell Mol Biol* 18(5), 670-674

Fagerhol, M. K. (1974) *Birth defects original article series* 10(4), 208-211 Fagerhol, M. K. & Laurell, C. B. (1967) *Clin Chim Acta* 16(2), 199-203

Rennard, S. I., Stocks, J. M., Stoller, J. K., Strange, C., Turino, G., Sandhaus, R. A. &

A. F., Hutcheon, M. & Keshavjee, S. (2004) *J Thorac Cardiovasc Surg* 127(5):1493-501.

Church, D. F. & Pryor, W. A. (1985) *Environ Health Perspect* 64, 111-126 Cichy, J., Potempa, J. & Travis, J. (1997) *J Biol Chem* 272(13):8250-8255.

Elborn, S. & O'Connor, C.M. (2006) *Ped Pul* 41(6), 584-591 Cox, D. W. & Billingsley, G. D. (1989) *Am J Hum Genet* 44(6), 844-854 Cox, D. W., Johnson, A. M. & Fagerhol, M.K. (1980) *Hum Genet* 53:429–433.

Cook, P. J. (1975) *Ann Hum Genet* 38(3):275-87.

(2001) *J Cell Physiol* 186(1), 73-81

(2005) *J Pediat (Rio J)* 81(6), 485-490

de Serres, F. J. (2002) *Chest* 122(5):1818-1829.

*Mol Biol* 24:727–732.

*Eng* 2(6), 407-415

*Med* 348(21):2059-73.

Flotte, T. R. (2002) *Chest* 121(3 Suppl), 98S-102S

676-681

579-585.

1349-1364

Silverman, E. K. *Hum Hered.* 2009;67(1):38-45.

de Serres, F. J. (2003) *Environ Health Perspect* 111(16), 1851-1854

10(1), 47-56

274(14), 9548-9555

Donnelly, S. C., Keatings, V., MacNee, W., Stolk, J., Hiemstra, P., Miniati, M.,


The Role of Alpha–1 Antitrypsin in Emphysema 67

Okayama, H., Brantly, M., Holmes, M. & Crystal, R. G. (1991) *Am J Hum Genet* 48(6), 1154-

Owen, C. A., Campbell, M. A., Sannes, P. L., Boukedes, S. S. & Campbell, E. J. (1995) *J Cell* 

Owen, M. C., Brennan, S. O., Lewis, J. H. & Carrell, R. W. (1983) *N Engl J Med* 309(12), 694-

Parmar, J. S., Mahadeva, R., Reed, B. J., Farahi, N., Cadwallader, K. A., Keogan, M. T., Bilton, D., Chilvers, E. R. & Lomas, D. A. (2002) *Am J Respir Cell Mol Biol* 26(6), 723-730 Parr, D. G., Guest, P. G., Reynolds, J. H., Dowson, L. J. & Stockley, R. A. (2007) *Am J Respir* 

Petrache, I., Fijalkowska, I., Medler, T. R., Skirball, J., Cruz, P., Zhen, L., Petrache, H. I.,

Roberts, E. A., Cox, D. W., Medline, A. & Wanless, I. R. (1984) *Am J Clin Pathol* 82(4), 424-427 Ryu, S. E., Choi, H. J., Kwon, K. S., Lee, K. N. & Yu, M. H. (1996) *Struct* 4(10), 1181-1192 Sandhaus, R. A., Turino, G., Stocks, J., Strange, C., Trapnell, B. C., Silverman, E. K., Everett,

Schindler, D. (1984) In Human Genetic Disorders: 16t' Miami Winter Symposium. In: S.

Schroeder, W. T., Miller, M. F., Woo, S. L. & Saunders, G. F. (1985) *Am J Hum Genet* 37(5),

Seersholm, N., Wilcke, J. T., Kok-Jensen, A. & Dirksen, A. (2000) *Am J Respir Crit Care Med*

Seyama, K., Nukiwa, T., Takabe, K., Takahashi, H., Miyake, K. & Kira, S. (1991) *J Biol Chem*

Silverman, E. K., Miletich, J. P., Pierce, J. A., Sherman, L. A., Endicott, S. K., Broze, G. J., Jr., &

Silverman, G. A., Bird, P. I., Carrell, R. W., Church, F. C., Coughlin, P. B., Gettins, P. G.,

Smith, R. M., Traber, L. D., Traber, D. L. & Spragg, R. G. (1989) *J Clinic Invest* 84(4), 1145-1154 Song, H. K., Lee, K. N., Kwon, K. S., Yu, M. H. & Suh, S. W. (1995) *FEBS Lett* 377(2), 150-154 Sørheim, I. C., Bakke, P., Gulsvik, A., Pillai, S. G., Johannessen, A., Gaarder, P. I., Campbell,

Irving, J. A., Lomas, D. A., Luke, C. J., Moyer, R. W., Pemberton, P. A., Remold-O'Donnell, E., Salvesen, G. S., Travis, J. & Whisstock, J. C. (2001) *J Biol Chem* 

E. J., Agustí, A., Calverley, P. M., Donner, C. F., Make, B. J., Rennard, S. I., Vestbo, J., Wouters, E. F., Paré, P. D., Levy, R. D., Coxson, H. O., Lomas, D. A., Hersh, C. P.

Silva, G. E., Sherrill, D. L., Guerra, S. & Barbee, R.A. (2003) *Chest* 123(5):1435-1440.

Campbell, E. J. (1989) *Am Rev Respir Dis* 140(4), 961-966

& Silverman, E. K. (2010) *Chest* 138(5):1125-1132. Stratikos, E. & Gettins, P. G. (1997). *Proc Natl Acad Sci USA* 94: 453-458.

Stein, P. E. & Carrell, R. W. (1995) *Nat Struct Biol* 2(2), 96-113 Stecenko, A. A. & Brigham, K. L. (2003) *Gene Ther* 10(2):95-9. Stockley, R. A. (2010) *Expert Opin Emerg Drugs* 15(4):685-94.

Ahmad, S. B., J. Schulz, W. Scott, and J. Whelan (ed). *Advances in Gene Technology*,

Flotte, T. R. & Tuder, R. M. (2006) *Am J Pathol* 169(4), 1155-1166

Patterson, G. A. & Cooper, J. D. (1995) *Chest Surg Clin N Am* 5(4):851-68.

1158

698

868-872

161(1):81-84.

266(19), 12627-12632

276:33293–33296.

*Biol* 131(3), 775-789

*Crit Care Med* 176(12):1215-21.

