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Discovering inflammatory biomarkers in chronic obstructive pulmonary disease and cystic fibrosis : a… Ngan, David Allen 2011

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 DISCOVERING INFLAMMATORY BIOMARKERS IN CHRONIC OBSTRUCTIVE PULMONARY DISEASE AND CYSTIC FIBROSIS: A CASE STUDY OF GRANZYME B  by  David Allen Ngan  B.Sc. (Hons), Trinity Western University, 2007  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE  in  THE FACULTY OF GRADUATE STUDIES  (Experimental Medicine)   THE UNIVERSITY OF BRITISH COLUMBIA  (Vancouver)    July 2011  © David Allen Ngan, 2011   ii Abstract  Granzymes, and particularly granzyme B (GzmB), are classically known to be involved in cell-mediated immunity and the induction of apoptosis through cell-specific targeting activity of cytotoxic T-lymphocytes in conjunction with perforin.  However, recent literature has emerged that describes a largely overlooked role for GzmB in potentially mediating disease progression.  This pathogenic role has been based on findings that GzmB can cleave extracellular matrix proteins while functioning independently of perforin.  In chronic inflammatory lung states such as chronic obstructive pulmonary disease (COPD) and cystic fibrosis (CF), there are important implications including the degradation of extracellular matrix leading to airway remodelling, reduced lung integrity, and emphysema. The generation of autoantigens may also result from cleavage of extracellular matrix proteins, contributing to inflammation.  We measured GzmB levels in plasma and lung tissue homogenates of COPD subjects by enzyme-linked immunosorbent assays (ELISAs) to determine the relationship with lung function, measured by FEV1 % predicted and FEV1/FVC ratio, and clinical COPD severity. We found that GzmB levels in the lung were positively associated with lung function.  The data raise the possibility that the GzmB we measured may be part of a protective inflammatory response in the microenvironment, or it may be pathogenic in the early but not the later phases of COPD.  In CF subjects, we measured levels of GzmB and other inflammatory biomarkers in plasma samples to determine their relationship with lung function parameters and hospitalization status.  While plasma levels of GzmB were not related to lung function or hospitalization status, we found that IL-6, IL-1β, and LPS levels were significantly higher in hospitalized patients, and CRP, IL-6, IL-1β, and LBP were significantly correlated with lung function impairment.  The results provide evidence that systemic inflammation is an independent factor associated with disease progression in CF and suggests an important role for chronic bacterial colonization in the lungs.  Further research is needed to validate the pathogenic contributions of GzmB in diseases with chronic inflammatory lung states and to delineate the mechanisms of such potential contributions.    iii Preface  A version of Chapter 2 of this thesis, “The Possible Role of Granzyme B in the Pathogenesis of Chronic Obstructive Pulmonary Disease,” has been published in the journal Therapeutic Advances in Respiratory Disease: • Ngan DA, Vickerman SV, Granville DJ, Man SF, Sin DD. The possible role of granzyme B in the pathogenesis of chronic obstructive pulmonary disease. Ther Adv Respir Dis 2009;3(3):113-129. I wrote and edited the majority of the manuscript, including additional revisions to address the reviewers’ comments, and created the figures.  SV Vickerman originally drafted the sections “Classical role of GzmB” and “Other granzymes.”  DJ Granville, SF Man, and DD Sin provided guidance and revisions for the manuscript.  The study in Chapter 4 was conducted with the approval of the University of British Columbia – Providence Health Care Research Ethics Board (UBC-PHC REB).  Certificate Number of the Ethics Certificate obtained: H09-00048.    iv Table of Contents  Abstract .................................................................................................................................... ii Preface ..................................................................................................................................... iii Table of Contents ................................................................................................................... iv List of Tables .......................................................................................................................... vi List of Figures ........................................................................................................................ vii List of Abbreviations and Acronyms ................................................................................. viii Acknowledgements ................................................................................................................ ix Dedication ................................................................................................................................ x 1 Introduction .......................................................................................................................... 1 2 The Possible Role of Granzyme B in the Pathogenesis of Chronic Obstructive Pulmonary Disease .................................................................................................................. 3 2.1 Introduction ........................................................................................................................... 3 2.2 Classical role of GzmB ......................................................................................................... 3 2.3 Potential pathogenic roles of GzmB in COPD ...................................................................... 6 2.3.1 Protease-antiprotease imbalance ....................................................................................... 7 2.3.2 Inflammation .................................................................................................................. 10 2.3.3 Accelerated aging ........................................................................................................... 13 2.4 Other granzymes ................................................................................................................. 15 2.5 Conclusion .......................................................................................................................... 16 3 Characterization of Granzyme B Protein Expression in Lung Tissue and Plasma of Chronic Obstructive Pulmonary Disease Patients ............................................................. 17 3.1 Introduction ......................................................................................................................... 17 3.2 Methods ............................................................................................................................... 17 3.2.1 Study populations and biological specimens .................................................................. 17 3.2.1.1 Advair, Biomarkers in COPD (Cohort 1) .............................................................. 18 3.2.1.2 Lung Tissue Research Consortium (Cohort 2)....................................................... 18 3.2.1.3 UBC James Hogg Research Centre Biobank and Registry (Cohort 3) .................. 18   v 3.2.2 Plasma preparation.......................................................................................................... 18 3.2.3 Lung tissue homogenization ........................................................................................... 18 3.2.4 Granzyme B assay .......................................................................................................... 19 3.2.5 Statistical analysis ........................................................................................................... 20 3.3 Results ................................................................................................................................. 20 3.3.1 Patient demographics ...................................................................................................... 20 3.3.2 Plasma GzmB and severity of COPD ............................................................................. 21 3.3.3 Plasma GzmB is inversely correlated with lung GzmB.................................................. 23 3.3.4 Lung tissue GzmB and severity of COPD ...................................................................... 24 3.4 Discussion ........................................................................................................................... 27 4 The Relationship of Systemic Inflammation to Hospitalization in Adult Patients with Cystic Fibrosis ....................................................................................................................... 30 4.1 Introduction ......................................................................................................................... 30 4.2 Methods ............................................................................................................................... 30 4.2.1 Study population and blood collection ........................................................................... 30 4.2.2 Clinical information ........................................................................................................ 31 4.2.3 Biomarker assays ............................................................................................................ 31 4.2.4 Statistical analysis ........................................................................................................... 32 4.3 Results ................................................................................................................................. 33 4.3.1 Patient demographics ...................................................................................................... 33 4.3.2 Circulating systemic inflammatory biomarkers and hospitalization status .................... 35 4.3.3 Systemic inflammation and lung function ...................................................................... 39 4.4 Discussion ........................................................................................................................... 40 5 Conclusion .......................................................................................................................... 44 References .............................................................................................................................. 46    vi List of Tables Table 3.1    Patient demographics and clinical characteristics of COPD subjects. ................. 21 Table 4.1    Patient demographics and clinical characteristics of CF subjects. ...................... 34 Table 4.2    Geometric means (and interquartile ranges) of biomarkers in CF patients who were and were not hospitalized. .................................................................................. 38 Table 4.3    Relationship between FEV1 percent predicted and biomarkers in CF subjects per 1 log increase in the biomarkers (n = 58). .................................................................. 40    vii List of Figures Figure 2.1    Classical representation of the direct induction of apoptosis through a granzyme B (GzmB)- and perforin (Prf)-mediated pathway. ........................................................ 5 Figure 2.2    Potential pathogenic roles of granzyme B (GzmB) in the pathogenesis of COPD. ....................................................................................................................................... 