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Novel mechanisms in interstitial lung disease

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AC MacKinnon, SL Farnworth, M Gibbons, SJ Forbes, T Sethi. University of Edinburgh, Edinburgh, UK

Galectin-3 is a beta-galactoside binding animal lectin of approximately 30 kDa, which is highly expressed in fibrotic tissue of diverse aetiologies. Mice deficient in galectin-3 develop reduced fibrosis in several models of organ fibrosis in vivo. Galectin-3 is secreted by macrophages and is a potent mitogen for fibroblasts in vitro. In the chronic inflammatory milieu macrophages interact with other cell types including cells of mesenchymal origin (fibroblasts), which transdifferentiate into matrix-secreting myofibroblasts, with resultant scar formation and disruption of tissue architecture. Our previous work has demonstrated that fibroblasts deficient in galectin-3 fail to differentiate into myofibroblasts in vitro and in vivo. In addition, myofibroblasts may arise from epithelial cells by a process of epithelial to mesenchymal transition (EMT). EMT of alveolar epithelial cells (AEC) has been widely observed in patients with interstitial pulmonary fibrosis (IPF). AEC convert into myofibroblasts following exposure to the profibrotic cytokine transforming growth factor beta (TGF-β) raising the possibility that epithelial cells may serve as a novel source of myofibroblasts in IPF. Primary epithelial cells from galectin-3 null mice show reduced differentiation into myofibroblasts in response to TGF-β in vitro, suggesting that galectin-3 may play a role in EMT in these cells. In the human lung epithelial cell line A549, inhibition of galectin-3 function with Bis-(3-deoxy-3-(3-methoxybenzamido)-β-d-galactopyranosyl)-sulfane a specific inhibitor of extracellular galectin-3 carbohydrate binding and siRNA-mediated depletion of galectin-3 and its membrane receptor CD98 inhibited TGF-β-mediated downstream signalling showing reduced activation of SMAD2/3. Inhibition of galectin-3/CD98 function reduced TGF-β-mediated expression of the myofibroblast marker alpha-smooth muscle actin and increased expression of the epithelial marker E-cadherin suggesting that galectin-3/CD98 regulates TGF-β-induced signalling and the development of EMT in alveolar epithelial cells. Therefore galectin-3 via its interaction with CD98 plays a key role in TGF-β-induced EMT in lung. Strategies to inhibit galectin-3 function may have potential as antifibrotic agents in IPF.


AC MacKinnon, SL Farnworth, T Sethi. University of Edinburgh, Edinburgh, UK

Macrophages stimulated with Th2 cytokines IL-4 and IL-13 have been classified as alternatively activated and are implicated in diverse disease pathologies such as asthma, organ fibrosis and granulomatous diseases. Alveolar macrophages from patients with idiopathic pulmonary fibrosis (IPF) exhibit an alternative macrophage phenotype, which might be a part of a positive feedback loop with lung fibroblasts perpetuating fibrotic processes. Galectin-3 is a carbohydrate-binding lectin present on macrophages. Disruption of the galectin-3 gene specifically restrains IL-4/IL-13-induced alternative macrophage activation in bone marrow-derived macrophages in vitro and in resident lung macrophages in vivo without affecting IFNγ/lipopolysaccharide-induced classic activation or IL-10-induced deactivation. Increased galectin-3 expression and secretion is a feature of alternative macrophage activation, IL-4 stimulates galectin-3 expression and release in parallel with other phenotypic markers of alternative macrophage activation. By contrast, classic macrophage activation induced by lipopolysaccharide inhibits galectin-3 expression and release. IL-4-mediated alternative macrophage activation is inhibited by siRNA targeted deletion of galectin-3 or its membrane receptor CD98 and by inhibition of phosphoinositol-3OH kinase. In addition, IL-4 induced alternative activation is blocked by Bis-(3-deoxy-3-(3-methoxybenzamido)-β-d-galactopyranosyl)-sulfane, a specific inhibitor of extracellular galectin-3 carbohydrate binding. These results demonstrate that a galectin-3 feedback loop drives alternative macrophage activation. Our previous studies have shown that mice deficient in galectin-3 display reduced fibrosis in several models of organ fibrosis. Mice treated intratracheally with bleomycin show increased expression of galectin-3 in broncoalveolar lavage fluid, which correlates with the onset of lung fibrosis. Pharmacological modulation of galectin-3 function may represent a novel therapeutic strategy in pathologies associated with alternatively activated macrophages such as IPF.


VA Varney, J Evans, DT Salisbury, H Parnell, S Ratnatheepan, R Tayar. St Helier Hospital, Carshalton, Surrey, UK

Introduction: Glutathione (GSH) is a major lung antioxidant with reduced levels described in interstitial lung disease (ILD) that are improved by N-acetylcysteine, a precursor of GSH. Gamma glutamyl transferase (γGT) is key in the catabolism of GSH and produces reactive oxygen species (ROS). In mice, the genetic absence of γGT protects the lung against bleomycin-induced fibrosis and therefore supports the proposed adverse effects of γGT in lung fibrosis. Red blood cells exposed to ROS easily undergo membrane lipid peroxidation with increases in mean cell volume (MCV). Lung oxidative stress will recruit monocytes (MΦ) via cytokines such as granulocyte macrophage colony-stimulating factor from the bone marrow. We have observed a consistant feature (not yet described) in cases of ILD in which MCV, γGT and MΦ are increased in new patients referred for treatment. Patients treated with oral septrin following our pilot study, have shown reductions in these parameters with clinical improvement by 3 months.

