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Asthma is a chronic inflammatory disease of the airways and viral infections account for the majority of exacerbations and may play a role in its pathogenesis. Bradykinin levels are increased in the lungs of asthmatics and inhaled bradykinin produces bronchoconstriction in asthmatic but not in normal patients. In this study, guinea-pigs were inoculated with parainfluenza and influenza virus to establish airways inflammation and hyperreactivity. The role of bradykinin in the parainfluenza model was examined by using the tissue kallikrein inhibitor, FE999024, and the bradykinin B2 receptor antagonist, MEN16132. Firstly, the effects of bradykinin inhalation in conscious guinea-pigs were characterized by using inhibitors of its breakdown and selective antagonists. Inhaled bradykinin produced a bronchoconstriction only after treatment with the inhibitors of angiotensin converting enzyme and/or neutral endopeptidase, captopril and phosphoramidon respectively. Inhaled bradykinin also increased inflammatory cell influx to the lungs when its breakdown was inhibited with both drugs. Cell influx and bronchoconstriction were blocked by the B2 receptor antagonists icatibant and MEN16132. These responses were therefore B2 receptor-mediated. In ovalbumin sensitized guinea-pigs, inhaled ovalbumin produced early and late asthmatic responses, inflammatory cell influx and airway hyperreactivity to histamine. These were inhibited by dexamethasone. Bradykinin caused bronchoconstriction without using metabolism inhibitors, indicating airways hyperreactivity to bradykinin. Parainfluenza-3 and influenza caused inflammatory cell influx and airways hyperreactivity to histamine. These were inhibited by FE999024, MEN16132 and dexamethasone. Parainfluenza-3 virus inoculated into sensitized guinea-pigs exacerbated the response to inhaled ovalbumin, with a prolonged bronchconstriction replacing early and late phases. This was resistant to dexamethasone. This study supports a role for bradykinin in virus-induced lung inflammation and the use of inhibitors of bradykinin for potential treatment of airway inflammation.

Numerous chronic lung diseases are characterised by oxidative stress or more accurately, perturbations in redox cycling and signalling. Cellular redox alterations lead to phenotypic adaptations such as increased cell proliferation, altered differentiation and induction of apoptosis. Biomarkers that reflect changes in cellular oxidation states as a function of normal redox signalling as well as following environmental insults would be extremely useful for studying disease processes in the lung in vitro and in vivo. One current area of interest in this field is the Peroxiredoxins (Prxs), a family of ubiquitously expressed antioxidant (AO) enzymes. The aim of this project was to assess the utility of measuring the changes in Prx expression levels and oxidation states as a quantitative biomarker of oxidative lung injury. Utilising normal human bronchial epithelial (NHBE) primary cells, which were cultured at an air-liquid interface (ALI), Prx status was analysed throughout culture morphogenesis, and following oxidative insult via glucose oxidase (GOx) and cigarette smoke (CS) exposures. Human in vivo correlation was also undertaken, using lung tissue from COPD (GOLD4) patients. Results were analysed using conventional toxicology, Western blotting (WB) and immunohistochemistry (IHC) techniques. Preliminary results revealed elevated basal levels of oxidative stress within the NHBE model, which was alleviated via the addition of AO to the media. Following GOx and CS exposures, there was increased Prx hyperoxidation in conjunction with variations in expression. A dose-dependant response to GOx and CS was observed in the NHBE cultures +AO. Variations in Prx expression levels and oxidation state were also detected throughout the in vivo correlation, with specific Prx responses observed in the different stages of airway remodelling. The Prxs were determined to be sensitive and reliable biomarkers of intracellular oxidative stress, injury severity and epithelial remodelling, thus indicating a role for the Prxs as an 'early stage' biomarker, with disruptions in redox state occurring in advance of phenotypic or morphological adaptations. Additionally, by eliminating the issue of interspecies reactivity, there may be a potential role for the NHBE-Prx system as an in vivo pre-screening tool.

