Article Text

Download PDFPDF

Disease pathogenesis in interstitial lung disease

Statistics from

Request Permissions

If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.


M. A. Gibbons, A. C. MacKinnon, R. Duffin, J. P. Iredale, T. Sethi, S. J. Forbes. MRC Centre for Inflammation Research, University of Edinburgh, Edinburgh, UK

Introduction and Objectives The pathogenesis of idiopathic pulmonary fibrosis remains a controversial subject. The prevailing hypothesis favours an aberrant wound healing response where epithelial injury stimulates myofibroblast differentiation with collagen deposition and resultant fibrosis. The role of the macrophage is controversial. We hypothesised that the profibrotic alternatively activated macrophage is critical to lung fibrosis progression. Using both the bleomycin and adenoviral transforming growth factor β (AdTGFβ) models we sought to determine the effects of depletion of circulating monocytes and alveolar macrophages on the degree of pulmonary fibrosis.

Methods Fibrosis was induced by intratracheal instillation of either 0.033 mg (0.05 U) of bleomycin or 3×108 plaque-forming units (PFU) of AdTGFβ. Circulating monocytes and alveolar macrophages were depleted at various time points by administration of liposomal clodronate by intraperitoneal or intratracheal injection, respectively. In the bleomycin model fibrosis was assessed at both early and late stages (days 18 and 32). In the AdTGFβ model fibrosis was assessed at 14 days. Fibrosis was assessed by collagen quantification, histological fibrosis score and quantitative PCR (qPCR). Markers of macrophage phenotype were assessed by immunohistochemistry and qPCR.

Results In the bleomycin model depletion of circulating monocytes or alveolar macrophages at various time points had no effect on early fibrosis. Depletion at later points reduced the degree of pulmonary fibrosis (fig 1). Reduction in fibrosis was associated with a reduction in markers of alternative macrophage activation. In the AdTGFβ model, depletion of circulating monocytes reduced the degree of pulmonary fibrosis.

Conclusions We have shown for the first time the critical role that monocytes and macrophages play in the pathogenesis of pulmonary fibrosis. Our data suggest that it is the profibrotic alternatively activated macrophage that is the key player involved in this process. These results enhance our knowledge of a devastating and untreatable disease. By improving our understanding of a controversial disease process we hope to enable better targeted therapies to be identified. Specifically, with the advent of cell therapy and cell manipulation, we may be able to deplete or modulate the behaviour of these profibrotic macrophages, and consequently halt, and even reverse, the degree of pulmonary fibrosis.

Abstract S67 Figure 1

Depletion of circulating monocytes or alveolar macrophages ameliorates lung fibrosis. Administration of liposomal clodronate (LipClod) (1) intratracheally (IT) on day 21 and (2) intraperitoneally (IP) on days 21, 22 and 23 ameliorates peak lung fibrosis (day 32) as measured by Sircol assay on whole left lung samples.


1K. McNulty, 1S. Aguilar, 1C. J. Scotton, 2D. Bonnet, 1S. M. Janes. 1Centre for Respiratory Research, University College London, London, UK, 2Haematopoietic Stem Cell Laboratory, London Research Institute, Cancer Research UK, London, UK

Introduction Pulmonary fibrosis is the end stage of various conditions, lacks satisfactory treatment options and results in significant morbidity and mortality. Alveolar epithelial cell (AEC) injury plays a central role in the pathogenesis of pulmonary fibrosis; restoration of epithelial integrity is required for re-establishment of normal alveolar architecture. Keratinocyte growth factor (KGF) assists epithelial repair; KGF stimulates AEC proliferation, enhances DNA repair and reduces apoptosis. Intratracheal delivery of KGF attenuates fibrosis in animal models of pulmonary fibrosis. Circulating bone marrow-derived cells (BMDCs) are known to home to injured lung. We sought to determine whether BMDCs could be used as vehicles to deliver KGF to injured lung, to attenuate fibrosis.