7 Figure 2.3    Two proposed mechanisms of action for granzyme B (GzmB). ........................ 10 Figure 3.1    Plasma levels of GzmB were not associated with lung function or GOLD stages of COPD in Cohort 1 (n = 100). ................................................................................. 22 Figure 3.2    Plasma levels of GzmB were not associated with lung function or GOLD stages of COPD in Cohort 2 (n = 27). ................................................................................... 23 Figure 3.3    Blood levels of GzmB in Cohort 2 (n = 27) were significantly inversely associated with corresponding lung levels of GzmB. ................................................. 24 Figure 3.4    Lung levels of GzmB were not associated with lung function or GOLD stages of COPD in Cohort 2 (n = 27). ........................................................................................ 25 Figure 3.5    Lung levels of GzmB were associated with lung function but not GOLD stages of COPD in Cohort 3 (n = 136). ................................................................................. 26 Figure 4.1    Plasma biomarker concentrations in hospitalized (n = 21) and non-hospitalized (n = 37) cystic fibrosis subjects. ................................................................................. 36 Figure 4.2    Plasma IL-6 was significantly correlated with plasma IL-1β in cystic fibrosis subjects (n = 58). ......................................................................................................... 37    viii List of Abbreviations and Acronyms A1AT: α1-antitrypsin BAL: bronchoalveolar lavage BMI: body mass index CCL18/PARC: chemokine C-C motif ligand 18 / pulmonary and activation-regulated chemokine CF: cystic fibrosis CFTR: cystic fibrosis transmembrane conductance regulator COPD: chronic obstructive pulmonary disease CRP: C-reactive protein CTL: cytotoxic T-lymphocyte ECM: extracellular matrix ELISA: enzyme-linked immunosorbent assay FEV1: forced expiratory volume in 1 second FEV1/FVC: ratio of forced expiratory volume in 1 second to forced vital capacity FVC: forced vital capacity GOLD: Global Initiative for Chronic Obstructive Lung Disease GzmB: granzyme B IL: interleukin LAL: Limulus amebocyte lysate LBP: lipopolysaccharide-binding protein LPS: lipopolysaccharide NFκB: nuclear factor-κB NK: natural killer OCT: optimal cutting temperature Prf: perforin sCD14: soluble cluster of differentiation 14 SP-D: surfactant protein D TLR: Toll-like receptor TNF-α: tumour necrosis factor-α Treg: regulatory T   ix Acknowledgements  I sincerely thank my research supervisor, Dr. Don Sin, for the tremendous opportunity and privilege of working in his research laboratory at the UBC James Hogg Research Centre.  Dr. Sin and Dr. Paul Man have together provided excellent supervision and encouragement, which has been an essential and inspiring experience in helping me develop my career as a scientist.  Dr. David Granville has also provided valuable advice, guidance, and direction as a member of my supervisory committee, and it has been helpful to gain insights from an expert in the field.  Dr. Janet McElhaney has contributed guiding comments and feedback that has helped enhance this thesis.  I have valued the technical assistance and guidance from Yuexin Li over the years as well as the help and support of Shawna Vickerman and the other Sin Lab members.  Dr. Mark Elliott has offered vital help in obtaining lung tissue specimens from the UBC James Hogg Research Centre Biobank and Registry.  I also acknowledge the assistance of Cystic Fibrosis Clinic study coordinators Vincent Zenarosa and Wen Wang for recruitment of subjects in the CF study as well as for blood collection.  Dr. Pearce Wilcox has helped in guiding the project as well.  This work is supported by the Canadian Institutes of Health Research and the British Columbia Lung Association.  We thank the Lung Tissue Research Consortium of the National Heart, Lung, and Blood Institute and the Advair, Biomarkers in COPD Investigators for access to samples used in our research.    x Dedication       To my mother    1 1     Introduction  Chronic obstructive pulmonary disease (COPD) is a highly prevalent inflammatory lung condition affecting 600 million people and accounting for 3 million deaths annually worldwide 1.  Over the next 20 years, the burden of COPD is expected to increase 1. Although cigarette smoking is a major risk factor for COPD, smokers constitute 60-70% of the COPD cases worldwide and the rest are lifetime never-smokers 2.  Furthermore, once COPD develops, smoking cessation reduces but does not abrogate the increased risk of morbidity and mortality.  Besides smoking, other factors for COPD include occupational and biomass exposures, childhood infections, diet, and genetic predispositions 2.  The pathogenesis of COPD is not well understood 3, and therapies that can effectively modify disease activity or progression are lacking 4.  COPD is characterized by expiratory airflow obstruction, hyperinflation, gas trapping, and gas exchange abnormalities that lead to breathlessness, cor pulmonale, and significant morbidity and mortality 5.  Over time, COPD contributes to significant extrapulmonary manifestations including cardiovascular disease and cancer, which, importantly, are the two leading causes of morbidity and mortality in patients with mild to moderate COPD 6.  Other systemic manifestations that have been attributed to COPD include cachexia, osteoporosis, and depression 6-10.  The two main pulmonary phenotypes of COPD are small airways disease and emphysema, and these may co-exist as observed in the majority of clinical cases 11. Biomass-related COPD tends to cause primarily airways disease, while α1-antitrypsin (A1AT) deficiency syndrome predominantly causes emphysema 12.  Leading hypotheses for the pathogenesis of COPD include protease-antiprotease imbalance, abnormal injury and repair processes due to inflammation, and accelerated aging 2, 13-15.  Recent literature has challenged the classical view that granzymes are predominantly involved in cell-mediated immunity and apoptosis through the cell-specific targeting activity of cytotoxic T-lymphocytes, and greater attention has been focused on the potential pathogenic role of particularly granzyme B (GzmB) in mediating disease progression in a wide variety of chronic diseases 16, 17.  The role of GzmB in the pathogenesis of diseases involving chronic inflammatory states in the lung, such as COPD and cystic fibrosis (CF), is uncertain and has not been fully explored.   2  In COPD, a greater proportion of cytotoxic T-lymphocytes expressing GzmB and perforin intracellularly has been reported in both the blood and bronchoalveolar lavage fluid, compared to non-smoking control subjects 18.  Furthermore, local expression of granzymes A and B has been observed in the type II pneumocytes of subjects with severe COPD 19.  In CF, inflammation in the respiratory tract has been characterized by neutrophils, macrophages, and T-cells, and elastin has been shown to be significantly reduced in the alveoli of CF subjects compared to healthy control subjects 20.  Airway remodelling related to impaired lung function has been described, and matrix breakdown has been implicated in this process 21 .  Collectively, these findings suggest that remodelling of the extracellular matrix and chronic inflammation have a fundamental role in lung injury and disease progression, and GzmB may be involved in these processes by virtue of its ability to degrade extracellular matrix proteins and mediate apoptosis.  This thesis will explore lung and blood concentrations of GzmB in COPD and CF and these will be correlated with clinical measures of disease progression and severity.  In particular, lung and blood levels of GzmB will be analyzed in relation to lung function, narrowly defined by changes in spirometry, and clinical diagnosis of COPD severity.  In CF, blood levels of GzmB and other inflammatory biomarkers will be examined in relation to lung function parameters and hospitalization status, an important clinical endpoint in CF.     3 2     The Possible Role of Granzyme B in the Pathogenesis of Chronic Obstructive Pulmonary Disease∗  2.1 Introduction  In the previous chapter, we introduced COPD as a disease thought to be caused by protease-antiprotease imbalance, abnormal injury and repair processes due to inflammation, and accelerated aging.  In this chapter, we review in greater detail one promising candidate molecule that we believe may play a unifying role in all three pathogenic mechanisms of COPD: granzyme B (GzmB).  2.2 Classical role of GzmB  The granzymes are a family of serine proteases, of which there are five known members in humans: A, B, H, K, and M 22.  Of these, granzyme B (GzmB) is the most completely characterized 23.  However, while the pro-apoptotic intracellular mechanisms of this granzyme have been thoroughly examined, the extracellular effects of GzmB action are less understood 24, 25.  It is thought that both the intracellular and extracellular functions of the granzymes play a significant role in disease, and that the latter may be particularly consequential in the pathophysiology of chronic inflammatory diseases 25 such as COPD.    GzmB is a 32-kDa protein 26-28, though its precise molecular mass may vary due to differences in the degree of glycosylation 29.  The protease is generated as an inactive zymogen that must be processed and activated prior to release from the originating cell 30. Although it is generally considered a monomer, there is evidence that GzmB may exist as a dimer under certain conditions 31, 32.  GzmB has classically been described in the context of cytotoxic T-lymphocytes (CTLs) and natural killer (NK) cells and their role in the targeted death of tumor and virally-infected cells.  More recently, GzmB has been found to be expressed by some non-cytotoxic lymphoid and non-lymphoid cells, including regulatory T (Treg) cells 33, 34, type II pneumocytes 19, basophils 35, macrophages 19, 36, 37, dendritic cells 38,  ∗  A version of Chapter 2 has been published.  Ngan DA, Vickerman SV, Granville DJ, Man SF, Sin DD. The possible role of granzyme B in the pathogenesis of chronic obstructive pulmonary disease. Ther Adv Respir Dis 2009;3(3):113-129.   4 mast cells 39, smooth muscle cells 37, keratinocytes 40, 41, syncytial trophoblasts 42, and Sertoli cells 42, though the precise function of GzmB in non-lymphoid cells has not been fully elucidated 23.  GzmB and perforin (Prf), a pore-forming protein similar in structure and function to the terminal complement molecule C9 43, are sequestered in cytotoxic granules within CTLs and NK cells.  Release of these molecules is induced by stimulation of the cells by foreign antigens presented on the surface of target cells 44.  Antigen recognition is followed by formation of the immunological synapse and polarization of these cytotoxic granules towards the target cell, poising them for release 45, 46.  Prf is essential in mediating the action of GzmB against targeted cells.  Accordingly, mice lacking Prf harbour CTLs and NK cells that are functionally incompetent against virus-infected and cancerous cells 47, 48.  Similarly, transfection of rat basophilic leukemia mast cells with cDNA for GzmB alone demonstrates an inability to kill thymoma and mastocytoma tumour targets, whereas co-transfection with both GzmB and Prf results in substantial cytotoxic activity 49.  