Asthma is a condition that is characterized by variable airflow obstruction, airway hyperresponsiveness (AHR), chronic airway inflammation and airway remodeling. There is microlocalisation of mast cells within the airway smooth muscle (ASM) bundle in asthma at a level significantly above health and other respiratory diseases. These cells are recruited and their survival promoted. However, their functional consequences have yet to be discovered. AHR in asthma is a result of increased responsiveness of the ASM to agonist and has found to increase with localized mast cell numbers. This phenomenon could be a result of an intrinsic abnormality or of local effects. So far, it has yet to be elucidated. We sort to examine this phenomenon of the presence of mast cells within the ASM and the increased responsiveness of the ASM; mechanisms involved in sustaining mast cell numbers, and the intrinsic differences of ASM between asthma and health. ASM had the ability to maintain survival and promote proliferation of co-cultured mast cells, a mechanism supported by the actions of stem cell factor (SCF), interleukin (IL)-6 and cell adhesion molecule (CADM)-1. There was a physical interaction in mast cell adhesion to ASM between CADM1 and the SCF receptor. The co-culture also enhanced constitutive mast cell degranulation. Intracellular calcium levels of ASM from asthmatic patients at rest were found to oscillate to a great degree. Following stimulation with agonist, the ASM gave a reduced intracellular calcium response. However, their contractile ability measured still remained greater than ASM isolated from non-asthmatic subjects. SCF, IL-6 and CADM1 supported the survival of mast cells co-cultured with ASM. Although ASM from asthmatic subjects produce a reduced intracellular calcium response to agonist, at baseline these cells are more activated and they still retain their increased contractile response. Mechanisms which may contribute to the altered airway physiology in asthma.

Historically, asthma and chronic obstructive pulmonary disease (COPD) represent polar ends of the spectrum of airways disease defined in part by distinctive profiles of airway inflammation; in practice, overlap can exist between asthma and COPD. This thesis examined the pattern of inflammatory cell infiltration and cytokine expression within the bronchus in COPD and asthma with further study of moderate-severe asthma. In addition using sputum, cytokine expression was further assessed in COPD and asthma and its relation to severity. Based on previous studies, this thesis examined the expression of specifically Interleukin (IL)-13 and Granulocyte Macrophage Colony Stimulating Factor (GMCSF). We demonstrated mast cell myositis in moderate and severe asthma which reflected increased disease symptoms. Preferential localization of inflammatory cells to airway smooth muscle (ASM) was absent in COPD. CD3+ T-cells infiltration of large airway glands was increased in COPD which may influence mucus hyper-secretion. We demonstrated IL-13 overexpression within the submucosa in moderate-severe asthma with specific increase in the ASM in severe disease. IL-13 expression was related to eosinophilic inflammation. In sputum, IL-13 protein was increased in mild and severe asthma reflecting IL-13 expression in ASM. There was a general absence of bronchus and sputum IL-13 in COPD. Sputum GMCSF was increased in moderate-severe asthma and mild-severe COPD. Parallel upregulation of GMCSF and associated receptor (GMCSFr) expression in the submucosa and ASM was present in severe asthma. GMCSF/GMCSFr expression did not exhibit preferential expression in the large airway of COPD. Our findings suggest inflammatory cell infiltration of the airway structures is present in asthma and COPD which may influence the phenotype. In addition IL-13 is important in severe asthma whilst GMCSF is expressed in asthma and COPD across a range of severity, but to a greater degree in severe asthma.