Methods Murine haematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) were transduced with a lentiviral vector conditionally expressing the KGF transgene under doxycycline control. Transduced HSCs and MSCs (or controls) were delivered to C57Bl/6 mice given oropharyngeal bleomycin to induce lung injury and fibrosis. Mice receiving HSCs underwent bone marrow transplantation 8 weeks prior to bleomycin exposure; HSCs were delivered systemically following irradiation. MSCs were given systemically 8 h and 3 days after bleomycin exposure. Mice were sacrificed at day 14 following bleomycin exposure. Lung tissue was processed for histopathological examination (Ashcroft Score), immunohistochemistry, flow cytometry and quantitiative reverse transcription-PCR (qRT-PCR).

Results Murine MSCs and HSCs were efficiently transduced with the lentiviral vector, and retained their differentiation capacity. MSCs engrafted at low levels in the lung and no significant difference was seen in KGF expression, AEC proliferation or lung architecture in mice given MSCs. However, MSC administration did result in a reduction in procollagen gene expression. Transplantation of transduced HSCs resulted in multilineage bone marrow engraftment. KGF–HSC-treated mice had significantly increased KGF expression in the lung. This was associated with significantly increased AEC proliferation, reduced profibrotic cytokines and improved fibrosis (fig 1). KGF–HSC-treated mice had significant improvements in weight loss and survival, suggesting the changes were physiologically relevant.

Conclusion We have shown that HSCs can be used as vectors to deliver KGF therapy to injured lung parenchyma. Genetically modified cell therapy represents a novel and exciting approach to the treatment of lung injury and fibrosis.

Abstract S68 Figure 1

Comparison of morphometric analysis using the Ashcroft Score, showing significant reduction in lung fibrosis in mice given haematopoietic stem cells (HSCs) expressing keratinocyte growth factor (KGF), compared with control mice. Bars represent means±SEM (p = 0.03; Mann–Whitney). BLM, bleomycin; KGF, HSCs expressing KGF.


1K. Weir, 1C. Rooney, 1A. Hood, 1K. Ruddock, 2I. Donnelly, 1M. R. Adamson, 3S. J. Bourke, 2C. McSharry, 1K. Anderson. 1Department of Respiratory Medicine, Crosshouse Hospital, Kilmarnock, UK, 2Division of Immunology, Infection & Inflammation, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow, UK, 3Royal Victoria Infirmary, Newcastle, UK

Introduction and Objectives A characteristic histological feature of hypersensitivity pneumonitis (HP) is the lipid-laden foamy alveolar macrophage suggestive of altered lipid metabolism associated with interstitial inflammation. We have recently described increased serum cholesterol concentration among pigeon fanciers. Our hypothesis is that there is an altered inflammatory profile of biomarkers associated with endothelial cell activation in HP.

Methods In 100 pigeon fanciers we quantified the serum concentration of a variety of inflammatory mediators and immunoglobulin G (IgG) antibody titre to inhaled avian antigens by immunoassay. These were compared with the clinical history of symptoms of HP and lung function.

Results All subjects had an IgG response to avian antigens (median (interquartile range) 29.0 μg/ml (12.5–54.0)), that was significantly inhibited by cigarette smoking (median μg/ml of never: ex:current 35:28:10, p<0.05). This antibody correlated with C-reactive protein (CRP) (r = 0.279, p = 0.015), with the cytokines interleukin-8 (IL-8), monokine induced by interferon γ (MIG), interferon-inducible protein 10 (IP10) and interferon α (IFNα) (each, p<0.05), and with E-selectin (r = 0.321, p = 0.004), intercellular adhesion molecule-1 (ICAM-1) (r = 0.242, p = 0.033) and soluble receptor for advanced glycation end-products (sRAGE) (r = −0.239, p = 0.035).

Conclusions These results suggest that it is sufficient to mount an antibody response to inhaled antigens in order to have associated systemic inflammation. This assertion may be taken further to include endothelial cell activation. Not all subjects were symptomatic or had abnormal lung function, therefore this suggests that subclinical inflammation is common and might help explain subacute development of HP.


A. Goodwin, J. Porte, G. Jenkins. Centre for Respiratory Research, Nottingham Biomedical Research Unit, Nottingham, UK

Introduction and Objectives Idiopathic pulmonary fibrosis (IPF) is a debilitating disease with a poor prognosis and current treatment is ineffective. Transforming growth factor β (TGFβ) is a pleiotropic cytokine with a central role in the pathogenesis of IPF. Lysophosphatidic acid (LPA) induces αvβ6-mediated TGFβ activation via the LPA receptor 2 (LPAR2) and is increased in the lungs of patients with IPF. Both the αvβ6 integrin and LPAR2 are upregulated in the epithelium overlying fibrotic lung tissue. However, regulation of these molecules is poorly understood, and how they may be dysregulated in disease is unknown. This study tested the hypothesis that LPA induces the β6 integrin subunit (Itgb6) and Lpar2 gene expression, via an autocrine loop of TGFβ activation.