It is believed that transfection of Prf facilitates GzmB access to the target cell, though the precise means by which it does so is currently under review. The conventional model of this process, wherein Prf forms a multimeric transmembrane pore in the target cell through which GzmB can pass, is generally accepted based on the structural and functional similarities between Prf and C9 43 and electron micrographs of plasma membrane-inserted polyperforin pores 50, 51.  However, this theory has been challenged by a lack of direct mechanistic evidence and the suggestion that the diameter of the perforin pore may be too small to facilitate passage of GzmB 52, though this argument is disputed by Kurschus et al. 53.  Furthermore, it has been suggested that GzmB and Prf form multimeric complexes with serglycin, the primary proteoglycan of cytotoxic granules that functions as a carrier molecule, and the delivery of GzmB-serglycin complexes by either Prf-serglycin or free Prf occurs without evident pore formation in the plasma membrane 54.  Irrespective of the exact mechanism, it is well established that Prf activity is required in some form for classical GzmB-mediated apoptosis (Figure 2.1). Recent studies propose two alternatives to the traditional perforin pore model.  One is a receptor-mediated endocytosis model in which GzmB is endocytosed in complex with the mannose-6 phosphate receptor and subsequently released from the endosome by Prf 53, 55-57. Alternatively, there is a receptor-independent model in which GzmB at the cell surface is   5 internalized during the cell-repair process in response to perforin pore-induced membrane damage 58.     Figure 2.1    Classical representation of the direct induction of apoptosis through a granzyme B (GzmB)- and perforin (Prf)-mediated pathway. Cell-specific release of GzmB in conjunction with Prf by effector cells such as cytotoxic T-lymphocytes (CTLs) facilitates entry of GzmB into the target cell through a polyperforin pore.  Cleavage of the pro- apoptotic molecule Bid by GzmB results in GzmB-truncated Bid (gtBid), an activated form that translocates to the mitochondrion and induces release of cytochrome c (Cyt c) through activation of pro- apoptotic proteins Bax and Bak.  Cyt c release enables formation of a dATP/Apaf-1/procaspase 9 complex called the apoptosome.  Autocatalytic proteolysis of procaspase 9 to caspase 9 initiates the downstream caspase cascade, leading to manifestation of the apoptotic phenotype.   Once inside the target cell, GzmB proceeds to initiate programmed cell death via the apoptosis-inducing caspase (cysteine-aspartic acid protease) cascade.  GzmB preferentially   6 cleaves after Asp/Glu residues 26 and, more specifically, after Asp in the sequence Ile/Val- Gly/Met/Glu-Xaa-Asp-Xaa-Gly 22.  Although GzmB is capable of cleaving numerous intracellular substrates 23, 59, the key targets of GzmB proteolysis relating to apoptosis include caspases as well as the pro-apoptotic, Bcl-2 homologue molecule Bid.  Multiple caspases have been identified as GzmB targets 59, but Adrain et al. 60 have shown that caspases 3, 7, 8, and 10 are directly activated by GzmB.  Regardless of the direct activation of the caspases by GzmB, only the proteolysis of Bid is essential to the initiation of apoptosis by GzmB 61.  Cleavage of inactive Bid by GzmB results in a truncated molecule, gtBid, which translocates to the mitochondrion and interacts with Bax and Bak to induce the release of cytochrome c 62-67.  Cytochrome c release leads to formation of a dATP/Apaf-1/procaspase 9 complex called the apoptosome, facilitating autocatalytic activation of procaspase 9 to caspase 9 68-70 and, ultimately, activation of caspases 3 and 7 71, 72.  The caspase cascade culminates with substrate proteolysis and consequent manifestation of the apoptotic phenotype 59.  2.3 Potential pathogenic roles of GzmB in COPD  There have been few studies into the specific role of GzmB in COPD.  It is speculated that the pathogenesis of emphysema is mediated by T-lymphocytes, primarily CD8+ CTLs 73. It has been noted that CD8+ T-lymphocytes and macrophages are prevalent in the lungs of healthy smokers and patients with mild stages of COPD, while neutrophilic inflammation prevails in the severe stages 74, 75.  One study found that the bronchoalveolar lavage (BAL) fluid of asymptomatic smokers and COPD patients had increased numbers of CD8+ T-cells and a 3- to 4-fold higher proportion of T-cells expressing GzmB and perforin compared to non-smoking control subjects 18, despite a suggested role for GzmB in enhancing the function of regulatory T cells, which control autoimmunity and suppress effector T cells 33.  The study also found that GzmB levels in the BAL fluid were significantly elevated in COPD compared to the control subjects and there was a correlation between the percentage of GzmB- expressing T-cells and bronchial epithelial cell apoptosis 18.  Higher levels of perforin have also been found in COPD airways 18, 76.  While it is generally accepted that GzmB and granzyme A (GzmA) expression is found primarily in CTLs and NK cells, local expression of the granzymes has also been reported in type II pneumocytes of severe COPD lungs 19.   7 Collectively, the data suggest that GzmB has a significant pathological role in COPD.  In the following discussion, we assess the potential of GzmB in the context of its contributions to three leading hypotheses for the pathogenesis of COPD (Figure 2.2).   Figure 2.2    Potential pathogenic roles of granzyme B (GzmB) in the pathogenesis of COPD. Hypotheses for the pathogenesis of COPD include accelerated aging, protease-antiprotease imbalance, and abnormal injury and repair processes due to sustained inflammation.  Strikingly, GzmB may be implicated in each of these circumstances.  Mediation in alveolar apoptosis may contribute to accelerated aging in the lungs.  Extracellular release of uninhibited GzmB exemplifies the protease-antiprotease imbalance concept where degradation of the extracellular matrix (ECM) can result from proteolytic activity.  Extracellular GzmB may also generate autoantigens and elicit aberrant inflammation, perpetuated by infiltration of inflammatory cells importing additional GzmB.  2.3.1 Protease-antiprotease imbalance  The discovery of an association between genetic α1-antitrypsin (A1AT) deficiency and emphysema in young individuals by Laurell and Eriksson in 1963 provided the foundation for the modern protease-antiprotease imbalance hypothesis of COPD   8 pathogenesis 77.  A1AT was subsequently classified as a member of the serine protease inhibitor (serpin) superfamily 78, 79.  Serpins, as their name suggests, include proteins with the ability to inhibit the endopeptidase enzymatic activity of serine proteases, though some members of this superfamily are known to lack such an inhibitory role 80.  A1AT, also known as SERPINA1, is a functional inhibitor of serine proteases that is particularly active against neutrophil elastase, as evidenced by previous kinetic studies 81.  While A1AT deficiency has been reported to account for fewer than 5% of diagnosed cases of COPD 82, observations of the consequences of this hereditary condition have provided valuable insights into understanding one pathophysiological mechanism of COPD.  Other proteases and antiproteases including matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) 83, 84, and SERPINE2 85, 86 have been implicated in the pathogenesis of COPD.  Numerous animal studies have suggested that the action of serpins against proteases such as neutrophil elastase may prevent the induction of pulmonary emphysema 87-89.  Neutrophil and macrophage elastases have been implicated as a contributor and requirement, respectively, in the development of emphysema in mice 90, 91. In human clinical trials, A1AT replacement therapy has shown some promise in a number of A1AT deficient individuals 92.  Interestingly, A1AT deficient patients can redevelop emphysema after lung transplantation, underscoring the importance of serpins in modulating disease progression 93.  In addition to neutrophil elastase, another potent protease that has been similarly linked with emphysema is proteinase 3 (PRTN3), with observed degradative effects on elastin in vitro and subsequent emphysema after intratracheal instillation in hamsters 94.  Of particular interest is the discovery of significant protein sequence and active site homology between PRTN3 and other serine proteases such as elastase, cathepsin G, and granzymes 95. With respect to the similarity with granzymes, the sequence homology is even described to occur at amino acid positions 9-16 (PHSRPYMA), a conserved motif region considered to be a hallmark of granzymes 95, 96.  Similar to other proteases in this class of molecules, GzmB is another potent protease and dysregulation of its expression or activity could potentially contribute to extracellular matrix (ECM) destruction and parenchymal remodelling if it is released in the absence of Prf and subsequently accumulates extracellularly.  While the majority of GzmB is released   9 unidirectionally towards target cells, GzmB can also be released nonspecifically, escaping into the extracellular environment 25, 97.  Several mechanisms have been proposed including escape from the immunological synapse during or after target cell killing, constitutive granzyme secretion by CTLs after degranulation, and induction of granzyme release from CTLs by extracellular stimuli 97.  In vitro experiments have shown that bystander cells in the proximity of the target cell can undergo apoptosis by means of a granule-dependent mechanism 98.  In vivo, local serum concentrations of GzmB are highly elevated in areas where a CTL response is occurring 99.  The effect on non-targeted cells may be due in part to the failure of endogenous GzmB inhibition mechanisms, including the lack of counteraction by proteinase inhibitor 9 (PI-9), a human serpin shown to directly inhibit GzmB activity in vitro and also neutrophil elastase through a similar mechanism 23, 100-102.  Also, while the formation of immunological synapses has been described in T-cells and NK cells 46, 103, many other cell types that express and secrete granzymes do not form immunological synapses, are not immune cells, and do not produce perforin to mediate GzmB internalization.  Without perforin-mediated delivery of GzmB to target cells, the non-specifically released GzmB would have no direct cytotoxic activity 49 and may accumulate in the extracellular milieu, leading to tissue destruction and parenchymal remodelling.  Importantly, it has been demonstrated in vitro that GzmB and GzmA are capable of cleaving ECM proteins 97, 104, 105, and though the amount of non-specifically released GzmB may be small, it may be sufficient to elicit matrix remodeling in chronic inflammatory states. Specifically, GzmB has been shown to cleave vitronectin, fibronectin, laminin, fibrillin-1, decorin, and aggrecan proteoglycan 104, 106-108, as well as co-localizing to elastin fibres 109 whose degradation has been associated with aging, atherosclerosis, and COPD 110.  Though the observation did not seem to be reflected at the protein level, a two-fold decrease in perforin mRNA was noted in peripheral CD8+ T-cells of emphysematous smokers compared to controls while GzmB mRNA levels were not significantly different, suggesting increased propensity for an extracellular role for GzmB in the diseased state 111.  This decreased expression of perforin in CD8+ T-cells has also been described in lesions of chronic pulmonary tuberculosis 112.  The major components of connective tissue in the lung are collagens, elastic fibres comprised of elastin and microfibrils, proteoglycans, and interstitial cells including myofibroblasts and fibroblasts 113.  