Asthma is characterized by variable airflow obstruction, airway hyperresponsiveness (AHR), inflammation and remodeling. Mast cells (MC) co-localise to airway structures such as epithelium and airway smooth muscle (ASM) - the latter is a key determinant of AHR. Our group has reported that the CXCL10/CXCR3 axis is important in MC migration towards ASM, MCs adhere to ASM predominately via CADM-1 and this interaction promotes MC survival and proliferation. Whether there are other important mechanisms driving MC localization, ASM migration, and how MCs affect ASM differentiation is uncertain. We sought to examine i) chemokine concentrations in airway secretions in eosinophilic bronchitis (EB), and asthma, and their effects on MC migration ii) CCR3 mediated ASM migration, iii) effects of MC–ASM co-culture on ASM differentiation. Bronchoalveolar lavage (BAL) CXCL10 and CXCL8 concentrations were increased in subjects with EB compared to asthmatics and controls; were chemotactic for MCs and was attenuated by CXCR1 or CXCR3 inhibition. CCL11 mediated ASM migration and wound healing, but had no effect on proliferation or survival. Co-culture with β-tryptase or MCs degraded CCL11, and inhibited CCL11-mediated ASM migration. In vitro co-culture of ASM cells with β-tryptase or MCs increased ASM-derived TGF-β1 secretion, α-smooth muscle actin (α-SMA) expression and agonist-provoked contraction. Promotion to a more contractile phenotype was inhibited by leupeptin and TGF-β1 neutralization, suggesting ASM differentiation was driven by the autocrine release of TGF-β1 in response to β-tryptase. Importantly, in vivo, in asthmatic bronchial biopsies, intensity of α-SMA expression was strongly related to the number of MCs within or adjacent to ASM bundles. In conclusion, CXCL8 and CXCL10 are important in MC migration to the epithelium, but CCL11 is unlikely to be important in ASM migration. MCs drive ASM differentiation to a more contractile phenotype via autocrine release of TGF-β1, which may contribute to the disordered airway physiology in asthma.

Background: Exertional breathlessness and fatigue are common disabling symptoms of patients with Chronic Obstructive Pulmonary Disease (COPD) and Chronic Heart Failure (CHF). The mechanisms behind these symptoms are similar including skeletal muscle dysfunction. Exercise training at least partially reverses the skeletal muscle abnormalities and improves exercise performance and health related quality of life in both conditions. Pulmonary rehabilitation, with exercise training as a core component, is an integral part of the management of COPD, but a service for CHF has not developed in the same way. The hypothesis, for the main studies described in this thesis, was that the successful model of pulmonary rehabilitation could be applied to patients with CHF and patients with COPD and CHF could be beneficially trained together. Methods: Two main studies were undertaken; 1) a randomised controlled trial of pulmonary rehabilitation (PR) vs. normal care (NC) in patients with CHF 2) a comparative observational study of PR between COPD and CHF. Alongside these studies, the outcome measures commonly used for COPD were applied to patients with CHF. Two pilot studies were performed investigating the effect of exercise training on other systemic manifestations of COPD and CHF. Results: Patients with CHF made significant improvements in exercise performance and health status with PR compared to NC. The improvements were similar to those seen in the patients with COPD. Measures of exercise performance and health status were applied successfully to patients with CHF. Conclusions: Patients with COPD and CHF can be successfully trained together demonstrating the feasibility of generic exercise rehabilitation for exertional breathlessness. Further work would need to investigate whether combined exercise programmes for COPD and CHF provides economies of scale for both populations. The work in this thesis highlights the possibility of organising services for chronic disease around a disability rather than an individual disease.

Background: Neutrophilic inflammation causes lung damage in cystic fibrosis (CF). Recent data from animal models and other chronic pulmonary inflammatory conditions suggest that the monocytes/macrophages may be an important driver of airway inflammation. CF may be associated with increased airway levels of chemoattractants for monocytes and resulting expansion of CD14++ small macrophages. I sought to assess the levels of monocyte chemoattractants CCL2 and CX3CL1 in the blood and airways of CF patients, and expression of their respective receptors CCR2 and CX3CR1 on monocytes. In a pilot study, I sought evidence for expansion of airway CD14++ small macrophages. Methods: Blood was obtained from 32 CF patients and 25 healthy controls; and induced sputum (IS) from 24 CF patients and 17 healthy controls. Flow cytometry was used to determine expression of CCR2 and CX3CR1 on CD14++ and CD14+CD16+ blood monocytes and to characterise IS airway macrophages. CCL2 and CX3CL1 levels in blood and IS were determined by ELISA. Results: Absolute count of total monocytes and monocytes subpopulations was not different between CF and controls. Serum CCL2, but not CX3CL1, was increased in CF patients (p=0.006). Similarly, CF was associated with increased IS CCL2, but not CX3CL1 (190.6 vs. 77.3 pg/mL; p=0.029). CCR2 was expressed on CD14++ monocytes but not on CD14+CD16+ monocytes. Both CD14+CD16+ and CD14++ cells expressed CX3CR1 but the expression was higher in CD14+CD16+ cells compared to the CD14++ cells. There was no difference in expression of both chemokine receptors by either monocyte subpopulation between CF and controls. Small macrophages were significantly increased in CF airways (p=0.018). Conclusion: CCL2, but not CX3CL1 is increased in the airway and blood of CF patients. Blood monocytes from CF patients are phenotypically competent to respond to CCL2, since they express normal levels of CCR2. Preliminary results suggest an expansion of small macrophages in CF airways.