Method: 149 patients (age range 41–92 years) with new diagnosis of ILD had baseline blood samples and computed tomography scans classified. The groups were compared with an age and gender-matched control population (n  =  160) measured in the same laboratory. Changes after oral septrin treatment for a minimum of 3 months have been looked at in 59 patients.

Results: Means ± SD: Comparisons with controls (unpaired t test). Pre and post-septrin treated (paired t test) (see table).

Conclusion: MCV, γGT & MΦ were significantly increased in ILD and may represent oxidative stress. These parameters were significantly reduced by septrin treatment suggesting reduced oxidative stress.

Abstract S55 Table


1S Bournazos, 1J Grinfeld, 1E Cole, 1K Hodgson, 1A Wass, 1S Maggs, 1A Lee, 1I Bournazou, 1K Alexander, 1I Dransfield, 1P McFarlane, 1N Hirani, 1AJ Simpson, 2SP Hart. 1University of Edinburgh, Edinburgh, UK, 2University of Hull/Hull York Medical School, Hull, UK

Background: Engagement of neutrophil Fcγ receptors by IgG complexes may contribute to the pathogenesis of idiopathic pulmonary fibrosis (IPF).1 Neutrophil FcγRIIIB (CD16B) occurs as two allelic variants (NA1 and NA2) with different binding affinities for particular IgG subclasses. We aimed to determine the association of these polymorphisms with IPF in a cohort of white UK patients.

Methods: We determined FcγRIIIB NA1/2 polymorphisms in 67 patients with IPF (diagnosed according to the ATS/ERS consensus classification) and in 110 disease-free controls using allele-specific PCR amplification.

Results: Significant skewing in the distribution of FcγRIIIB genotypes was observed between IPF patients and disease-free controls. In the IPF cohort there was higher frequency of the NA1/NA1 genotype (0.18 vs 0.04; p = 0.003) and lower NA2/NA2 genotype frequency (0.33 vs 0.52; p = 0.002). Overall, NA1 allele frequency was increased in IPF patients compared with controls (0.43 vs 0.26, χ2  =  5.6; p = 0.018). IPF was associated with presence of the NA1 allele (odds ratio (OR) 2.27; 95% CI 1.13 to 3.16, p = 0.02), whereas the presence of the NA2 allele may be protective against IPF (OR 0.19; CI 0.05 to 0.63, p = 0.006).

Conclusions: We have demonstrated an association between FcγRIIIB polymorphisms and IPF. Given the increased binding affinity of the NA1 isoform for IgG1 and IgG3, these findings support a role for IgG complexes in the pathogenesis of IPF.


EI McIlroy, LA Borthwick, K Brougham, C Ward, J Lordan, JA Kirby, PA Corris, AJ Fisher. Applied Immunobiology and Transplantation Research Group, Institute of Cellular Medicine, Newcastle University, Newcastle upon Tyne, UK

Introduction: Epithelial to mesenchymal transition (EMT) has been implicated in fibrotic remodelling seen in conditions such as idiopathic pulmonary fibrosis and posttransplant obliterative bronchiolitis (OB). The role of inflammation in driving fibrosis remains a subject of debate. Previous work in our laboratory has shown that tumour necrosis factor alpha (TNFα) accentuated transforming growth factor beta (TGFβ) 1 driven EMT. We hypothesised that the pro-inflammatory cytokines IL-1β and IL-8 may also accentuate TGF-β1 driven EMT and contribute to the dysregulated repair in damaged lung tissues.

Aims: To evaluate the effect of IL-1β and IL-8 on TGF-β1 driven EMT in lung epithelium using a standardised epithelial wound model.

Materials and Methods: A549 cells and primary bronchial epithelial cells were cultured for 72 h with TGF-β1 ± IL-1β, IL-8 or TNFα and EMT assessed. Morphology was demonstrated with haematoxylin and eosin stain. Western blot analysis and confocal microscopy were used to study epithelial and mesenchymal protein expression. Pro-matrix metalloproteinase (MMP) production was assessed by gelatine zymography. Extracellular matrix deposition was examined using TCA protein precipitation. Confluent monolayers of A549 cells were injured with a standardised 1 mm wound and treated with TGF-β1 ± IL-1β, IL-8 or TNFα and the quality and rate of wound closure assessed by morphometry.

Results: TGF-β1 dramatically downregulates the expression of the epithelial markers cytokeratin-19 and E-cadherin (22% and 59%, respectively) and upregulates vimentin expression (60%), fibronectin expression (426%) and deposition (170%) and pro-MMP-9 secretion (270%) compared with control cells (p<0.05, n  =  3). Co-treatment of the cells with TGF-β1 plus TNFα or TGF-β1 plus IL-1β significantly upregulated vimentin expression (29% and 41%, respectively), fibronectin expression (60% and 10%, respectively) and pro-MMP-9 secretion (81% and 39%) compared with TGF-β1 alone (p<0.05, n  =  3). Co-treatment with TGF-β1 plus IL-8 had no significant effect compared with TGF-β1 alone (p>0.05, n  =  3). At high concentrations (10 ng/ml) TGF-β1 inhibits wound closure. Co-treatment with TGF-β1 plus TNFα or TGF-β1 plus IL-1β causes accelerated wound closure; however, the quality of the wound repair is highly dysregulated.

Conclusion: IL-1β, but not with IL-8, accentuated TGF-β1 driven EMT in lung epithelial cells. Furthermore, the presence of the pro-inflammatory cytokines IL-1β or TNFα promotes dysregulated wound repair with fibrosis.

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