Both structural robustness and elasticity   10 are important influences on the mechanical aspects of pulmonary function 114, 115.  The accumulation of GzmB in the extracellular environment during chronic inflammation in COPD could be an attractive explanation for ECM degradation and remodelling and, consequently, the emphysematous phenotype in the lung since compromised elastic fibre architecture and function are pathological features of COPD 116, 117 (Figure 2.3).   Figure 2.3    Two proposed mechanisms of action for granzyme B (GzmB). In the classical view, GzmB is released by effector cells such as cytotoxic T-lymphocytes (CTLs) towards a target cell.  Through a perforin-mediated mechanism, GzmB enters the cell and activates the apoptotic machinery, leading to controlled cell death.  GzmB may also be released nonspecifically and uninhibited to the extracellular environment.  The consequences would include extracellular matrix (ECM) remodeling, generation of autoantigens, and adhesion-dependent apoptosis, resulting in a chronic inflammatory state and emphysematous phenotype in the lungs.  2.3.2 Inflammation  The inflammation hypothesis of COPD pathogenesis is based on observations that lung and systemic inflammation increases with disease progression 10, 118 and during   11 exacerbations 119.  Inflammation has been associated with poor health, increased risk of hospitalization, and death 120, 121.  Animal experiments have demonstrated that changes in the expression of inflammatory or anti-inflammatory cells or molecules such as CD8+ T-cells 122, interleukins (IL) 1β/13/18 123-125, chemokine receptors CCR6 and CXCR2 126, 127, tumour necrosis factor (TNF)-α and its receptors 128, and platelet-derived growth factor 129, can lead to emphysema 130.  However, anti-inflammatory agents in the form of corticosteroids have failed to make a major impact on COPD clinical outcomes 131, 132 and it is yet unclear whether inflammation is a cause or effect of COPD though it is evident that the chronic inflammatory state is detrimental to disease progression.  There is debate as to whether GzmB can be neutralized by the main serpins of the lung, such as A1AT, but recent research suggests that GzmB inhibition does not occur and therefore this protease may not be adequately controlled in inflamed tissues 133.  This would be problematic in patients with stable COPD where an increased number of CD8+ T- lymphocytes has been observed in bronchial biopsy specimens 134.  Poor lung function has previously been associated with greater numbers of airway CD8+ T-cells 118, 135, 136, and perforin and GzmB expression is upregulated in infiltrating airway CD8+ lymphocytes of patients with COPD and other lung conditions 76, 137, 138.  Studies in mice have suggested a pivotal role for CD8+ T-cells in controlling the nature of the inflammatory response elicited in the lungs, including recruitment of macrophages 122, 139.  A study of the inflammatory cell profile in the lungs of smokers with emphysema revealed a predominance of lymphocytes and specifically CD8+ T-cells versus the large number of neutrophils in the smoking and non- smoking patient controls 73.  Moreover, it has been shown by immunohistochemistry that lung-infiltrating CD8+ T-cells and CD57+ NK cells do express GzmB and can be considered activated 19.  The percentage of CD8+ lymphocytes in BAL fluid does not differ significantly between patients with COPD, smokers without COPD, and never-smokers 140, 141.  However, a greater proportion of CD8+ lung lymphocytes isolated from BAL fluid produced intracellular inflammatory cytokines in COPD patients compared to controls, including IL-2, IL-4, IL-10, and IL-13 140.  Both soluble GzmB and the percentage of T-cells with intracellular GzmB have been observed to be increased in COPD versus controls 18.  Smokers with COPD exhibit higher proportions of CD8+ T-cells in the paratracheal lymph nodes than   12 those without COPD, and it has been speculated that the immune response may originate or focus in these local lymph nodes 142.  While the exact antigenic stimulus is unclear at this time, autoimmunity is widely believed to be involved 142, 143.  The activity of non-specifically released GzmB has been implicated in several autoimmune disorders including Sjogren’s syndrome, scleroderma, and systemic lupus erythematosus 144-154.  Fragments of the ECM generated by proteolysis have been shown to possess chemotactic properties and contribute to inflammation 155-160, and this may be relevant to COPD as anti-elastin autoantibodies have been discovered that may cause chronic lung inflammation and emphysema 161.  Perpetuity of inflammation may result as a consequence of the infiltration of inflammatory cells importing additional GzmB.  In the blood, elevated levels of the systemic inflammation marker IL-6 in COPD patients have been described, though the percentage of CD8+ or CD4+ T-lymphocytes and NK cells expressing GzmB and perforin did not differ between emphysematous smoking COPD patients and both smoking and non-smoking control subjects 111.  Conversely, in another study, intracellular GzmB has been detected in a greater percentage of T-cells from the blood of COPD patients compared to never-smokers 18.  One recurrent idea seems to be the phenotypic variation of the CD8+ T-cells of COPD subjects, where activation and the expression of surface antigens and cytokines differs with controls despite apparently equal proportions of peripheral CD8+ T-cells in COPD versus normal subjects 162-164.  Activation of peripheral CD8+ T-cells has been significantly correlated with disease severity as assessed by forced expiratory volume in 1 second (FEV1) % predicted values for pulmonary function 162, and GzmB synthesis and secretion may be involved.  The recruitment of lymphocytes and other inflammatory cells to the lungs and various sites in the body may at least partially be regulated by GzmB, which can potentially compromise the vascular endothelium and increase permeability 165.  In vitro, treatment with mouse GzmB was found to disrupt intercellular junctions and cause visible gaps in the culture monolayer of mouse endothelioma cells 166.  In these cells, GzmB treatment also resulted in irregular, disjointed patterns of cell adhesion molecules including vascular endothelial cadherin (VE-cadherin), platelet/endothelial cell adhesion molecule-1 (PECAM- 1), and junctional adhesion molecule-A (JAM-A) as visualized by immunostaining 166.  Thus,   13 by inhibiting key adhesion molecules, GzmB may have a role in controlling leukocyte transmigration and the inflammatory process 167-169.  2.3.3 Accelerated aging  The accelerated aging hypothesis of COPD pathogenesis is a recent concept and is based on the principle that inflammatory and oxidant stresses from cigarette smoke hasten physiological aging in the lungs by activating apoptotic pathways 13.  Animal experiments support this notion in showing that overexpression of IFN-γ induced DNA damage, apoptosis, and emphysema while both chemical caspase inhibition and genetic knockout of caspase 3 were able to simultaneously reduce apoptosis and prevent emphysema 170, 171.  In humans, COPD lung samples consistently exhibit overexpression of apoptotic and senescent markers in alveolar cells compared to control lungs of smokers without COPD 172, 173. Alveolar dilatation and impairment of gas exchange is linked with lung aging, and the expression of GzmB in type II pneumocytes and alveolar macrophages may represent one contributing means by which alveolar cell apoptosis occurs 19, 174.  Aged lungs and COPD lungs share many striking similarities, including reduction of lung function, reduction of vital capacity, comparable inflammatory profile, shortened telomere length, and decrease in expression of antioxidant and anti-aging molecules 172, 175, 176 .  Additionally, senescence-prone mice have been found to be more susceptible to cigarette smoke and exhibit reduced levels of the antioxidant glutathione than senescence-resistant mice 177.  T-cell senescence has been reported in COPD and autoimmune disorders such as multiple sclerosis and rheumatoid arthritis, with an increased percentage of CD4+ CD28- T- cells observed 178, 179.  Of perhaps even greater interest was the further finding that this subset of CD4+ T-cells contained higher amounts of intracellular GzmB and perforin than their typical counterparts that have not lost the CD28 surface marker 178.  GzmB has recently been associated with the pathogenesis of atherosclerosis, and knockout of GzmB in an apolipoprotein E (ApoE)-knockout mouse model lessened the extent of atherosclerosis, medial thinning in vessels, and arterial and skin aging typical of this strain 180, 181.  ApoE and GzmB double-knockout mice seem to age more slowly, possibly due to an improved ability to maintain elasticity and integrity of the extracellular matrix in the absence of GzmB 180.  The prevention of this form of degeneration would be critically   14 important in modulating the evident aging process and structural changes in COPD lungs.  In humans, facial wrinkling has been associated with decreased lung function, increased risk of COPD, and increased risk of more significant emphysema, suggesting a similar underlying initiating process 182.  While the role of GzmB in COPD beyond the respiratory system is not well known, coronary atherosclerosis and rheumatoid arthritis, disorders that share many similar pathologic features with COPD 183, 184, are affected by atypical GzmB expression that might contribute to extracellular matrix destruction and result in disease 36, 37, 104, 181, 185, 186.  The role of GzmB as a pro-apoptotic mediator facilitates its direct involvement in COPD as a disease of accelerated aging.  Pronounced inflammation is a feature of numerous diseases related to aging including Alzheimer’s disease, rheumatoid arthritis, osteoporosis, cardiovascular disease, and COPD 175.  It is noted by Rahman and Adcock 187 that reactive oxygen species, such as those derived from cigarette smoke, drive inflammation via activation of redox-sensitive transcription factors, including nuclear factor-κB (NFκB) and activator protein-1, which in turn promote the upregulation of several pro-inflammatory molecules.  The consequent enhanced inflammation would purportedly perpetuate the accelerated aging process through the activation of apoptotic pathways by mediators such as GzmB.  In rheumatoid arthritis, a demonstration of dose-dependent induction of apoptosis by GzmB has been described, indicating the potency of the protease 188.  In addition to the classical cell-specific targeting mechanism of apoptosis that requires perforin mediation, GzmB has been found to induce human coronary artery smooth muscle cell death in vitro by cleaving extracellular proteins and facilitating adhesion-dependent apoptosis 105.  A parallel effect attributed to the aftermath of proteolytic extracellular matrix degradation has been observed in cultured mouse embryonic fibroblasts, as it was demonstrated that 24 hours of treatment with mouse GzmB adversely influenced natural cell morphology, adherence, and viability in a dose-dependent manner 166, 189.  Lung fibroblasts of COPD patients have been shown to have impaired proliferation, providing further evidence of accelerated aging and implying altered extracellular matrix regeneration and remodelling is associated with effects of GzmB 190.    15 2.4 Other granzymes  Although we have taken GzmB to be the principal focus of this chapter, other granzymes (A, H, K, and M in humans) are also present in immune cells in lower abundance, albeit with the exception of granzyme A (GzmA) 24.  