Piitulainen, E. & Eriksson, S. (1999) *Eur Respir J* 13(2), 247-251 Rahman, I. & MacNee, W. (1996) *Free Rad Biol Med* 21(5), 669-681

S. E. & Stoller, JK; *Chest* 134(4):831-834.

Cambridge University Press, Cambridge


Li, Z., Alam, S., Wang, J., Sandstrom, C. S., Janciauskiene, S. & Mahadeva, R. (2009) *Am J* 

Lomas, D. A., Evans, D. L., Stone, S. R., Chang, W. S. & Carrell, R. W. (1993) *Biochem*

Mahadeva, R., Atkinson, C., Li, Z., Stewart, S., Janciauskiene, S., Kelley, D. G., Parmar, J., Pitman, R., Shapiro, S. D. & Lomas, D. A. (2005) *Am J Path* 166(2), 377-386 Mahadeva, R., Westerbeek, R. C., Perry, D. J., Lovegrove, J. U., Whitehouse, D. B., Carroll, N.

Mahadeva, R., Chang, W. S., Dafforn, T. R., Oakley, D. J., Foreman, R. C., Calvin, J., Wight,

Mahadeva, R., Dafforn, T. R., Carrell, R. W. & Lomas, D. A. (2002) *J Biol Chem* 277(9), 6771-

Mal, H., Guignabert, C., Thabut, G., d'Ortho, M. P., Brugière, O., Dauriat, G., Marrash-

Mallya, M., Phillips, R. L., Saldanha, S. A., Gooptu, B., Brown, S. C., Termine, D. J., Shirvani,

Meyer, K. C., Nunley, D. R., Dauber, J. H., Iacono, A. T., Keenan, R. J., Cornwell, R. D. &

Miravitlles, M., Herr, C., Ferrarotti, I., Jardi, R., Rodriguez-Frias, F., Luisetti, M. & Bals, R.

Morrison, H. M., Kramps, J. A., Burnett, D. & Stockley, R. A. (1987) *Clin Sci* (Lond). 72:373-

Mulgrew, A. T., Taggart, C. C., Lawless, M. W., Greene, C. M., Brantly, M. L., O'Neill, S. J., &

Oakeshott, J. G., Muir, A., Clark, P., Martin, N. G., Wilson, S. R. & Whitfield, J. B. (1985) *Ann* 

Ogushi, F., Fells, G. A., Hubbard, R. C., Straus, S. D. & Crystal, R. G. (1987) *J Clin Invest*

R., Ross-Russell, R. I., Webb, A. K., Bilton, D. & Lomas, D. A. (1998) *Eur Respir J*

Chahla, R., Rangheard, A. S., Lesèche, G. & Fournier, M. (2004) *Am J Respir Crit Care* 

A. M., Wu, Y., Sifers, R. N., Abagyan, R. & Lomas, D. A. (2007) *J Med Chem*

Lomas, D. A., Evans, D. L., Finch, J. T. & Carrell, R. W. (1992) *Nature* 357(6379), 605-607 Long, G. L., Chandra, T., Woo, S. L., Davie, E. W., & Kurachi, K. (1984) *Biochem* 23(21), 4828-

Louie, S. G., Sclar, D. A. & Gill, M. A. (2005) *Ann Pharmacother* 39(11):1861-1869.

Larsson, C. (1978) *Acta Med Scand* 204(5), 345-351

19;32(2):500-8.

48370-280

11(4):873-9

*Med* 170(7):811-814

50(22):5357-5363.

(2010) *Eur Respir J* 35(5):960-8

Morse, J. O. (1978) *N Engl J Med* 299(20), 1099-1105

*Hum Biol* 12(2), 149-160

80(5), 1366-1374

6774

381

Laurell, C. B. & Eriksson, S. (1963) *Scand J Clin Invest* 15;132-140.

Lomas, D. A. & Mahadeva, R. (2002) *J Clin Invest* 110(11), 1585-1590

Lu, Q., Harrington, E. O. & Rounds, S. (2005) *Keio J Med* 54(4), 184-189

D. G., & Lomas, D. A. (1999) *J Clin Invest* 103(7), 999-1006

Love, R. B. (2001) *Am J Respir Crit Care Med* 164(1):97-102.

Morgan, K., Scobie, G. & Kalsheker, N. (1992) *Eur J Clin Invest* 22(2), 134-137

Modrykamien, A. & Stoller, J. K. (2009) *Pharmacother* 10(16):2653-2661. Moraga, F. & Janciauskiene, S. (2000) *J Biol Chem* 275(11), 7693-7700

McElvaney, N. G. (2004) *Chest* 125(5), 1952-1957 Needham, M., & Stockley, R. A. (2004) *Thorax* 59(5), 441-445

*Physiol Lung Cell Mol Physiol* 297:L388-400.

Luisetti, M. & Seersholm, N. (2004) *Thorax* 59(2):164-9. Mahadeva, R. & Lomas, D. A. (1998) *Thorax* 53(6), 501-505

	- E. J., Agustí, A., Calverley, P. M., Donner, C. F., Make, B. J., Rennard, S. I., Vestbo, J., Wouters, E. F., Paré, P. D., Levy, R. D., Coxson, H. O., Lomas, D. A., Hersh, C. P. & Silverman, E. K. (2010) *Chest* 138(5):1125-1132.

**4** 

*USA* 

Frank Guarnieri

**The Dichotomy Between Understanding** 

 *School of Medicine, Virginia Commonwealth University, Richmond, VA, Department of Biomedical Engineering, Boston University, Boston, MA,* 

*Paka Pulmonary Pharmaceuticals, Acton, Department of Physiology and Biophysics, MA,* 

The long history of investigations into the causes and potential treatments of emphysema encompasses a vast array of chemical and biological research disciplines. A key finding that played a major role in initiating these inquiries occurred in 1963 when Laurell and Eriksson[1] found that individuals with a genetic deficiency in serum alpha-1-antitrypsin (AAT) were prone to develop pulmonary emphysema[2]. This genetic linkage was given a mechanistic basis when Turino and colleagues in 1969 discovered that patients with reduced inhibition of pancreatic elastase also lacked serum AAT and were prone to develop severe pulmonary emphysema[3]. Subsequent studies in the early 1970s confirmed that excessive elastase activity due to lack of AAT was in fact the genetic mechanism responsible for the onset of emphysema [4-7]. A key environmental connection was made with the discovery that cigarette smoke increased macrophage secretion of elastase[8] in the lungs, oxidized AAT[9], and that the chemical irritants in smoke recruited neutrophils to the lungs via chemotaxis[10-12]. This integrated genetic-environmental understanding firmly established elastase inhibition as a mechanistic target for preventing the alveolar destruction

The validation of elastase[13, 14 ] as a protein target for treating emphysema, motivated three different therapeutic approaches, 1) infusing patients with AAT purified from serum[15], 2) development of small molecule inhibitors[13, 16, 17], 3) novel association of small peptides[18] and synthetic inhibitors [19] with albumin microspheres. The first approach is a biological therapeutic, the second approach is a chemical therapeutic, and the third approach is a prescient recognition that *in vivo* efficacy will likely require long lung residence time pharmacodynamics. The Pharmaceutical industry launched several major multi-decade programs to develop orally available small molecule inhibitors, while apparently completely ignoring the concurrent academic medical research beginning to unravel the complex biology of emphysema and its indication that oral delivery of small molecules was unlikely to have any therapeutic benefit. Interestingly, a completely different basic research discipline, X-ray crystallography, had a seminal impact on the class of molecules from which Zeneca derived their clinical candidate. The first structure[20] was solved In 1976 by Alber, Petsko, and Tsernoglou, which showed atomic resolution details of

**1. Introduction** 

characteristic of emphysema.

