Gail E. Johnson

Phenotype of airway epithelial cells suggests epithelial to mesenchymal cell transition in clinically stable lung transplant recipients

Background: Obliterative bronchiolitis in chronic rejection of lung allografts is characterised by airway epithelial damage and fibrosis. The process whereby normal epithelium is lost and replaced by fibroblastic scar tissue is poorly understood, but recent findings suggest that epithelial cells can become fibroblasts through epithelial-mesenchymal transition (EMT). It is hypothesised that EMT occurs in lung allografts and plays a potential role in airway remodelling. Methods: Sixteen stable lung transplant recipients underwent bronchoscopy with bronchoalveolar lavage (BAL), endobronchial biopsies, and bronchial brushings. Biopsy sections were stained for the fibroblast marker S100A4. Brushings were cultured on collagen, stained with anti-S100A4, and examined for further EMT markers including matrix metalloproteinase (MMP) zymographic activity and epithelial invasion through collagen coated filters. Results: A median 15% (0–48%) of the biopsy epithelium stained for S100A4 in stable lung transplant recipients and MMP-7 co-localisation was observed. In non-stimulated epithelial cultures from lung allografts, S100A4 staining was identified with MMP-2 and MMP-9 production and zymographic activity. MMP total protein and activity was increased following stimulation with transforming growth factor (TGF)-β1. Non-stimulated transplant epithelial cells were invasive and penetration of collagen coated filters increased following TGF-β1 stimulation. Conclusions: This study provides evidence of EMT markers in lung allografts of patients without loss of lung function. The EMT process may represent a final common pathway following injury in more common diseases characterised by airway remodelling.

Epithelial to mesenchymal transition (EMT) and airway remodelling after human lung transplantation

Background: Aberrant epithelial repair is a key event in the airway remodelling which characterises obliterative bronchiolitis (OB) in the transplanted lung. The potential for airway epithelium from lung transplant recipients to undergo epithelial to mesenchymal cell transition (EMT) was assessed in culture and in vivo in lung allograft tissue. Methods: Change in epithelial and mesenchymal marker expression was assessed after stimulation with transforming growth factor β1 (TGF-β1) alone or in combination with tumour necrosis factor α (TNFα) and compared with untreated controls. The ability of cells to deposit extracellular matrix, secrete matrix metalloproteinases (MMPs) and invade collagen was investigated. Immunolocalisation of epithelial and mesenchymal markers was compared in airway tissue from stable recipients and those with OB. Results: Untreated cells maintained epithelial morphology and phenotype. TGF-β1 reduced expression of epithelial markers, increased expression of vimentin and fibronectin, promoted collagen I and fibronectin deposition and increased MMP-9 production. Co-treatment with TNFα dramatically accentuated phenotypic and some functional features of EMT. Airway epithelial biopsies from recipients with OB demonstrated significantly increased staining for mesenchymal markers and significantly reduced E-cadherin staining compared with stable recipients. Conclusions: These observations demonstrate the ability of human airway epithelium to undergo EMT and suggest this phenomenon may be a potential link between inflammatory injury and TGF-β1-driven airway remodelling in the development of OB.

Raised interleukin-17 is immunolocalised to neutrophils in cystic fibrosis lung disease

Interleukin (IL)-17 is pivotal in orchestrating the activity of neutrophils. Neutrophilic inflammation is the dominant pathology in cystic fibrosis (CF) lung disease. We investigated IL-17 protein expression in the lower airway in CF, its cellular immunolocalisation and the effects of IL-17 on CF primary bronchial epithelial cells. Immunohistochemistry was performed on explanted CF lungs and compared with the non-suppurative condition pulmonary hypertension (PH). Airway lavages and epithelial cultures were generated from explanted CF lungs. Immunoreactivity for IL-17 was significantly increased in the lower airway epithelium in CF (median 14.1%) compared with PH (2.95%, p = 0.0001). The number of cells staining positive for IL-17 in the lower airway mucosa was also increased (64 cells·mm−1 compared with 9 cells·mm−1 basement membrane, p = 0.0005) and included both neutrophils in addition to mononuclear cells. IL-17 was detectable in airway lavages from explanted CF lungs. Treatment of epithelial cultures with IL-17 increased production of IL-8, IL-6 and granulocyte macrophage colony-stimulating factor. In conclusion, immunoreactive IL-17 is raised in the lower airway of people with CF and localises to both neutrophils and mononuclear cells. IL-17 increases production of pro-neutrophilic mediators by CF epithelial cells, suggesting potential for a positive feedback element in airway inflammation.