These other granzymes are poorly characterized and not well understood.  However, considering the potentially significant role that GzmB plays in COPD, they warrant a brief overview.  It has been reported that GzmA is capable of inducing cell death via a GzmB- and caspase-independent pathway 23, 191, 192, though it is unclear whether this process takes place under physiological conditions.  GzmA also exhibits ECM cleavage abilities including degradation of type IV collagen 23-25, 97, 193, but a role in the pathogenesis of COPD in this regard is questionable due to immediate extracellular inactivation of GzmA by antithrombin III in blood plasma 194.  However, Vernooy et al. 19 demonstrated that type II pneumocytes express both GzmB and GzmA and found that expression of the latter is increased in patients with very severe COPD compared to control subjects.  In vitro experiments have shown that recombinant rat GzmA causes rounding and reduced adhesion of type II pneumocytes as well as stimulation of IL-8, a neutrophil chemoattractant, factors that could contribute to alveolar destruction and enhanced inflammation, respectively 195.  Recent research suggests that the role of GzmA in promoting inflammation by inducing production of pro-inflammatory cytokines in monocytic and other cells may be more important than its cytotoxic ability 196.  Evidence for secretion of cytokines such as IL-1β, IL-6, IL-8, and TNF-α by human monocytes and lung fibroblasts upon stimulation by GzmA supports this pro-inflammatory, non-cytotoxic role 197, 198. Apoptosis-like, caspase-independent cell death has been induced by granzyme H (GzmH) in vitro, though it is better characterized by its ability to cleave certain viral proteins, and no normal cellular substrates have been identified for this granzyme 23, 24.  Granzyme K (GzmK) causes cell death in a caspase-independent manner similar to GzmA, and may also have ECM cleavage abilities 23, 24.   Bratke et al. 199 investigated the role of GzmK in human lung disease, and while they found no difference in the levels in BAL fluid of COPD subjects compared to controls, they did note upregulation of GzmK in cases of acute airway inflammation.  Based on these results, the role of GzmK in COPD exacerbations is a future avenue of investigation.  The ability of granzyme M (GzmM) to independently induce cell death in vivo is controversial, although it may play a role in enhancing GzmB activity   16 through inactivation of the intracellular GzmB inhibitor PI-9 23, 24.  Neither GzmH nor GzmM have been implicated in ECM cleavage 25, and there is no current evidence linking them to COPD or other lung diseases.  The emphasis of current research on granzymes other than GzmB has shifted towards the pro-inflammatory and non-cytotoxic potential of these proteases in mediating host immune defense 200.  2.5 Conclusion  Although the present experimental data are incomplete, GzmB likely plays an important role in the pathogenesis of COPD.  Traditionally viewed only as a molecule for targeted induction of apoptosis, evidence for an extracellular role has been emerging including a proposed association with matrix remodeling, generation of autoantigens, and adhesion-dependent apoptosis.  These events are germane in the pathogenesis of COPD and indicate that GzmB is one member of a small group of molecules with the potential to fit into several of the known pathogenic pathways.  In this regard, GzmB should be considered a highly relevant molecule.  The pathogenic contributions of this molecule are a fruitful area for future investigation, and these findings will determine if GzmB represents a promising new target for drug and biomarker discovery in COPD.    17 3     Characterization of Granzyme B Protein Expression in Lung Tissue and Plasma of Chronic Obstructive Pulmonary Disease Patients  3.1 Introduction  As discussed in the previous chapters, there are several reasons to speculate an important role for GzmB in the pathogenesis of COPD.  The capacity of GzmB to be involved in protease-antiprotease imbalances, sustained inflammation, and accelerated aging, current steps in the pathogenesis of COPD, suggests a central role for the molecule. Extracellularly-released GzmB is of primary interest for the proposed association with remodelling of the extracellular matrix, generation of autoantigens, and adhesion-dependent apoptosis.  These events may cause a chronic inflammatory state and emphysematous phenotype in the lungs.  To explore the relationship of GzmB to disease progression in COPD, narrowly defined by changes in spirometry, expression levels of GzmB in the systemic circulation can be determined.  Measuring the concentration of GzmB in lung tissue may also provide important information on the potential effects of local expression.  We hypothesized that nonspecific, extracellular release of GzmB may occur in chronic obstructive pulmonary disease that contributes to pathogenesis, and thus the expression levels of GzmB would correlate with disease progression as defined by changes in spirometry.  To test this hypothesis, we measured the concentration of GzmB in the blood and lung tissues of COPD patients.  3.2 Methods  3.2.1 Study populations and biological specimens  Three study populations were included in this study with subjects having had a clinical diagnosis of COPD according to Global Initiative for Chronic Obstructive Lung Disease (GOLD) international guidelines 2.  Demographic and clinical data for the subjects in this study were obtained by chart review.  All study subjects gave informed consent.  This study was conducted with the approval of the University of British Columbia – Providence Health Care Research Ethics Board (UBC-PHC REB).   18 3.2.1.1 Advair, Biomarkers in COPD (Cohort 1)  We used the blood samples of 50 COPD subjects diagnosed with GOLD Stage II disease and 50 subjects with GOLD Stage III or IV disease, for a total of 100 subjects, from the Advair, Biomarkers in COPD study 201.  The spirometric criteria of this study were FEV1 of less than 80% of predicted and an FEV1 to FVC ratio of less than 0.70 (post- bronchodilator). 3.2.1.2 Lung Tissue Research Consortium (Cohort 2)  We obtained 27 paired, frozen plasma and lung tissue core specimens of COPD subjects from the Lung Tissue Research Consortium of the National Heart, Lung, and Blood Institute.  The lung tissue specimens had been flash-frozen in liquid nitrogen.  GOLD Stage III disease was not represented in this sample. 3.2.1.3 UBC James Hogg Research Centre Biobank and Registry (Cohort 3)  Frozen, optimal cutting temperature (OCT) compound-embedded lung tissue core specimens were obtained from the UBC James Hogg Research Centre (JHRC) Biobank at St. Paul’s Hospital (Vancouver, British Columbia, Canada), a bank of surgically resected human lung tissues 202.  The sample of 136 COPD subjects comprised a full spectrum of disease severity from all four GOLD stages.  3.2.2 Plasma preparation  Venous blood samples were collected by venipuncture into BD Vacutainer Blood Collection Tubes containing ethylenediaminetetraacetic acid (EDTA) anticoagulant (Becton, Dickinson and Company) using standard techniques.  Plasma was prepared from the collected blood samples by centrifugation and stored at -80°C.  3.2.3 Lung tissue homogenization  Lung tissue core specimens were placed in plastic weighing boats over dry ice and cut with razor blades.  The mass of tissue used was determined by weighing on a top-loading laboratory balance.  To normalize the amount of lung tissue used among the study subjects, a standardized amount of total protein extraction buffer was added (Cohort 2: 7 mL of buffer   19 per gram of lung tissue; Cohort 3: 5 mL of buffer per gram of lung tissue).  The specimens from Cohort 2 required greater dilution because they had been flash-frozen without OCT compound as an added diluent.  Total protein extraction buffer consisted of 20 mM HEPES, 1 mM EDTA, 250 mM sucrose, 100 mM sodium fluoride, 100 mM sodium pyrophosphate, and 10 mM sodium orthovanadate.  The prepared buffer was adjusted to pH 7.4, and 1% Triton and 0.1% protease inhibitor cocktail (Sigma-Aldrich P8340, St. Louis, MO) were freshly added to the protein extraction buffer prior to use.  Lung tissue specimens were homogenized using a Qiagen TissueLyser LT according to instructions from the manufacturer’s handbook (“Protocol: Purification of Protein from Animal and Human Tissues”).  For each sample, 5 mm mean diameter stainless steel beads were used to mechanically disrupt specimens in 2 mL microcentrifuge tubes for 5 minutes at the 50-Hz setting.  The homogenized suspension was centrifuged at 10,000 × g for 5 minutes at 4°C.  The supernatant containing soluble proteins was drawn and stored at -80°C for use in assays.  3.2.4 Granzyme B assay  GzmB was measured in plasma from collected blood samples and the prepared lung tissue homogenates using a high-sensitivity enzyme-linked immunosorbent assay (ELISA) kit that was commercially available (eBioscience, San Diego, CA) and based on a monoclonal anti-human GzmB antibody.  Test protocol instructions from the manufacturer’s product manual were followed.  The lower detection limit of the kit was 0.2 pg/mL, and the coefficients of variation were 5.29%, 10.25%, 6.09%, and 4.81% for Cohort 1 (plasma), Cohort 2 (plasma), Cohort 2 (lung tissue homogenates), and Cohort 3 (lung tissue homogenates), respectively.  Plasma samples from Cohorts 1 and 2 were diluted 2 times with dilution buffer prior to the assay.  Lung tissue homogenates from Cohort 2 were diluted 100 times with dilution buffer prior to the assay, and those for Cohort 3 were diluted 20 times due to the presence of OCT compound as an added diluent.  Spike recovery testing was performed in human serum by the kit manufacturer, and they reported that there was no detectable interference or cross-reactivity with other proteases such as proteinase 3, tryptase, cathepsin G, granzyme A, human neutrophil elastase, trypsin, and chymotrypsin.    20 3.2.5 Statistical analysis  Data for the GzmB measurements were analyzed after natural log transformation due to their skewed distribution.  The relationships of GzmB to lung function (FEV1 as a percentage of predicted, FEV1/FVC ratio) and between blood GzmB and lung GzmB were assessed using univariate linear regression.  GzmB levels were compared between GOLD stages using a Student’s t-test for independent samples, or using one-way analysis of variance (ANOVA) with Tukey’s post-hoc test for multiple comparisons.  P-values less than 0.05 were considered significant (two-tailed test).  All analyses were conducted using GraphPad Prism 5 (La Jolla, CA).  3.3 Results  3.3.1 Patient demographics  The baseline clinical characteristics of the study subjects are summarized in Table 3.1.  Lung function data from spirometry and complete smoking history were not available for some of the study subjects, including pack-years data for Cohort 1.  In Cohort 3, many subjects (84.56%) from whom lung tissue core specimens were obtained had diagnoses of lung cancer (for example, adenocarcinoma, bronchioloalveolar carcinoma, large cell carcinoma, non-small cell carcinoma, and squamous cell carcinoma), though nonneoplastic regions remote from the tumours would have been sampled 202.  In the plasma and lung tissue homogenates of Cohorts 1-3, no significant association was found between GzmB and age, sex, BMI, current smoking status, or pack-years.   21 Table 3.1    Patient demographics and clinical characteristics of COPD subjects.  