**and Treating Emphysema** 


### **The Dichotomy Between Understanding and Treating Emphysema**

#### Frank Guarnieri

*Paka Pulmonary Pharmaceuticals, Acton, Department of Physiology and Biophysics, MA, School of Medicine, Virginia Commonwealth University, Richmond, VA, Department of Biomedical Engineering, Boston University, Boston, MA, USA* 

#### **1. Introduction**

68 Emphysema

Stockley, R. (2003) Antiproteinases and antioxidants. In: Gibson GJ, G. D., Costabel U, Sterk, P, and Corrin B. (ed). *Respir Med*, Third Ed., Elsevier Science limited, London Stockley, R. A., Parr, D. G., Piitulainen, E., Stolk, J., Stoel, B. C. & Dirksen, A. (2010) *Respir* 

Stockley, R. A., Bayley, D. L., Unsal, I. & Dowson, L. J. (2002) *Am J Respir Crit Care Med*

Stoller, J. K., Fallat, R., Schluchter, M. D., O'Brien, R. G., Connor, J. T., Gross, N., O'Neil, K.,

Stoller, J. K., Fallat, R., Schluchter, M. D., O'Brien, R. G., Connor, J. T., Gross, N., O'Neil, K.,

Stoller, J. K., Gildea, T. R., Ries, A. L., Meli, Y. M. & Karafa, M. T; National Emphysema Treatment Trial Research Group. (2007) *Ann Thorac Surg* 83(1):241-51.

Taggart, C., Cervantes-Laurean, D., Kim, G., McElvaney, N. G., Wehr, N., Moss, J. & Levine,

Takahashi, H., Nukiwa, T., Satoh, K., Ogushi, F., Brantly, M., Fells, G., Stier, L., Courtney, M.

Tanash, H. A., Riise, G. C., Hansson, L., Nilsson, P. M. & Piitulainen, E. *J Heart Lung* 

Tutic, M., Bloch, K. E., Lardinois, D., Brack, T., Russi, E. W. & Weder, W. (2004) *J Thorac* 

Wencker, M., Banik, N., Hotze, L. A., Kropp, J., Biersack, M. J., Ulbich, E. & Konietzko, N.

Zhang, Z., Farrell, A. J., Blake, D. R., Chidwick, K. & Winyard, P. G. (1993) *FEBS Lett* 321(2-

Zhou, A., Stein, P. E., Huntington, J. A., Sivasothy, P., Lomas, D. A. & Carrell, R. W. (2004) *J* 

Zorzetto, M., Russi, E., Senn, O., Imboden, M., Ferrarotti, I., Tinelli, C., Campo, I., Ottaviani,

& Probst-Hensch, N; SAPALDIA Team. (2008) *Clin Chem* 54(8):1331-1338.

S., Scabini, R., von Eckardstein, A., Berger, W., Brändli, O., Rochat, T., Luisetti, M.

Wewers, M. D., Casolaro, M. A. & Crystal, R. G. (1987) *Am Rev Respir Dis* 135(3), 539-543 Wilczynska, M., Fa, M., Karolin, J., Ohlsson, P. I., Johansson, L. B. & Ny, T. (1997) *Nat Struct* 

Sandhaus, R. & Crystal, R. G. 2003 /*Chest* 2009 136(5 Suppl):e30

Sandhaus, R. & Crystal, R. G. (2003) *Chest* 123(5):1425-34

*Res* 11:136

165(11), 1494-1498

Sveger, T. (1976) *N Engl J Med* 294(24), 1316-1321

*Cardiovasc Surg* 128(3):408-13. Vogt, W. (1995) *Free Rad Biol Med* 18(1), 93-105

*Biol* 4(5):354-7.

3), 274-278

*Mol Biol* 342(3), 931-941

R. L. (2000) *J Biol Chem* 275: 27258-27265.

*Transplant* 2011 Aug 5. [Epub ahead of print]

*Am J Respir Crit Care Med* 1998;154:A400.

Wong, P. S. & Travis, J. (1980) *BBRC* 96(3), 1449-1454

Wewers, M. D. & Gadek, J. E. (1987) *Ann Intern Med* 107(5), 761-763

Takahashi, H. & Crystal, R. G. (1990) *Am J Hum Genet* 47(3), 403-413

& Crystal, R. G. (1988) *J Biol Chem* 263(30), 15528-15534

Tobin, M. J., Cook, P. J. & Hutchison, D. C. (1983) *Brit J Dis chest* 77(1), 14-27

The long history of investigations into the causes and potential treatments of emphysema encompasses a vast array of chemical and biological research disciplines. A key finding that played a major role in initiating these inquiries occurred in 1963 when Laurell and Eriksson[1] found that individuals with a genetic deficiency in serum alpha-1-antitrypsin (AAT) were prone to develop pulmonary emphysema[2]. This genetic linkage was given a mechanistic basis when Turino and colleagues in 1969 discovered that patients with reduced inhibition of pancreatic elastase also lacked serum AAT and were prone to develop severe pulmonary emphysema[3]. Subsequent studies in the early 1970s confirmed that excessive elastase activity due to lack of AAT was in fact the genetic mechanism responsible for the onset of emphysema [4-7]. A key environmental connection was made with the discovery that cigarette smoke increased macrophage secretion of elastase[8] in the lungs, oxidized AAT[9], and that the chemical irritants in smoke recruited neutrophils to the lungs via chemotaxis[10-12]. This integrated genetic-environmental understanding firmly established elastase inhibition as a mechanistic target for preventing the alveolar destruction characteristic of emphysema.