Airway hyperresponsiveness (AHR) is a central feature of the asthma paradigm. Non Asthmatic Eosinophilic Bronchits (EB) has emerged as a powerful disease control model to study the immunopathological mechanisms of AHR in asthma. EB is characterised by cough, AHR is absent despite ongoing eosinophilic airway inflammation. This thesis compares features of structural and cellular remodeling in the airway wall in asthma and EB as well as the static geometry of proximal conducting airways in these two polar conditions. We have demonstrated for the first time that structural remodeling, notably increased airway smooth muscle (ASM) mass, vascular remodeling and the expression of VEGF, number of submucosal glands and collagen 3 deposition; occur to similar degrees in both asthma and EB. None of these structural components were associated with AHR in asthma. In contrast we found that the number of mast cells within the ASM independently correlated with the degree of AHR. Coupled with these findings, asthma was characterised by reduced patency of the proximal airway lumen due to airway wall thickening and the degree of thickening correlated with the degree of AHR. In contrast EB was characterised by maintained proximal airway luminal patency despite an increase in the area of the airway wall. Our findings suggest that AHR is dissociated from airway wall structural remodeling in asthma and associated with mast cell infiltration of the ASM. Finally we have shown that fibrocytes are present in the ASM in asthma in contrast to EB and may contribute to the increased ASM mass seen in asthma. Future studies should explore the mechanisms that promote reduced luminal patency in asthma and preserve luminal patency in EB, as well as the functional impact of mast cell infiltration of the ASM and fibrocytes in asthma upon ASM dynamics.

Background: The ciliated respiratory epithelium that covers the surface of human airway forms an immunologically active natural barrier to invasion and injury by inhaled noxious agents. Ciliary dysfunction and or epithelial damage compromise this innate defence mechanism. Aim: To study the ciliary function and epithelial ultrastructure of adult patients with asthma and paediatric lung transplant recipients. To study the response of bronchial epithelial cells of patients with atopic severe asthma, to allergen and bacteria. Methods: Digital high speed video microscopy was used to study the ciliary function on bronchoscopic bronchial epithelial brushings. Transmission electron microscopy was used to study the detailed epithelial ultrastructure. Cytokines and chemokines released by primary bronchial epithelial cells were measured using SECTOR Imager 6000 (MSD, USA). Results: Ciliary dysfunction and ultrastructural abnormalities are closely related to asthma severity. Ciliary dysfunction is a feature of moderate to severe asthma and profound ultrastructural abnormalities are restricted to severe disease. Primary bronchial epithelial cells of patients with atopic severe asthma and healthy controls are capable of releasing chemokines and cytokines in response to Dermatophagoides Pteronyssinus allergen 1 and Streptococcus pneumoniae in a dose and time dependent manner. Ciliary dysfunction is a feature of native airway epithelium in paediatric Cystic Fibrosis lung transplant recipients. The allograft epithelium shows profound ultrastructural abnormalities in both Cystic Fibrosis and non-suppurative lung disease lung transplant recipients. Summary: The phenotype of secondary ciliary dyskinesia and the differential cytokine/chemokine response of the epithelium of patients with severe asthma seen in this study extend our current paradigm of severe asthma and present a new therapeutic target. The damaged allograft epithelium seen in paediatric lung transplant recipients may increase risk of microbial colonisation of the allograft airway, which may play a role in the development of Bronchiolitis Obliterans Syndrome (BOS).