Cohort 1 (n = 100) Cohort 2 (n = 27) Cohort 3 (n = 136) Age, years ± SD  67.42 ± 7.94 62.04 ± 11.16 62.95 ± 10.41 Sex, male n (%)  72 (72.00) 15 (55.56) 72 (52.94) BMI, kg/m2 ± SD  28.23 ± 6.29 26.59 ± 4.45 24.92 ± 5.07 FEV1, % predicted ± SD  46.62 ± 22.54 55.67 ± 29.83 75.49 ± 24.97* FVC, % predicted ± SD  73.36 ± 19.63 71.70 ± 24.15 89.40 ± 20.15* FEV1/FVC ± SD  0.48 ± 0.17 0.53 ± 0.19 0.65 ± 0.14* Current Smokers, n (%)  35 (35.00) 3 (15.79)* 77 (58.33)* Pack-Years, ± SD n/a 58.38 ± 40.82* 42.82 ± 28.56* * Data for some subjects not available  3.3.2 Plasma GzmB and severity of COPD  In two independent cohorts (Cohorts 1 and 2), we did not find a significant association between plasma concentration of GzmB and lung function as measured by FEV1 % predicted and FEV1/FVC ratio (Figure 3.1 A, B; Figure 3.2 A, B).  There was also no significant difference between the GOLD stages of COPD severity (Figure 3.1 C; Figure 3.2 C, D).  In Cohort 1, there was a trend towards a higher concentration of plasma GzmB in more advanced cases of COPD (GOLD Stages III and IV) compared to less severe cases (GOLD Stage II) (P = 0.1511).  In Cohort 2, this trend was reversed.  Subjects with GOLD Stage I disease had a higher plasma concentration of GzmB than subjects with GOLD Stages II and IV disease, but the smaller sample size may have influenced the results.    22   Figure 3.1    Plasma levels of GzmB were not associated with lung function or GOLD stages of COPD in Cohort 1 (n = 100). (A) Plasma GzmB vs. FEV1 % predicted.  β ± SE = -0.011 ± 0.007; GzmB ln-transformed.  (B) Plasma GzmB vs. FEV1/FVC ratio.  β ± SE = -1.366 ± 0.955; GzmB ln-transformed.  (C) Comparison of GOLD Stage II to GOLD Stages III and IV together.  Geometric means (interquartile ranges): 24.33 (14.05, 43.22) pg/mL; 38.50 (17.70, 74.08) pg/mL.   23   Figure 3.2    Plasma levels of GzmB were not associated with lung function or GOLD stages of COPD in Cohort 2 (n = 27). (A) Plasma GzmB vs. FEV1 % predicted.  β ± SE = 0.004 ± 0.007; GzmB ln-transformed.  (B) Plasma GzmB vs. FEV1/FVC ratio.  β ± SE = 0.040 ± 1.133; GzmB ln-transformed.  (C) Comparison across all GOLD stages except GOLD Stage III, which was not available.  Geometric means (interquartile ranges): 40.74 (18.26, 90.88) pg/mL; 89.33 (64.25, 174.67) pg/mL; 44.29 (16.63, 131.05) pg/mL; 40.62 (25.09, 97.67) pg/mL.  (D) Comparison of GOLD Stage I to GOLD Stages II and IV together.  Geometric means (interquartile ranges): 89.33 (64.25, 174.67) pg/mL; 42.41 (22.06, 122.7) pg/mL.  3.3.3 Plasma GzmB is inversely correlated with lung GzmB  The blood and lung specimens from Cohort 2 were paired, and we were able to analyze the relationship between the concentrations of GzmB in lung tissue and plasma. There was an inverse relationship between lung tissue GzmB levels and plasma GzmB levels   24 (Figure 3.3) (P = 0.0464).  This indicated that a greater amount of GzmB in the lungs corresponded to a lesser amount of GzmB in the circulation in the 27 subjects.   Figure 3.3    Blood levels of GzmB in Cohort 2 (n = 27) were significantly inversely associated with corresponding lung levels of GzmB. β ± SE = -0.356 ± 0.170; GzmB ln-transformed.  3.3.4 Lung tissue GzmB and severity of COPD  In Cohort 2, we did not find a significant association between the concentration of GzmB in lung tissue homogenates and lung function as measured by FEV1 % predicted and FEV1/FVC ratio (Figure 3.4 A, B).  However, in Cohort 3 (an independent cohort with a larger sample size), we found that lung tissue GzmB was associated with lung function (Figure 3.5 A, B) (P < 0.05).  Subjects with impaired lung function had lower concentrations of GzmB in their lung tissue.  We did not find a significant difference in lung tissue GzmB between the GOLD stages of COPD severity in Cohorts 2 and 3 (Figure 3.4 C, D; Figure 3.5 C, D).   25  Figure 3.4    Lung levels of GzmB were not associated with lung function or GOLD stages of COPD in Cohort 2 (n = 27). (A) Lung GzmB vs. FEV1 % predicted.  β ± SE = 0.002 ± 0.007; GzmB ln-transformed.  (B) Lung GzmB vs. FEV1/FVC ratio.  β ± SE = 0.185 ± 1.043; GzmB ln-transformed.  (C) Comparison across all GOLD stages except GOLD Stage III, which was not available.  Geometric means (interquartile ranges): 12149.62 (8880.42, 16622.32) pg/mL; 15431.96 (10820.3, 18689.03) pg/mL; 4850.81 (3330.94, 6655.25) pg/mL; 8765.09 (7239.27, 9310.1) pg/mL.  (D) Comparison of GOLD Stage I to GOLD Stages II and IV together.  Geometric means (interquartile ranges): 15431.96 (10820.3, 18689.03) pg/mL; 6520.57 (3774.62, 9012.67) pg/mL.   26  Figure 3.5    Lung levels of GzmB were associated with lung function but not GOLD stages of COPD in Cohort 3 (n = 136). (A) Lung GzmB vs. FEV1 % predicted.  β ± SE = 0.006 ± 0.003; GzmB ln-transformed.  (B) Lung GzmB vs. FEV1/FVC ratio.  β ± SE = 1.266 ± 0.520; GzmB ln-transformed.  (C) Comparison across all GOLD stages.  Geometric means (interquartile ranges): 2456.63 (1640.78, 3602.87) pg/mL; 2245.70 (1222.58, 3768.97) pg/mL; 1840.27 (1379.87, 2550.75) pg/mL; 2340.48 (1644.96, 3371.15) pg/mL; 1574.82 (994.21, 3657.05) pg/mL.  (D) Comparison of GOLD Stage I to GOLD Stages II-IV.  Geometric means (interquartile ranges): 2245.70 (1222.58, 3768.97) pg/mL; 1800.64 (1332.40, 3061.73) pg/mL.    27 3.4 Discussion  Our data suggest that plasma levels of GzmB are not related to lung function, conveniently but narrowly defined by FEV1 as a percentage of predicted and FEV1/FVC ratio, or GOLD clinical diagnosis of COPD severity.  This is supported by a study that has found peripheral T-lymphocytes are not generally activated and do not express cytotoxic markers such as GzmB 111.  However, we did find that plasma GzmB levels were inversely correlated with lung tissue levels of GzmB (Figure 3.3).  We speculate that this may be due to preferential homing of GzmB-containing cells into the lungs from vascular tissues with inflammatory stimulus in the lungs.  It has been shown that, compared to non-smoking controls, COPD patients have more cytotoxic T-lymphocytes (CTLs) in smaller than in larger airways 203, 204, suggesting that these cells are specifically recruited towards the areas of tissue destruction and emphysema.  These cells would be in an activated state and express GzmB, and would be drawn away from the peripheral CD8+ T-cell pool.  The consequence would be a greater amount of GzmB in the lung tissue and correspondingly lesser levels expressed in the blood, which was our finding.  In the largest cohort in which we measured GzmB in lung tissue homogenates, we found that GzmB levels were positively associated with lung function, a result contrary to our original hypothesis (Figure 3.5 A, B).  We expected to find that greater protein expression levels of GzmB would correlate with impaired lung function and disease progression, according to the model of nonspecific, extracellularly-released GzmB playing an important role in COPD pathogenesis.  There are a number of possible explanations for this observed outcome.  First, sampling error may have been an important limitation of our study. Our sample sizes were small, and the randomized selection of study subjects may have led to sampling bias that does not reflect the characteristics of the whole population of COPD patients.  In most cases, lung specimens were obtained from patients who had undergone surgical lung resection as part of treatment for lung cancer and would be expected to have a higher baseline expression of GzmB.  Our lung tissue core specimens may also have contributed to sampling error, as we performed our measurements in single cores from each subject and the cores are taken from randomized locations of a heterogeneous lung.  Further, viability of the sampled lung tissue may need to be taken into account.  Lung tissue specimens taken from more severe stages of COPD are expected to have a greater extent of   28 emphysema compared to less severe stages, including extensive damage to the alveoli and associated capillaries.  Our experimental method normalized the measurements by mass of lung tissue (the concentrations of the prepared homogenates were dependent on mass) without regard for viability, and it is likely that lung tissue from more advanced COPD cases was mostly emphysematous.  Consequently, GzmB-expressing cells may have limited access to the lung, resulting in apparently decreased GzmB expression in those lung specimens. This would have affected the comparison with less severe stages of COPD, where destruction of the lung tissue would have been less of an issue and GzmB expression may appear to be higher.  In a preliminary pilot study, we attempted to adjust for the amount of tissue from which GzmB was extracted by normalizing our measurements using the concentration of total protein (bicinchoninic acid protein assay; Thermo Fisher Scientific, Rockford, IL) or DNA (Hoechst 33258 fluorometric DNA quantitation assay; Sigma-Aldrich, St. Louis, MO) in each sample.  Lung GzmB levels and lung function were positively associated before and after normalizing for protein concentration, and the trend also remained after normalizing for DNA concentration though the relationship was not statistically significant (data not shown).  An additional consideration is that the use of steroid medications in advanced cases of COPD is known to result in toxicity to cytotoxic T-lymphocytes and a lowered lymphocyte count 205.  With a decreased number of GzmB-expressing cells in the lungs, this could support our observation that the lung tissue concentration of GzmB is decreased in more advanced COPD (Figure 3.5 A, B).  Another possibility is that GzmB may actually be protective in the microenvironment of the lung parenchyma.  Higher levels of GzmB in lung tissue could reflect a favourable state, as the precise contributions of CTLs and other cytotoxic cells to COPD pathogenesis via GzmB are speculative and remain to be fully elucidated.  A limitation of our study is that our methods did not allow us to determine the specific localization of the GzmB we measured or the originating source, though CTLs, natural killer cells, and type II pneumocytes have been identified in previous studies as being important in COPD 19.  Also, while cell culture supernatant and serum have been tested with the GzmB assay, lung tissue homogenates have not been specifically validated and this should be recognized while interpreting these data.  Moreover, as this was a cross-sectional study designed to explore potential associations, we cannot make definite conclusions on causality or directionality of the relationships we observed.  In future studies, investigation of   29 the bioactivity of GzmB in these samples may be beneficial as well as concurrent measurement of perforin, which may help reveal whether biologically active GzmB acts extracellularly in COPD.  It is possible that GzmB may play an important role in the pathogenesis of COPD early in its disease course but not in the later stages when the lungs are mostly destroyed and the inflammatory process may be “burnt out”.  In this study, we found that GzmB levels in the lung tissue of COPD patients is positively correlated with lung function as measured by both FEV1 % predicted and FEV1/FVC ratio.  These data raise the possibility that GzmB plays an active role in early phases of COPD, and further work is needed to fully understand the complexities of the inflammatory process in COPD and how it may be related to disease progression.    30 4     The Relationship of Systemic Inflammation to Hospitalization in Adult Patients with Cystic Fibrosis  4.1  Introduction  Cystic fibrosis (CF) is a progressive, debilitating disease that affects nearly 30,000 Americans and occurs with a frequency of about 1 in 3500 births 206.  It is characterized by persistent lung infection and lung function impairment.  