The validation of elastase[13, 14 ] as a protein target for treating emphysema, motivated three different therapeutic approaches, 1) infusing patients with AAT purified from serum[15], 2) development of small molecule inhibitors[13, 16, 17], 3) novel association of small peptides[18] and synthetic inhibitors [19] with albumin microspheres. The first approach is a biological therapeutic, the second approach is a chemical therapeutic, and the third approach is a prescient recognition that *in vivo* efficacy will likely require long lung residence time pharmacodynamics. The Pharmaceutical industry launched several major multi-decade programs to develop orally available small molecule inhibitors, while apparently completely ignoring the concurrent academic medical research beginning to unravel the complex biology of emphysema and its indication that oral delivery of small molecules was unlikely to have any therapeutic benefit. Interestingly, a completely different basic research discipline, X-ray crystallography, had a seminal impact on the class of molecules from which Zeneca derived their clinical candidate. The first structure[20] was solved In 1976 by Alber, Petsko, and Tsernoglou, which showed atomic resolution details of

The Dichotomy Between Understanding and Treating Emphysema 71

days later [37], 2) dose related changes in pulmonary function after 4 weeks [38], 3) 14 day lung residence time of the enzyme complexed with alpha-macroglobulin [39], 4) resistance to AAT inactivation in the presence of activated neutrophils [40], 5) uptake by alveolar macrophages with subsequent re-release of elastase [41]. Additionally, activated neutrophils

While the complex biology of emphysema most likely precludes treatment using a simple small molecule elastase inhibitor strategy, important physiological parameters essential for developing an effective therapeutic modality were reported by Stone and Lucey between 1988 and 1991. These investigators showed that, 1) one intratracheal dose of elastase causes maximum damage after 4 weeks [43], 2) a potent elastase inhibitor given intratracheally in 170-fold molar excess has a lung half-life of 4 minutes (Figure 2) and actually results in worse emphysema relative to animals given saline with no elastase [44], 3) covalently linking an active small molecule to a polymer of hydroxyethyl-aspartamide (stationary phase for hydrophilic column chromatography) results in a lung half-life of 441 minutes and amelioration of elastase induced emphysema [45]. This collection of results indicates that long lung residence time is an essential component of any meaningful emphysema treatment and that elastase must be down-regulated continuously for at least 4 weeks.

Fig. 2. This is a recreation of the data from Phil Stone's lab showing that small molecule elastase inhibitors have a lung half-life of 4-5 minutes. It is important to understand that these experiments were conducted by intratracheally instilling the small molecule elastase inhibitor and thus 100% of the dose was initially deposited into the lungs. Small molecules administered

orally will result in only a tiny fraction of the dose ever actually entering the lungs.

can secrete elastase for over 12 days [42].

**2. Lessons from the Stone lab** 

elastase digesting a substrate. Sawyer and colleagues deposited the first high resolution crystal structure of porcine pancreatic elastase[21] into the protein data bank, and in 1982 Hughes and colleagues[22] solved a structure of the enzyme bound to a trifluoroacetyl dipeptide inhibitor (deposited in the PDB in 1986), thus making high resolution structures with and without bound ligand available to the research community. The trifluoroacetyl motif (shown in Figure 1) became a cornerstone of Zeneca's small molecule research program[23-32], which resulted in the clinical candidate ICI 200,880[33]that was halted due to lack of efficacy in Phase II clinical trials.

Fig. 1. Co-crystal structure taken from the protein data bank file 2EST. This structure shows the catalytic serine (shown in space fill) performing a nucleophilic attack on the carbon of the ketone attached to a trifluoromethyl group – the fluorines are shown in light blue. The highly electronegative fluorine atoms significantly enhance the electropositive nature of this carbon and hence trifluoromethyl-ketone molecules have a high affinity for elastase.

Even as the first therapeutics were being developed, a report on neutrophil lung recruitment via elastin peptide chemotaxis [34] gave the first indications of the complexity and immunological involvement [35] in the development of pulmonary diseases. Elastase digests elastin resulting in peptide fragments that elicit circulating neutrophils to enter the lungs. These neutrophils secrete fresh elastase causing new lung damage, new elastin peptide fragments and recruitment of new neutrophils again secreting fresh elastase into the lungs in a destructive feedback loop. These studies already presented evidence that inhibiting elastase in the short term would be insufficient to treat emphysema. Compounding the complexity, early elastase inhibitors administered intraperitoneally that showed promise in stemming emphysema, cleared rapidly [36] *in vivo* with concomitant renal nephropathy [17]. The complex interplay between lung injury and immune response that begins with a single intratrachael instillation of elastase motivated many detailed studies aimed at elucidating the basic biology of emphysema progression. Early key findings on the long term effects on lung tissue of only one exposure to elastase includes, 1) ultrastructural changes occurring 16 days later [37], 2) dose related changes in pulmonary function after 4 weeks [38], 3) 14 day lung residence time of the enzyme complexed with alpha-macroglobulin [39], 4) resistance to AAT inactivation in the presence of activated neutrophils [40], 5) uptake by alveolar macrophages with subsequent re-release of elastase [41]. Additionally, activated neutrophils can secrete elastase for over 12 days [42].

#### **2. Lessons from the Stone lab**

70 Emphysema

elastase digesting a substrate. Sawyer and colleagues deposited the first high resolution crystal structure of porcine pancreatic elastase[21] into the protein data bank, and in 1982 Hughes and colleagues[22] solved a structure of the enzyme bound to a trifluoroacetyl dipeptide inhibitor (deposited in the PDB in 1986), thus making high resolution structures with and without bound ligand available to the research community. The trifluoroacetyl motif (shown in Figure 1) became a cornerstone of Zeneca's small molecule research program[23-32], which resulted in the clinical candidate ICI 200,880[33]that was halted due

Fig. 1. Co-crystal structure taken from the protein data bank file 2EST. This structure shows the catalytic serine (shown in space fill) performing a nucleophilic attack on the carbon of the ketone attached to a trifluoromethyl group – the fluorines are shown in light blue. The highly electronegative fluorine atoms significantly enhance the electropositive nature of this

Even as the first therapeutics were being developed, a report on neutrophil lung recruitment via elastin peptide chemotaxis [34] gave the first indications of the complexity and immunological involvement [35] in the development of pulmonary diseases. Elastase digests elastin resulting in peptide fragments that elicit circulating neutrophils to enter the lungs. These neutrophils secrete fresh elastase causing new lung damage, new elastin peptide fragments and recruitment of new neutrophils again secreting fresh elastase into the lungs in a destructive feedback loop. These studies already presented evidence that inhibiting elastase in the short term would be insufficient to treat emphysema. Compounding the complexity, early elastase inhibitors administered intraperitoneally that showed promise in stemming emphysema, cleared rapidly [36] *in vivo* with concomitant renal nephropathy [17]. The complex interplay between lung injury and immune response that begins with a single intratrachael instillation of elastase motivated many detailed studies aimed at elucidating the basic biology of emphysema progression. Early key findings on the long term effects on lung tissue of only one exposure to elastase includes, 1) ultrastructural changes occurring 16

carbon and hence trifluoromethyl-ketone molecules have a high affinity for elastase.

to lack of efficacy in Phase II clinical trials.