Ceramide Is Increased in the Lower Airway Epithelium of People with Advanced Cystic Fibrosis Lung Disease

RATIONALE Ceramide accumulates in the airway epithelium of mice deficient in cystic fibrosis transmembrane conductance regulator, resulting in susceptibility to Pseudomonas aeruginosa infection and inflammation. OBJECTIVES To investigate quantitatively ceramide levels in the lower airway of people with cystic fibrosis compared with pulmonary hypertension, emphysema, and lung donors. METHODS Immunohistochemistry was performed on the lower airway epithelium of explanted lungs (eight cystic fibrosis, emphysema, and pulmonary hypertension, respectively) and eight donor lungs using ceramide, neutrophil elastase, and myeloperoxidase antibodies. High-performance liquid chromatography-mass spectrometry was performed on tissue from five lungs with cystic fibrosis and five with pulmonary hypertension. MEASUREMENTS AND MAIN RESULTS Staining for ceramide was significantly increased in the lower airway epithelium of people with cystic fibrosis (median, 14.11%) compared with pulmonary hypertension (3.03%; P = 0.0009); unused lung donors (3.44%; P = 0.0009); and emphysema (5.06%; P = 0.01). Ceramide staining was increased in emphysematous lungs compared with pulmonary hypertension (P = 0.0135) and unused donors (P = 0.0009). The number of neutrophil elastase- and myeloperoxidase-positive cells in the airway was positively correlated with the percentage of epithelium staining for ceramide (P = 0.001). Ceramide staining was significantly increased in lungs colonized with Pseudomonas aeruginosa (10.1%) compared with those not colonized (3.14%; P = 0.0106). Significantly raised levels of ceramides C16:0, C18:0, and C20:0 were detected by mass spectrometry in lungs with cystic fibrosis compared with pulmonary hypertension. Differences in C22:0 were not significant. CONCLUSIONS Immunoreactive ceramide is increased in the lower airway epithelium of people with advanced cystic fibrosis. Detected by mass-spectrometry ceramide species C16:0, C18:0, and C20:0 but not C22:0 are increased.

Simvastatin attenuates release of neutrophilic and remodeling factors from primary bronchial epithelial cells derived from stable lung transplant recipients

Obliterative bronchiolitis (OB), the major cause of chronic lung allograft dysfunction, is characterized by airway neutrophilia, inflammation, and remodeling, with progressive fibroproliferation and obliteration of small airways that ultimately leads to patient death. Statins have potential anti-inflammatory effects and have been demonstrated to confer a survival advantage in lung transplant patients. We postulated that the beneficial effects of simvastatin in lung transplantation are in part due to inhibition of the epithelial production of key mediators of neutrophil chemotaxis, inflammation, and airway remodeling. Our objective was to assess the effect of simvastatin on a unique population of primary bronchial epithelial cells (PBECs) derived from stable lung allografts, with specific reference to airway neutrophilia and remodeling. PBEC cultures were stimulated with IL-17 or transforming growth factor (TGF)-beta, with and without simvastatin. Supernatant levels of factors critical to driving airway neutrophilia and remodeling were measured. IL-17 upregulated IL-8, IL-6, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), and VEGF, whereas TGF-beta increased IL-6, GM-CSF, matrix metalloproteinase (MMP)-2, and MMP-9. Simvastatin attenuated effects of both IL-17 and TGF-beta. We have demonstrated the ability of simvastatin to attenuate release of airway neutrophilic and remodeling mediators and to inhibit their upregulation by TGF-beta and IL-17. These data illustrate the potential of simvastatin to alleviate neutrophilic airway inflammation and remodeling in the transplanted lung and may have additional relevance to other neutrophilic airway conditions, such as chronic obstructive pulmonary disease.

Primary airway epithelial cell culture from lung transplant recipients.

Long-term survival in lung transplantation is limited by the development of obliterative bronchiolitis, a condition characterised by inflammation, epithelial injury, fibroproliferation and obliteration of bronchioles leading to airflow obstruction. To investigate the role of the bronchial epithelium in the pathogenesis of obliterative bronchiolitis the current study aimed to establish primary bronchial epithelial cell cultures (PBEC) from lung allografts. Four to six bronchial brushings were obtained from sub-segmental bronchi of lung allografts. Cells were seeded onto collagen-coated plates and grown to confluence in bronchial epithelial growth medium. Bronchial brushings (n = 33) were obtained from 27 patients. PBECs were grown to confluence from 12 out of 33 (39%) brushings. Failure to reach confluence was due to early innate infection. Bacteria were usually isolated from both bronchoalveolar lavage and culture media, but a separate population was identified in culture media only. Primary culture of bronchial epithelial cells from lung transplant recipients is feasible, despite a high rate of early, patient-derived infection. Latent infection of the allograft, identified only by bronchial brushings, may itself be a persistent stimulus for epithelial injury. This technique facilitates future mechanistic studies of airway epithelial responses in the pathogenesis of obliterative bronchiolitis.