It also affects other organs including the sinuses, gastrointestinal tract, endocrine glands, and the bone 207-210.  Although all CF cases are caused by a mutation in the gene for the CF transmembrane conductance regulator, there is considerable heterogeneity in the rate at which the disease progresses 211.  The traditional risk factors for rapid progression include reduced body mass index, colonization of the airways with pathogenic bacteria such as Pseudomonas aeruginosa, and female sex 212- 214 .  More recently, some have suggested that systemic inflammation may be another important risk factor for poor health outcomes in CF independent of these traditional risk factors 208, 213, 215, 216.  However, the studies that have evaluated this issue have produced inconsistent results and have measured different components of the immune system, making cross comparisons difficult.  Moreover, none of these studies have evaluated these biomarkers on hard clinical outcomes such as exacerbations or hospitalizations, which are important endpoints in CF.  In this study, we determined the relationship of plasma inflammatory biomarkers to lung function and to the risk of hospitalizations in adult patients with CF.  The plasma biomarkers were carefully chosen to represent innate or adaptive immunity, by-products of Gram-negative pathogens, or lung-based proteins.  4.2 Methods  4.2.1 Study population and blood collection  We enrolled 58 consecutive adult patients from the Cystic Fibrosis (CF) Clinic at St. Paul’s Hospital (Vancouver, British Columbia, Canada), who were clinically stable at the time of assessment.  For inclusion, patients had to have one or more clinical features consistent with the CF phenotype 217 as well as a genotype with two identifiable disease- causing CF transmembrane conductance regulator (CFTR) mutations, sweat chloride   31 measurements greater than 60 mmol/L on two occasions, and nasal potential difference results consistent with CF.  Patients who had an exacerbation within the previous 4 weeks were excluded from the study.  This study was conducted with the approval of the University of British Columbia – Providence Health Care Research Ethics Board (UBC-PHC REB). Following informed consent, we collected venous blood samples and performed spirometry using standard techniques, in accordance with guidelines from the American Thoracic Society 218.  Demographic and clinical data were obtained by chart review.  4.2.2 Clinical information  The subjects’ infection status was determined by microbial review within the preceding 3 years of study entry. Those who had at least one sputum culture positive for Pseudomonas aeruginosa were considered to be chronically colonized by this organism. We also performed chart review and retrieved data from the hospital database to determine whether the patients had a hospitalization for CF exacerbation in the previous 5 years.  4.2.3 Biomarker assays  Plasma was prepared from the collected blood samples and a select number of circulating inflammatory proteins were measured using high-sensitivity enzyme-linked immunosorbent assay (ELISA) kits that were commercially available. These included cytokines involved in the early phase inflammatory response such as interleukin (IL)-6, and IL-1β (R&D Systems, Minneapolis, MN); acute phase response proteins such as C-reactive protein (CRP; R&D Systems); proteins involved in adaptive immunity such as GzmB (eBioscience, San Diego, CA); pneumo-proteins (i.e. proteins synthesized predominantly in the lungs) such as chemokine C-C motif ligand 18 / pulmonary and activation-regulated chemokine (CCL18/PARC; R&D Systems) and surfactant protein D (SP-D; BioVendor, Brno, Czech Republic) 219, 220; and proteins involved in lipopolysaccharide (LPS) signalling such as LPS-binding protein (LBP; Hycult Biotech, Uden, The Netherlands) and soluble cluster of differentiation 14 (sCD14; R&D Systems). The coefficients of variation for these kits were 7.42%, 12.33%, 3.14%, 5.80%, 1.24%, 2.17%, 1.67%, and 5.85% respectively, and the lower detection limits were 0.039 pg/mL, 0.057 pg/mL, 0.010 ng/mL, 0.2 pg/mL, 0.01 ng/mL, and 0.2 ng/mL, 4.4 ng/mL, and 0.125 ng/mL respectively.  LPS levels were measured   32 using a commercially-available kinetic chromogenic Limulus amebocyte lysate (LAL) assay kit (Lonza Walkersville, Walkersville, MD) following plasma dilution and heat inactivation pre-treatment steps to diminish interference from plasma proteins.  The coefficient of variation for the assay was 2.24%, and the lower detection limit was 0.5 pg/mL.  4.2.4 Statistical analysis  Data for the biomarker measurements were analyzed after natural log transformation due to their skewed distribution.  The relationships of the biomarkers to lung function (FEV1 as a percentage of predicted) and to each other were assessed using linear regression. Multivariate linear regression analysis was performed to assess the role of possible confounding factors such as age, sex, and pseudomonal status.  Plasma biomarker levels were compared between those who did and did not experience a hospitalization using a Student’s t-test for independent samples.  Fisher’s exact test was used to analyze categorical data.  We used a logistic regression model to determine the relationship of plasma biomarkers to the risk of hospitalization, independent of possible confounders such as age, sex, BMI, FEV1 as a percentage of predicted, and colonization with Pseudomonas aeruginosa.  To ensure parsimony and enhance the robustness of the model, we used a stepwise approach to select only those covariates that significantly impacted on the relationship (the significance level for entering, P ≤ 0.05 and the significance level for stay, P ≤ 0.05).  C-statistics were also calculated for the various models to determine the incremental benefit of adding biomarker measurement to the overall discriminative ability of traditional risk factors for hospitalization (e.g. BMI and FEV1).  The relationship of plasma biomarkers to FEV1 % predicted was ascertained using multiple regression analysis.  We used a similar stepwise approach in selecting the appropriate covariates into this model as we did for the hospitalization analysis. To facilitate interpretation and cross-comparisons of beta-coefficients of plasma biomarkers from the regression model, we standardized the beta-coefficients to their standard deviation. Thus, the beta-coefficients are presented per 1 SD increase in the plasma biomarker expression.  P-values less than 0.05 were considered significant (two-tailed test).  All analyses were conducted using SAS (Carey, NC) version 9.1.    33 4.3 Results  4.3.1 Patient demographics  The age of the study subjects ranged from 18 to 61 years (Table 4.1).  The baseline clinical characteristics of the study subjects are summarized in Table 4.1 according to whether or not they had been hospitalized for a CF exacerbation in the previous 5 years.  Of the 58 subjects, 21 had been hospitalized (36.21% of total) and 37 had not.  Reduced FEV1, reduced BMI, and presence of Pseudomonas in sputum cultures were all significant risk factors for CF hospitalizations. 42 of the subjects were classified as Pseudomonas+ based on sputum microbiology (72.41% of total) and 16 were Pseudomonas–.  A greater portion of hospitalized patients had been administered Tobramycin versus non-hospitalized patients.    34 Table 4.1    Patient demographics and clinical characteristics of CF subjects.  Total (n = 58) Hospitalized (n = 21) Non- hospitalized (n = 37) P-value Age, years ± SD  30.45 ± 10.32 31.43 ± 7.97 29.89 ± 11.51 0.5901 Sex, male n (%)  34 (58.62) 10 (47.62) 24 (64.86) 0.2000 BMI, kg/m2 ± SD  23.24 ± 3.25 21.71 ± 2.49 24.10 ± 3.34 0.0060 FEV1, % predicted ± SD  71.93 ± 24.80 59.29 ± 20.38 79.11 ± 24.44 0.0027 Current medications   Azithromycin, n (%)   Ciprofloxacin, n (%)   Dornase alfa, n (%)   Ibuprofen, n (%)   Prednisone, n (%)   Tobramycin, n (%)   Inhaled steroids, n (%)    18 (31.03)  2 (3.45)  24 (41.38)  2 (3.45)  0 (0)  13 (22.41)  37 (63.79)   10 (47.62)  0 (0)  12 (57.14)  1 (4.76)  0 (0)  9 (42.86)  15 (71.43)   8 (21.62)  2 (5.41)  12 (32.43)  1 (2.70)  0 (0)  4 (10.81)  22 (59.46)   0.0745  0.5299  0.0967  1.0000  –  0.0082  0.4078 Diabetes, n (%)  22 (37.93) 10 (47.62) 12 (32.43) 0.2747 Pseudomonas, n (%) 42 (72.41) 20 (95.24) 22 (59.46) 0.0048     35 4.3.2 Circulating systemic inflammatory biomarkers and hospitalization status  The geometric mean plasma levels of IL-6, IL-1β, and LPS of hospitalized CF subjects were 3.56 pg/mL, 0.187 pg/mL, and 1.296 ng/mL, respectively.  These were all significantly higher than in non-hospitalized subjects (1.68 pg/mL, 0.098 pg/mL, and 0.970 ng/mL, respectively) (Figure 4.1).  After adjustment for BMI and FEV1 % predicted, the relationships remained significant.  There was a significant association between plasma IL-6 levels and IL-1β levels (Figure 4.2).  Geometric mean plasma concentrations of CRP, GzmB, CCL18, SP-D, LBP, and sCD14 did not significantly differ between the hospitalized and non-hospitalized groups (Table 4.2).  The c-statistic of BMI and FEV1 together was 0.794. There was a modest improvement in the c-statistic by adding IL-6, IL-1β, or LPS.   36  Figure 4.1    Plasma biomarker concentrations in hospitalized (n = 21) and non-hospitalized (n = 37) cystic fibrosis subjects. * Unadjusted P-values shown; P = 0.0441, 0.0395, and 0.0263, respectively (A-C), adjusted for BMI and FEV1 % predicted.  (A) Geometric mean plasma levels of IL-6 were higher in hospitalized than in non- hospitalized subjects.  (B) Plasma levels of IL-1β were also higher in hospitalized subjects.  (C) The geometric mean plasma concentration of lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, was significantly higher in hospitalized subjects (1.296 ng/mL) than in non-hospitalized subjects (0.970 ng/mL).   37  Figure 4.2    Plasma IL-6 was significantly correlated with plasma IL-1β in cystic fibrosis subjects (n = 58). β ± SE = 0.595 ± 0.105; IL-6 and IL-1β ln-transformed.  R2 = 0.3666; P < 0.0001.   38 Table 4.2    Geometric means (and interquartile ranges) of biomarkers in CF patients who were and were not hospitalized. Biomarker Total (n = 58)* Hospitalized (n = 21)* Non- Hospitalized (n = 37)* P-value Adjusted P-value† c- statistics CRP, µg/mL  3.02 (1.54, 6.14) 3.45 (2.03, 8.03) 2.81 (1.26, 5.59) 0.4979 0.5169 0.792  IL-6, pg/mL  2.20 (1.06, 4.17) 3.56 (2.79, 5.82) 1.68 (0.95, 3.09) 0.0036 0.0441 0.827  IL-1β, pg/mL  0.156 (0.057, 0.286) 0.187 (0.057, 0.496) 0.098 (0.057, 0.198) 0.0075 0.0395 0.825  SP-D, ng/mL  84.33 (64.04, 112.29) 87.47 (75.28, 106.07) 82.61 (63.72, 113.24) 0.6046 0.3161 0.800  CCL18, ng/mL  59.48 (41.38, 75.28) 60.05 (38.67, 73.54) 59.16 (44.48, 75.87) 0.9189 0.9900 0.794  GzmB, pg/ml 109.15 (25.48, 389.84) 90.71 (25.36, 384.87) 121.23 (26.24, 389.84) 0.4586 0.9498 0.793  LPS, ng/mL  1.08 (0.88, 1.29) 1.296 (0.99, 1.37) 0.970 (0.80, 1.26) 0.0125 0.0263 0.837  sCD14, µg/mL 1.10 (0.87, 1.34) 1.11 (0.96, 1.33) 1.10 (0.78, 1.34) 0.8902 0.5743 0.788  LBP, µg/mL 27.58 (21.97, 39.87) 30.79 (23.74, 41.22) 25.90 (18.83, 35.72) 0.1669 0.8984 0.792 * Data are presented as geometric mean (25th, 75th percentile)  † Adjusted for BMI and FEV1 % predicted (based on stepwise regression), which collectively had a c- statistic (or area under the curve) value of 0.794    39 4.3.3 Systemic inflammation and lung function  CRP, IL-6, IL-1β, and LBP were significantly correlated with lung function impairment in both univariate and multivariate analysis (Table 4.3).  LPS was significantly related to FEV1 only in the multivariate analysis, raising the possibility that it was a false positive.  