While the complex biology of emphysema most likely precludes treatment using a simple small molecule elastase inhibitor strategy, important physiological parameters essential for developing an effective therapeutic modality were reported by Stone and Lucey between 1988 and 1991. These investigators showed that, 1) one intratracheal dose of elastase causes maximum damage after 4 weeks [43], 2) a potent elastase inhibitor given intratracheally in 170-fold molar excess has a lung half-life of 4 minutes (Figure 2) and actually results in worse emphysema relative to animals given saline with no elastase [44], 3) covalently linking an active small molecule to a polymer of hydroxyethyl-aspartamide (stationary phase for hydrophilic column chromatography) results in a lung half-life of 441 minutes and amelioration of elastase induced emphysema [45]. This collection of results indicates that long lung residence time is an essential component of any meaningful emphysema treatment and that elastase must be down-regulated continuously for at least 4 weeks.

Fig. 2. This is a recreation of the data from Phil Stone's lab showing that small molecule elastase inhibitors have a lung half-life of 4-5 minutes. It is important to understand that these experiments were conducted by intratracheally instilling the small molecule elastase inhibitor and thus 100% of the dose was initially deposited into the lungs. Small molecules administered orally will result in only a tiny fraction of the dose ever actually entering the lungs.

The Dichotomy Between Understanding and Treating Emphysema 73

peptide now called surfactant protein B. What is even more amazing is that subsequent biophysical studies demonstrated that the first 25 amino acids possesses essentially identical surface active properties[58, 59] to the whole protein (Figure 3). Further confirmation of the importance of the first domain of surfactant protein B comes from Discovery Labs with their Phase III clinical studies that one dose of (Lys-Leu-Leu-Leu)4 [60] a mimetic of SP-B 1-25 added

The long, involved, complicated history of emphysema integrates genetics, protein and small molecule therapies, medicinal chemistry, crystallography, biophysics, and several other research disciplines. Interestingly, all of this complexity can lead to a rather simple conclusion - that covalently linking Zeneca's clinical candidate to the first 25 residues of surfactant peptide B (Figure 4) would be an effective long acting anti-emphysema treatment if delivered intratracheally. When these studies were carried out[62], one dose of the SP-B (1-25)-Zeneca peptide-small molecule construct completely protected rodents exposed to near lethal doses of the human neutrophil elastase for 4 weeks (Figures 5&6). Of course it remains to be seen whether or not this simple idea will prove to be efficacious in humans, because recent studies have demonstrated that AAT plays a complex multifactorial role in the recruitment of neutrophils into the lungs. For example, Li[63] and colleagues have demonstrated that oxidized AAT induces lung epithelial cells to release IL-8, resulting in CXCR1 mediated neutrophil chemotaxis into the lungs, while Bergin[64] and coworkers have shown that glycosylated AAT sequesters IL-8 disrupting activation of CXCR1 and neutrophil mobilization. To further complicate matters, calpain[65] induces TNF-alpha mediated neutrophil chemotaxis and AAT binds to and inhibits calpain[66] thus preventing lung neutrophil infiltration by yet another mechanism. Even with all of this complexity and its implications that antioxidant therapy may be beneficial, the long established destructive role of unchecked elastase activity makes this enzyme a central target for inhibiting the progression of the alveolar wall destruction characteristic of emphysema as evidenced by the extensive pharmaceutical development that has gone into this endeavor, which includes

to cow lavage dramatically reduces mortality in severely preterm infants [61].

small molecules from ONO[67], Merck[68], Zeneca[24], and Glaxo[69] (Figure 7).

Fig. 4. A small molecule from the Zeneca family of fluoro-peptidomimetics covalently linked

to the N-terminal of the first 25 residues of surfactant peptide B.

**4. Conclusion** 

#### **3. Combining inhibitors with surfactant replacement therapy**

Even though Zeneca's clinical candidate ICI 200,880 was halted in Phase II clinical trials for lack of human efficacy, the molecule possessed two essential features of a drug, 1) high affinity anti-elastase activity, and 2) it was deemed safe to give to humans as evidenced by passing Phase I clinical trials. When the small molecule chemistry work of Zeneca is combined with the *in vivo* biology work of Phil Stone, the logical conclusion is that an efficacious *in vivo* emphysema treatment requires that ICI 200,880 somehow be recast so that it spreads across the vast surface area of the lungs and resists being expelled into systemic circulation. If such a recasting could be achieved, the long lung residence time could result in an immune response, thus ultimately negating the treatment. So the next logical step places the strong constraint that any adjuvant molecule used to do the recasting must naturally reside in the lungs. A natural lung molecule that has the intrinsic properties of spreading across the vast surface area of the lungs is a defining property of the lung surfactants.

Fig. 3. NMR structure of residues 1-25 from the N-terminal of surfactant peptide B taken from the protein data bank file 1DFW. An important feature of this peptide is its amphiphilic structure as illustrated by having one face composed of hydrophobic residues and the opposite face composed of charged and hydrophilic amino acids.

Human lung surfactant is a complex mixture of lipids and peptides that was extensively studied in the 1980s when it was realized that delivering surfactant harvested from animals to the lungs of severely pre-term infants is a life-saving [46-52] procedure. Early biophysical studies of lung surfactant indicated that it was ~90% lipids and ~10% protein by weight [53]. Detailed analysis showed that the protein component was actually made up of 4 different molecules, 2 larger hydrophilic proteins and 2 smaller hydrophobic proteins [54, 55]. Remarkably, when 1% or 0.1% by weight of the smallest of these proteins isolated from lavage fluid was added to synthetic phospholipids, both mixtures essentially eliminated dynamic surface tension in biophysical experiments [56, 57], a result that the investigators admitted was truly startling. The protein with such astounding surface active properties is a 79 residue peptide now called surfactant protein B. What is even more amazing is that subsequent biophysical studies demonstrated that the first 25 amino acids possesses essentially identical surface active properties[58, 59] to the whole protein (Figure 3). Further confirmation of the importance of the first domain of surfactant protein B comes from Discovery Labs with their Phase III clinical studies that one dose of (Lys-Leu-Leu-Leu)4 [60] a mimetic of SP-B 1-25 added to cow lavage dramatically reduces mortality in severely preterm infants [61].