A randomised controlled trial of azithromycin therapy in bronchiolitis obliterans syndrome (BOS) post lung transplantation

Background We conducted a placebo-controlled trial of azithromycin therapy in bronchiolitis obliterans syndrome (BOS) post lung transplantation. Methods We compared azithromycin (250 mg alternate days, 12 weeks) with placebo. Primary outcome was FEV1 change at 12 weeks. Results 48 patients were randomised; (25 azithromycin, 23 placebo). It was established, post randomisation that two did not have BOS. 46 patients were analysed as intention to treat (ITT) with 33 ‘Completers’. ITT analysis included placebo patients treated with open-label azithromycin after study withdrawal. Outcome The ITT analysis (n=46, 177 observations) estimated mean difference in FEV1 between treatments (azithromycin minus placebo) was 0.035 L, with a 95% CI of −0.112 L to 0.182 L (p=0.6). Five withdrawals, who were identified at the end of the study as having been randomised to placebo (four with rapid loss in FEV1, one withdrawn consent) had received rescue open-label azithromycin, with improvement in subsequent FEV1 at 12 weeks. Study Completers showed an estimated mean difference in FEV1 between treatment groups (azithromycin minus placebo) of 0.278 L, with 95% CI for the mean difference: 0.170 L to 0.386 L (p=<0.001). Nine of 23 ITT patients in the azithromycin group had ≥10% gain in FEV1 from baseline. No patients in the placebo group had ≥10% gain in FEV1 from baseline while on placebo (p=0.002). Seven serious adverse events, three azithromycin, four in the placebo group, were deemed unrelated to study medication. Conclusions Azithromycin therapy improves FEV1 in patients with BOS and appears superior to placebo. This study strengthens evidence for clinical practice of initiating azithromycin therapy in BOS. Trial registration number EU-CTR, 2006-000485-36/GB.

Pseudomonas aeruginosa accentuates epithelial to mesenchymal transition in the airway

Epithelial-to-mesenchymal transition (EMT) has been implicated in the dysregulated epithelial wound repair that contributes to obliterative bronchiolitis (OB) after lung transplantation. Acquisition of Pseudomonas aeruginosa in the transplanted airway has been shown to be a risk factor for the development of OB. We investigated the potential of P. aeruginosa to drive EMT in primary bronchial epithelial cells (PBECs) isolated from lung transplant recipients. Changes in the expression of epithelial and mesenchymal markers was assessed in cells challenged with clinical isolates of P. aeruginosa or co-cultured with P. aeruginosa-activated monocytic cells (THP-1) in the presence or absence of transforming growth factor (TGF)-&bgr;1. P. aeruginosa did not drive or accentuate TGF-&bgr;1-driven EMT directly. Co-culturing P. aeruginosa-activated THP-1 cells with PBECs did not drive EMT. However, co-culturing P. aeruginosa-activated THP-1 cells with PBECs significantly accentuated TGF-&bgr;1-driven EMT. P. aeruginosa, via the activation of monocytic cells, can accentuate TGF-&bgr;1-driven EMT. These in vitro observations may help explain the in vivo clinical observation of a link between acquisition of P. aeruginosa and an increased risk of developing OB.

Effect of azithromycin on primary bronchial epithelial cells derived from stable lung allografts

Obliterative bronchiolitis (OB), the main cause of lung allograft failure, is characterised by airway inflammation, neutrophilia, remodelling and fibrosis. Recent studies of the macrolide antibiotic azithromycin in OB, including one from our centre, have reported improved lung function with rescue therapy.1 The mechanism for this improvement remains unclear, but a recent study suggests that neutrophilic inflammation may be an important predictor of clinical response.2 Macrolides have anti-inflammatory properties in several pulmonary conditions. We hypothesised that the clinical benefit observed with azithromycin in patients undergoing lung transplantation may be related to the inhibition of factors key to airway neutrophilic inflammation, remodelling and fibrosis. The bronchial epithelium is in a pivotal …

Azithromycin attenuates effects of lipopolysaccharide on lung allograft bronchial epithelial cells

BACKGROUND The bronchial epithelium is a source of mediators that may play a role in the airway inflammation and remodeling of post-transplant obliterative bronchiolitis (OB). Traditional strategies have failed to have an impact on OB. Recent studies have suggested a role for azithromycin in managing the condition. In this study we aimed to determine the effect of azithromycin on LPS-mediated epithelial release of factors relevant to airway neutrophilia and remodeling in a unique population of primary bronchial epithelial cells (PBECs) derived from stable lung allografts. METHODS PBECs were established from bronchial brushings of stable lung transplant recipients and treated with lipopolysaccharide (LPS, 0.1, 1 and 10 microg/ml) for 48 hours. Interleukin-8 (IL-8), granulocyte macrophage colony-stimulating factor (GM-CSF) and vascular endothelial growth factor (VEGF) protein levels were measured by Luminex analyzer. PBECs were then incubated with LPS and azithromycin, and protein levels were again determined. RESULTS LPS caused a significant increase in IL-8 and GM-CSF at concentrations of 1 and 10 microg/ml, with no effect on VEGF release. Azithromycin caused a significant decrease in the LPS-stimulated IL-8 and GM-CSF release. CONCLUSIONS LPS upregulates release of IL-8 and GM-CSF from PBECs derived from stable lung allografts. Sub-microbicidal concentrations of azithromycin attenuate this and may, therefore, alleviate infection-driven neutrophilic airway inflammation and remodeling in the allograft airway.