Methods Immortalised human bronchial epithelial cells (iHBECs) were stimulated with TGFβ1 or LPA. TGFβ (1D11) and αvβ6 integrin (6.3G9) blocking antibodies were used to inhibit αvβ6-mediated TGFβ activation. The transcription inhibitor actinomycin D was used for mRNA stability studies. Integrin β6 subunit (Itgb6) and LPAR2 (Lpar2) mRNA expression were assessed by real-time PCR.

Results Both TGFβ and LPA increased Itgb6 and Lpar2 gene expression in a time- and concentration-dependent manner. TGFβ and β6 blocking antibodies completely blocked LPA-induced Itgb6 gene expression, implicating αvβ6-mediated TGFβ activation. Conversely, TGFβ and β6 subunit blocking antibodies partially inhibited LPA-induced Lpar2 gene expression, indicating both TGFβ-dependent and -independent mechanisms. TGFβ did not enhance Itgb6 or Lpar2 mRNA stability, indicating that TGFβ increased gene transcription.

Conclusions LPA increases Itgb6 and Lpar2 expression via an autocrine loop of TGFβ activation. Lpar2 expression also involves TGFβ-independent mechanisms. Further dissection of this pathway may identify novel therapeutic targets in IPF.


M. Fletcher, P. Boshier, R. T. Smolenski, P. A. Kirkham, H. Keun, G. Hanna, M. Takata, N. Marczin. Imperial College London, London, UK

Introduction Glutathione-S-transferase (GST) polymorphisms are associated with a spectrum of acute and chronic lung pathologies, suggesting that GST expression and activity are important in lung inflammation. We have previously found that GST inhibition by ethacrynic acid (EA) caused cytotoxicity of mouse lung epithelial (MLE) cells and rendered them more susceptible to stress-related injury, such as exposure to hydrogen peroxide (H2O2). To explore potential mechanisms of such injury we investigated the influence of EA, with/without H2O2, on intracellular energy and redox status, carbonyl stress and on global metabolic profiles.

Methods MLE cells were exposed to EA alone, or in combination with H2O2 for up to 5 h. Following cell extraction, adenine nucleotide content was measured by reversed-phase high-performance liquid chromatography (HPLC) and ultraviolet (UV) detection, and ratios for ATP/ADP (reflecting energy status) and NAD/ADPR (reflecting redox status) were calculated. Excess formation of carbonyl groups in proteins (“carbonyl stress”) was monitored spectrophotometrically following the reaction between 2,4-dinitrophenylhydrazine (DNP) and protein carbonyls. Global metabolic response was evaluated by nuclear magnetic resonance (NMR) spectroscopy, followed by individual assessment of >200 spectral regions.

Results EA alone had no influence on intracellular energy status, yet induced a significant concentration-dependent decrease in NAD/ADPR ratios (53.8±18.2 and 11.2±4.2% of control) after 1 h exposure with 0.2 and 0.3 mM EA, respectively. H2O2 exposure alone significantly reduced intracellular ATP/ADP and NAD/ADPR ratios (29.4±8.8 and 36.5±10.5% of control, respectively); however, these responses were unaffected by EA. In contrast, EA potentiated the H2O2-induced increase in protein carbonyls (from two- to fourfold) and substantially enhanced the stress-induced increase/decrease of a number of important metabolites. Among these, the most prominent changes were in oxidised glutathione, phenylalanine, glutamine, lactate and pyruvate (1.8- to 7.7-fold changes).

Conclusion We have demonstrated that EA is unlikely to compromise cellular energy state, but significantly reduces cellular redox status. Furthermore, our findings suggest that the presence of EA may enhance H2O2-induced cell injury via potentiation of protein carbonylation and by metabolic derangements. These observations highlight the importance of GST in the cellular response to oxidative stress and may help to understand the metabolic determinants of oxidative lung cell injury and adaptation.

View Abstract