#### **4. Conclusion**

72 Emphysema

Even though Zeneca's clinical candidate ICI 200,880 was halted in Phase II clinical trials for lack of human efficacy, the molecule possessed two essential features of a drug, 1) high affinity anti-elastase activity, and 2) it was deemed safe to give to humans as evidenced by passing Phase I clinical trials. When the small molecule chemistry work of Zeneca is combined with the *in vivo* biology work of Phil Stone, the logical conclusion is that an efficacious *in vivo* emphysema treatment requires that ICI 200,880 somehow be recast so that it spreads across the vast surface area of the lungs and resists being expelled into systemic circulation. If such a recasting could be achieved, the long lung residence time could result in an immune response, thus ultimately negating the treatment. So the next logical step places the strong constraint that any adjuvant molecule used to do the recasting must naturally reside in the lungs. A natural lung molecule that has the intrinsic properties of spreading across the vast surface area of the

Fig. 3. NMR structure of residues 1-25 from the N-terminal of surfactant peptide B taken

amphiphilic structure as illustrated by having one face composed of hydrophobic residues

Human lung surfactant is a complex mixture of lipids and peptides that was extensively studied in the 1980s when it was realized that delivering surfactant harvested from animals to the lungs of severely pre-term infants is a life-saving [46-52] procedure. Early biophysical studies of lung surfactant indicated that it was ~90% lipids and ~10% protein by weight [53]. Detailed analysis showed that the protein component was actually made up of 4 different molecules, 2 larger hydrophilic proteins and 2 smaller hydrophobic proteins [54, 55]. Remarkably, when 1% or 0.1% by weight of the smallest of these proteins isolated from lavage fluid was added to synthetic phospholipids, both mixtures essentially eliminated dynamic surface tension in biophysical experiments [56, 57], a result that the investigators admitted was truly startling. The protein with such astounding surface active properties is a 79 residue

from the protein data bank file 1DFW. An important feature of this peptide is its

and the opposite face composed of charged and hydrophilic amino acids.

**3. Combining inhibitors with surfactant replacement therapy** 

lungs is a defining property of the lung surfactants.

The long, involved, complicated history of emphysema integrates genetics, protein and small molecule therapies, medicinal chemistry, crystallography, biophysics, and several other research disciplines. Interestingly, all of this complexity can lead to a rather simple conclusion - that covalently linking Zeneca's clinical candidate to the first 25 residues of surfactant peptide B (Figure 4) would be an effective long acting anti-emphysema treatment if delivered intratracheally. When these studies were carried out[62], one dose of the SP-B (1-25)-Zeneca peptide-small molecule construct completely protected rodents exposed to near lethal doses of the human neutrophil elastase for 4 weeks (Figures 5&6). Of course it remains to be seen whether or not this simple idea will prove to be efficacious in humans, because recent studies have demonstrated that AAT plays a complex multifactorial role in the recruitment of neutrophils into the lungs. For example, Li[63] and colleagues have demonstrated that oxidized AAT induces lung epithelial cells to release IL-8, resulting in CXCR1 mediated neutrophil chemotaxis into the lungs, while Bergin[64] and coworkers have shown that glycosylated AAT sequesters IL-8 disrupting activation of CXCR1 and neutrophil mobilization. To further complicate matters, calpain[65] induces TNF-alpha mediated neutrophil chemotaxis and AAT binds to and inhibits calpain[66] thus preventing lung neutrophil infiltration by yet another mechanism. Even with all of this complexity and its implications that antioxidant therapy may be beneficial, the long established destructive role of unchecked elastase activity makes this enzyme a central target for inhibiting the progression of the alveolar wall destruction characteristic of emphysema as evidenced by the extensive pharmaceutical development that has gone into this endeavor, which includes small molecules from ONO[67], Merck[68], Zeneca[24], and Glaxo[69] (Figure 7).

Fig. 4. A small molecule from the Zeneca family of fluoro-peptidomimetics covalently linked to the N-terminal of the first 25 residues of surfactant peptide B.

The Dichotomy Between Understanding and Treating Emphysema 75

Fig. 7. Small molecule elastase inhibitors from ONO, Merck, Zeneca, and Glaxo.

*Antitrypsin.* Science, 1964. 146: p. 1678-9.

Arch Environ Health, 1973. 27(3): p. 196-200.

[1] Laurell, C.B. and S. Eriksson, *The electrophoretic alpha-iglobulin pattern of serum in alpha-1 antitrypsin deficiency.* Scand. J. Clin. Lab. Invest., 1963. 15: p. 132-140. [2] Kueppers, F., W.A. Briscoe, and A.G. Bearn, *Hereditary Deficiency of Serum Alpha-L-*

[3] Turino, G.M., et al., *Serum elastase inhibitor deficiency and alpha 1-antitrypsin deficiency in patients with obstructive emphysema.* Science, 1969. 165(894): p. 709-11. [4] Lieberman, J., *Involvement of leukocytic proteases in emphysema and antitrypsin deficiency.*

[5] Mittman, C., T. Barbela, and J. Lieberman, *Antitrypsin deficiency and abnormal protease* 

[6] Pierce, J.A., A.Z. Eisen, and H.K. Dhingra, *Relationship of antitrypsin deficiency to the pathogenesis of emphysema.* Trans Assoc Am Physicians, 1969. 82: p. 87-97. [7] Talamo, R.C., et al., *Symptomatic pulmonary emphysema in childhood associated with hereditary alpha-1-antitrypsin and elastase inhibitor deficiency.* J Pediatr, 1971. 79(1): p. 20-6. [8] Harris, J.O., et al., *Comparison of proteolytic enzyme activity in pulmonary alveolar* 

[9] Janoff, A. and H. Carp, *Possible mechanisms of emphysema in smokers: cigarette smoke* 

[10] Gadek, J.E., G.A. Fells, and R.G. Crystal, *Cigarette smoking induces functional antiprotease deficiency in the lower respiratory tract of humans.* Science, 1979. 206(4424): p. 1315-6. [11] Janoff, A., et al., *Cigarette smoke inhalation decreases alpha 1-antitrypsin activity in rat lung.*

[12] Rodriguez, R.J., et al., *Elastase release from human alveolar macrophages: comparison between* 

*smokers and nonsmokers.* Science, 1977. 198(4314): p. 313-4.

*macrophages and blood leukocytes in smokers and nonsmokers.* Am Rev Respir Dis, 1975.

*condensate suppresses protease inhibition in vitro.* Am Rev Respir Dis, 1977. 116(1): p.

*inhibitor phenotypes.* Arch Environ Health, 1973. 27(3): p. 201-6.