The use of standardized beta-coefficient (i.e. the change in FEV1% predicted per 1 standard deviation increase in the plasma concentrations of the biomarker) allows for comparison of the beta-coefficients across biomarkers.  CRP had the highest standardized beta-coefficient, followed by LBP, IL-6 and IL-1β, suggesting that CRP is most strongly associated with FEV1.    40 Table 4.3    Relationship between FEV1 percent predicted and biomarkers in CF subjects per 1 log increase in the biomarkers (n = 58). Biomarker Unadjusted Adjusted* Standardized Beta- Coefficient (per 1 SD increase in the levels of biomarker) β ± SE P-value β ± SE P- value CRP, mg/L  -10.61 ± 2.65 <0.001 -7.04 ± 1.98 0.008 -0.3134 IL-6, pg/mL  -10.72 ± 3.11 0.001 -5.53 ± 2.66 0.043 -0.2159 IL-1β, pg/mL  -6.97 ± 3.36 0.042 -4.65 ± 2.67 0.002 -0.1784 SP-D, µg/mL  -17.70 ± 7.95 0.030 -9.83 ± 5.93 0.103 -0.1584 CCL18, pg/mL  -6.33 ± 5.93 0.291 -3.01 ± 4.23 0.480 -0.0672 GzmB, pg/mL 4.09 ± 2.27 0.077 -0.33 ± 1.89 0.863 -0.0187  LPS, pg/mL  1.07 ± 7.44 0.886 13.30 ± 5.41 0.017 0.0239 sCD14, µg/mL 1.88 ± 9.67 0.847 -10.26 ± 6.96 0.150 -0.1414  LBP, µg/mL -22.28 ± 6.63 0.001 -16.02 ± 4.74 0.001 -0.2946 * Adjusted for age, pseudomonal status, and history of hospitalization, which were significantly related to reduced lung function  4.4 Discussion  We investigated the relationship of systemic inflammation to lung function impairment and risk of hospitalizations among clinically stable CF patients.  This study produced several important findings.  First, in addition to the traditional risk factors such as reduced BMI, poor lung function as measured by FEV1 percent predicted, and presence of Pseudomonas aeruginosa in sputum cultures, we found that plasma levels of two early phase inflammatory cytokines, IL-6 and IL-1β, were significantly related to the risk of   41 hospitalization in patients with CF, independent of the traditional risk factors.  However, there was no significant relationship of GzmB, a marker of adaptive immunity, lung-based proteins such as CCL18/PARC and SP-D, or acute-phase reactants such as CRP, LBP, and sCD14 221 with the risk of hospitalization.  Together, these data suggest that early phase inflammatory cytokines may be good candidate plasma biomarkers of health outcomes in CF. In CF, the innate immune response is most likely directed towards bacteria chronically colonizing the airways such as P. aeruginosa.  While it has been noted that the first acquisition of P. aeruginosa does not result in immediate, drastic effects to measurable clinical outcomes, chronic airway infection and colonization by this microorganism is associated with greater morbidity and mortality 222.  Once the transition to chronic pseudomonal infection commences, permanent eradication of P. aeruginosa becomes a challenge due to phenotypic adaptations of the organism to the CF airway and persistent re- infection 223, 224.  An in vivo study using a murine model infected with airborne Stenotrophomonas maltophilia, another serious opportunistic pathogen in CF, has shown that the introduction of the organism to the lungs elicits a strong local host inflammatory response 225 .  Induced sputum samples from CF patients feature increased pro-inflammatory mediators such as IL-6, IL-1β, IL-8, and tumour necrosis factor (TNF)-α compared to controls 226, and in BAL fluid, IL-8 levels are positively correlated with bacterial counts in addition to being higher than those of controls 227.  Further, cell culture experiments involving airway epithelial cells homozygous for the CFTR ∆F508 mutation have demonstrated that, unlike normal lung airway epithelial cells, these cells respond with IL-6 hypersecretion primarily mediated by pattern recognition receptor activation (e.g. LPS and flagellin) rather than cytokine receptor stimulation (e.g. IL-17A) 228.  This supports the notion that inflammation within the CF lung is driven at least in part by pulmonary bacterial infection.  Our second important finding was that plasma LPS derived from Gram-negative bacteria is significantly higher in those who were hospitalized for a CF exacerbation than those who were not.  LPS is an immunologically active antigen, which can cause an intense inflammatory process in the lung and elsewhere by activating monocytes, macrophages, and endothelial cells.  Its presence in the systemic circulation may enhance the systemic inflammatory response in CF, as previously seen in a murine model 229.  We postulate that some of the LPS expression in the systemic circulation may be derived from the lungs   42 through a process called translocation.  It is conceivable that the diseased respiratory tract in CF may facilitate translocation of bacterial components or pro-inflammatory cytokines from the lungs to the systemic circulation where it incites an inflammatory response.  This biological plausibility is supported by a study in rabbits where it was shown that it is physically possible for LPS to undergo pulmonary-to-systemic translocation under certain conditions, specifically in mechanical ventilation strategies 230.  While our methods did not allow us to determine the originating source of plasma LPS, we speculate that the LPS we measured is likely derived from P. aeruginosa in the lungs or other Gram-negative, CF- related bacteria.  Future studies will be needed to test this hypothesis and to ascertain whether the difference in plasma LPS concentration that we observed is biologically significant. Chronically elevated levels of circulating LPS may not be adequately neutralized and cleared by serum lipoproteins, and this may induce systemic stimulation of innate responses, production of cytokines, and, in turn, triggering of a multitude of adverse conditions such as bone loss, CF-related diabetes, and cachexia 209, 231, 232.  Third, biomarkers that are related to innate immunity or early acute phase reactants such as IL-6, IL-1β, CRP, and LBP are significantly associated with reduced lung function in CF patients independent of age, pseudomonal status, or history of hospitalization, suggesting that systemic inflammation is an independent factor for disease progression in CF.  Whether the rise in these biomarkers is the result or the cause of impaired lung function is not certain. Our data have extended the findings of a previous study that examined a cohort of adult CF patients aged 30 years or greater.  Levy et al. found an association between lower FEV1 percent predicted and higher serum CRP levels, but they did not adjust for sex or pseudomonal status of the patients, and the study cohort was limited to an older population 216 .  Furthermore, their retrospective study design prevented them from obtaining serum samples and performing pulmonary function tests within a close proximity of time.  While our current study did not make comparisons to healthy controls, previous studies have found that median plasma or serum concentrations of IL-6, IL-1β, IL-1 receptor antagonist (IRAP) 233 , neutrophil granule proteins, and CRP 234 were higher in CF patients versus healthy subjects.  Collectively, the use of plasma markers of systemic inflammation, especially IL-6 and CRP, provides additional indicators of clinical status and may add to our understanding of the relationship between inflammation and the severity of lung disease in CF patients.   43  There were important limitations to our study.  This is a cross-sectional study, which precludes firm conclusions on causality or directionality of the relationship.  While we postulate that systemic inflammation drives disease progression, it is entirely possible that disease progression is responsible for systemic inflammation, and a comprehensive prospective longitudinal study would be needed to address this issue.  Longitudinal data may also provide insight into plasma biomarker profiles during acute exacerbations and following antibiotic treatment.  Additionally, we did not measure other pro-inflammatory biomarkers such as TNF-α and IL-8 or those with known anti-inflammatory effects such as IL-10, which has been shown in a mouse model to reduce the inflammatory response to P. aeruginosa 235. Future investigation into these regulators of the inflammatory response could provide a clearer picture of the complex interactions involved that lead from excessive inflammation to disease progression.  Notwithstanding the limitations, we found that more intense systemic inflammation, mediated by the innate immune system, is associated with CF hospitalizations and with disease progression in CF as defined by changes in spirometry.  IL-6 and IL-1β, in particular, are promising systemic biomarkers for disease progression and hospitalization in CF.  A large prospective study testing these promising biomarkers would be of great value in determining their usefulness as a clinical tool in managing patients with CF.   44 5     Conclusion  In our case studies in COPD, we found that GzmB protein expression may be more important in lung tissue.  In the blood, we did not find a significant association with either lung function measures or clinical diagnosis of COPD severity by GOLD stages.  In lung tissue homogenates, levels of GzmB were positively associated with lung function, measured by both FEV1 % predicted and FEV1/FVC ratio.  The complexity of the inflammatory process in COPD is poorly understood, including the effect of potential lymphocyte count reductions in patients with advanced COPD due to the administration of steroid medications in their treatment.  In addition, pre-existing lung cancer in the study subjects may have influenced the GzmB data and altered the apparent relationship with COPD.  We also cannot rule out that GzmB is not pathogenic in COPD, and instead it could either be protective in the microenvironment of the lung parenchyma or pathogenic in the early but not the later phases of COPD.  The levels we measured may be indicative of an appropriate local inflammatory response by cytotoxic cells and not necessarily represent GzmB that would act in a harmful, pathogenic manner.  In CF, while plasma levels of GzmB were not related to lung function or hospitalization status, we found that IL-6, IL-1β, and LPS levels were significantly higher in hospitalized patients.  CRP, IL-6, IL-1β, and LBP were significantly correlated with lung function impairment both before and after adjusting for age, pseudomonal status, and hospitalization history.  These are biomarkers related to innate immunity and early acute phase reactants, and the results provide evidence to indicate that systemic inflammation is an independent factor associated with disease progression in CF.  Hospitalized CF patients have more intense systemic inflammation and a measurably higher concentration of circulating LPS in the blood, suggesting an important role for chronic bacterial colonization in the lungs and possible translocation of bacterial products to the systemic circulation.  The potential pathogenic role of GzmB remains as an interesting area for future investigation.  We have found that GzmB protein expression in COPD lung tissue varies with lung function measures, a clinical marker of disease progression.  While greater levels of GzmB were associated with better lung function and less severe disease in our studies, further research is needed to validate whether there are pathogenic contributions from GzmB   45 in diseases with chronic inflammatory lung states and to delineate the mechanisms of such potential contributions.   46 References 1. Murray CJ, Lopez AD. Alternative projections of mortality and disability by cause 1990-2020: Global Burden of Disease Study. 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