**5. References** 

111(5): p. 579-86.

Science, 1979. 206(4424): p. 1313-4.

65-72.

Fig. 5. Emphysema is induced in rodents by intratracheally instilling human neutrophil elastase. When elastase is administered with a potent Zeneca small molecule inhibitor, the rodents develop emphysema after 4 weeks to the same degree as rodents given no inhibitor. The small molecule was in 70-fold molar excess concentration relative to elastase.

Fig. 6. When this exact same small molecule was covalently linked to the fragment of surfactant peptide B as shown in Figure 4, one dose given in 30 fold molar excess completely protected the animal for 4 weeks. All animals were dosed with a mixture of HNE and either the Zeneca small molecule or the Zeneca small molecule covalently attached to the surfactant peptide and sacrificed after 4 weeks.

Fig. 7. Small molecule elastase inhibitors from ONO, Merck, Zeneca, and Glaxo.

#### **5. References**

74 Emphysema

Fig. 5. Emphysema is induced in rodents by intratracheally instilling human neutrophil elastase. When elastase is administered with a potent Zeneca small molecule inhibitor, the rodents develop emphysema after 4 weeks to the same degree as rodents given no inhibitor.

The small molecule was in 70-fold molar excess concentration relative to elastase.

Fig. 6. When this exact same small molecule was covalently linked to the fragment of

the Zeneca small molecule or the Zeneca small molecule covalently attached to the

surfactant peptide and sacrificed after 4 weeks.

surfactant peptide B as shown in Figure 4, one dose given in 30 fold molar excess completely protected the animal for 4 weeks. All animals were dosed with a mixture of HNE and either


The Dichotomy Between Understanding and Treating Emphysema 77

[31] Damewood, J.R., Jr., et al., *Nonpeptidic inhibitors of human leukocyte elastase. 2. Design,* 

[32] Warner, P., et al., *Non-peptidic inhibitors of human leukocyte elastase. 1. The design and synthesis of pyridone-containing inhibitors.* J Med Chem, 1994. 37(19): p. 3090-9. [33] Edwards, P.D. and P.R. Bernstein, *Synthetic inhibitors of elastase.* Med Res Rev, 1994.

[34] Senior, R.M., G.L. Griffin, and R.P. Mecham, *Chemotactic activity of elastin-derived* 

[35] Hunninghake, G.W. and J.E. Gadek, *Immunological aspects of chronic non-infectious* 

[36] Ip, M.P., et al., *The effects of small doses of oligopeptide elastase inhibitors on elastase-induced* 

[37] Morris, S.M., et al., *Ultrastructural changes in hamster lung four hours to twenty-four days* 

[38] Raub, J.A., et al., *Dose response of elastase-induced emphysema in hamsters.* Am Rev Respir

[39] Stone, P.J., et al., *Role of alpha-macroglobulin-elastase complexes in the pathogenesis of elastase-induced emphysema in hamsters.* J Clin Invest, 1982. 69(4): p. 920-31. [40] Zaslow, M.C., et al., *Human neutrophil elastase does not bind to alpha 1-protease inhibitor that* 

[41] McGowan, S.E., et al., *The fate of neutrophil elastase incorporated by human alveolar* 

[42] Werb, Z. and S. Gordon, *Elastase secretion by stimulated macrophages. Characterization and* 

[43] Lucey, E.C., et al., *An 18-month study of the effects on hamster lungs of intratracheally administered human neutrophil elastase.* Exp Lung Res, 1988. 14(5): p. 671-86. [44] Stone, P.J., E.C. Lucey, and G.L. Snider, *Induction and exacerbation of emphysema in* 

[45] Lucey, E.C., et al., *A polymer-bound elastase inhibitor is effective in preventing human neutrophil elastase-induced emphysema.* Ann N Y Acad Sci, 1991. 624: p. 341-2. [46] Halliday, H.L., *Clinical experience with exogenous natural surfactant.* Dev Pharmacol Ther,

[47] Halliday, H.L., et al., *Acute effects of instillation of surfactant in severe respiratory distress* 

[48] Long, W., et al., *A controlled trial of synthetic surfactant in infants weighing 1250 g or more* 

[49] Robertson, B., *Surfactant replacement in neonatal and adult respiratory distress syndrome.*

[50] Robertson, B., *Neonatal respiratory distress syndrome and surfactant therapy; a brief review.*

*with respiratory distress syndrome. The American Exosurf Neonatal Study Group I, and the Canadian Exosurf Neonatal Study Group.* N Engl J Med, 1991. 325(24): p. 1696-703.

*after exposure to elastase.* Anat Rec, 1981. 201(3): p. 523-35.

*macrophages.* Am Rev Respir Dis, 1983. 127(4): p. 449-55.

*syndrome.* Arch Dis Child, 1989. 64(1 Spec No): p. 13-6.

*regulation.* J Exp Med, 1975. 142(2): p. 361-77.

Am Rev Respir Dis, 1990. 141(1): p. 47-52.

Eur J Anaesthesiol, 1984. 1(4): p. 335-43.

Eur Respir J Suppl, 1989. 3: p. 73s-76s.

*pulmonary diseases of the lower respiratory tract in man.* Clin Immunol Rev, 1981. 1(3):

*emphysema in hamsters: a dose-response study.* Am Rev Respir Dis, 1981. 124(6): p. 714-7.

*has been exposed to activated human neutrophils.* Am Rev Respir Dis, 1983. 128(3): p.

*hamsters with human neutrophil elastase inactivated reversibly by a peptide boronic acid.*

*ketones.* J Med Chem, 1994. 37(20): p. 3303-12.

*peptides.* J Clin Invest, 1980. 66(4): p. 859-62.

p. 3313-26.

14(2): p. 127-94.

Dis, 1982. 125(4): p. 432-5.

1989. 13(2-4): p. 173-81.

p. 337-74.

434-9.

*active 3-amino-6-phenylpyridin-2-one trifluoromethyl ketones.* J Med Chem, 1994. 37(20):

*synthesis, and in vitro activity of a series of 3-amino-6-arylopyridin-2-one trifluoromethyl* 


[13] Janoff, A. and R. Dearing, *Prevention of elastase-induced experimental emphysema by a* 

[14] Kleinerman, J., et al., *The effect of the specific elastase inhibitor, alanyl alanyl prolyl alanine* 

[15] Gadek, J.E., et al., *Replacement therapy of alpha 1-antitrypsin deficiency. Reversal of protease-*

[16] Lange, F., et al., *Comparative effects of reversible and irreversible specific elastase inhibitors on elastase-induced emphysema.* Bull Eur Physiopathol Respir, 1980. 16 Suppl: p. 407-13. [17] Ranga, V., et al., *Effects of oligopeptide chloromethylketone administered after elastase: renal* 

[18] Martodam, R.R., et al., *Albumin microspheres as carrier of an inhibitor of leukocyte elastase:* 

[19] Gudapaty, S.R., et al., *The prevention of elastase-induced emphysema in hamsters by the* 

[20] Alber, T., G.A. Petsko, and D. Tsernoglou, *Crystal structure of elastase-substrate complex at* 

[21] Sawyer, L., et al., *The atomic structure of crystalline porcine pancreatic elastase at 2.5 A* 

[22] Hughes, D.L., et al., *Crystallographic study of the binding of a trifluoroacetyl dipeptide anilide* 

[23] Veale, C.A., et al., *Orally active trifluoromethyl ketone inhibitors of human leukocyte elastase.* J

[24] Edwards, P.D., et al., *Discovery and biological activity of orally active peptidyl trifluoromethyl ketone inhibitors of human neutrophil elastase.* J Med Chem, 1997. 40(12): p. 1876-85. [25] Edwards, P.D., et al., *Nonpeptidic inhibitors of human neutrophil elastase. 7. Design,* 

[26] Edwards, P.D., et al., *Peptidyl alpha-ketoheterocyclic inhibitors of human neutrophil elastase.* 

[27] Veale, C.A., et al., *Nonpeptidic inhibitors of human leukocyte elastase. 5. Design, synthesis,* 

[28] Veale, C.A., et al., *Non-peptidic inhibitors of human leukocyte elastase. 4. Design, synthesis,* 

[29] Bernstein, P.R., et al., *Nonpeptidic inhibitors of human leukocyte elastase. 6. Design of a* 

[30] Bernstein, P.R., et al., *Nonpeptidic inhibitors of human leukocyte elastase. 3. Design, synthesis,* 

*microspheres.* Am Rev Respir Dis, 1985. 132(1): p. 159-63.

*inhibitor with elastase.* J Mol Biol, 1982. 162(3): p. 645-58.

*trifluoromethyl ketones.* J Med Chem, 1995. 38(1): p. 98-108.

Med Chem, 1997. 40(20): p. 3173-81.

Med Chem, 1996. 39(5): p. 1112-24.

*ketones.* J Med Chem, 1995. 38(1): p. 86-97.

1995. 38(20): p. 3972-82.

38(1): p. 212-5.

*-- 55 degrees C.* Nature, 1976. 263(5575): p. 297-300.

Suppl: p. 399-405.

68(5): p. 1158-65.

124(5): p. 613-8.

118(2): p. 137-208.

2128-32.

p. 381-7.

*synthetic elastase inhibitor administered orally.* Bull Eur Physiopathol Respir, 1980. 16

*chloromethylketone, on elastase-induced emphysema.* Am Rev Respir Dis, 1980. 121(2):

*antiprotease imbalance within the alveolar structures of PiZ subjects.* J Clin Invest, 1981.

*toxicity and lack of prevention of experimental emphysema.* Am Rev Respir Dis, 1981.

*potential therapeutic agent for emphysema.* Proc Natl Acad Sci U S A, 1979. 76(5): p.

*intratracheal administration of a synthetic elastase inhibitor bound to albumin* 

*resolution: comparisons with the structure of alpha-chymotrypsin.* J Mol Biol, 1978.

*synthesis, and in vitro activity of a series of pyridopyrimidine trifluoromethyl ketones.* J

*3. In vitro and in vivo potency of a series of peptidyl alpha-ketobenzoxazoles.* J Med Chem,

*and X-ray crystallography of a series of orally active 5-aminopyrimidin-6-one-containing* 

*and in vitro and in vivo activity of a series of beta-carbolinone-containing trifluoromethyl* 

*potent, intratracheally active, pyridone-based trifluoromethyl ketone.* J Med Chem, 1995.

*X-ray crystallographic analysis, and structure-activity relationships for a series of orally* 

*active 3-amino-6-phenylpyridin-2-one trifluoromethyl ketones.* J Med Chem, 1994. 37(20): p. 3313-26.


**5** 

*Nagano, Japan* 

**Combined Pulmonary Fibrosis** 

 **and Emphysema (CPFE)** 

*1Department of Clinical Laboratory Sciences, Shinshu University School of Health Sciences.* 

Keisaku Fujimoto1 and Yoshiaki Kitaguchi2

*21st Department of Internal Medicine, Shinshu University School of Medicine.* 

Combined pulmonary fibrosis and emphysema (CPFE) is one of smoking-related lung

Emphysema is characterized by the permanent abnormal enlargement of airspaces distal to the terminal bronchioles, accompanied by destruction of their walls. The characteristics of emphysema do not, by definition, include thickening of the alveolar septa and fibrosis. However, coincidental idiopathic pulmonary fibrosis (IPF) and emphysema was firstly reported in 1990 by Wiggins et al (Wiggins J, et al., 1990) in London. Smoking-related interstitial lung diseases (SRILD) include desquamative interstitial pneumonia (DIP), respiratory bronchiolitis-related interstitial lung disease (RB-ILD), pulmonary Langerhans' cell histiocytosis (LCH) and idiopathic pulmonary fibrosis (IPF) (Ryu JH, et al., 2001). Tobacco smoking is also major course of emphysema and chronic obstructive pulmonary disease (COPD). Smoking is a common risk factor for both emphysema and pulmonary fibrosis. Recently, the occurrence of both emphysema and pulmonary fibrosis in the same patient has received increased attention as the syndrome of combined pulmonary fibrosis and emphysema (CPFE) (Cottin V, et al., 2005). It has been demonstrated that CPFE syndrome is not rare because on a series of 110 patients with IPF, 28% of them with at least 10% of the lung affected with emphysema, and thus are considered to have CPFE (Mejia M, et al., 2009).

In Japan Hiwatari *et al* (Hiwatari N, et al., 1993) reported nine patients with pulmonary emphysema and IPF among 152 pulmonary emphysema patients in 1993. Those patients were all men and heavy smokers. Odani *et al* (Odani K, et al., 2004) reported 31 patients combined with pulmonary emphysema and IPF among 14900 patients who underwent chest CT from January 1996 to March 2001 at Kochi Medical School Hospital in Japan. The CT of all patients showed the coexistence of emphysema with upper lung field predominance and diffuse parenchymal lung disease with significant pulmonary fibrosis predominantly in the lower lung fields (Figure 1). Centriacinar emphysema was present in 24 of the 31 (77%)

**1. Introduction** 

**2. Clinical characteristics of CPFE** 

diseases.

