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)-β, 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-β increased IL-6, GM-CSF, matrix metalloproteinase (MMP)-2, and MMP-9. Simvastatin attenuated effects of both IL-17 and TGF-β. We have demonstrated the ability of simvastatin to attenuate release of airway neutrophilic and remodeling mediators and to inhibit their upregulation by TGF-β 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.
lung transplantation is the only treatment option for a large number of patients with severe, end-stage lung disease. Although early outcomes following lung transplantation have improved, the long-term survival of lung transplant recipients is limited by the development of obliterative bronchiolitis (OB), a progressive process characterized by fibroproliferation and obliteration of respiratory bronchioles, associated with lymphocytic bronchiolitis, neutrophilia, airway mesenchymal cell accumulation, and increased deposition of extracellular matrix in the small airway lumen (6).
The current understanding of the pathophysiology of OB is limited, but airway neutrophilia is a recognized characteristic feature (7). The airway epithelium is considered to play a crucial role in the airway neutrophilic inflammation and remodeling of lung allografts, by the elaboration of cytokines, chemokines, and growth factors and, more recently, by the process of epithelial-mesenchymal transition (EMT) (6, 7, 45, 54). Current strategies in the management of OB are based on augmented immunosuppression through switching regimens, augmenting with corticosteroids, or cytolytic therapy and are, in the main, ineffective.
In a recently published study, the 6-yr survival of lung transplant recipients taking simvastatin was 91% compared with 54% in control subjects. In the same study, simvastatin also had a positive impact on episodes of acute rejection and incidence of OB at 1 yr (21). Despite these initial clinical data, the mechanisms of action of statins in preventing allograft dysfunction have yet to be elucidated.
In this study, we sought to ascertain the effects of simvastatin on the ex vivo release of factors known to have a pivotal role in neutrophil recruitment and function and of factors known to be involved in airway remodeling. We utilized a unique set of primary bronchial epithelial cells (PBECs), which we recently successfully established from lung transplant recipients (14). We believe these PBECs to be an advance on conventional, commercially available bronchial epithelial cell lines, being derived directly from the patient group of interest. We focused on epithelial cell release of interleukin (IL)-8, IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2, and MMP-9, well-described mediators of airway inflammation and remodeling. We sought to ascertain the effect of simvastatin on epithelial cell release of these compounds at baseline and following stimulation by IL-17 and transforming growth factor (TGF)-β. IL-17 is postulated to play a critical role in orchestrating airway neutrophilic inflammation, and its presence was recently demonstrated in the transplant airway (46, 49), whereas TGF-β, the prototypical instigator of airway remodeling and fibroproliferation, has been postulated to play a significant role in the pathogenesis of OB (10, 11, 54).
This study was approved by the Local Research Ethics Committee, and informed consent was obtained from all participants.
Bronchoscopy and bronchial sampling.
Our posttransplant patients undergo surveillance bronchoscopy at 1 wk and 1, 3, 6, and 12 mo, as well as further bronchoscopy if indicated on clinical grounds. All patients underwent pulmonary function testing and an assessment of clinical status made, based on standardized criteria (12). Bronchoscopy was performed in accordance with international guidelines (4).
Bronchoalveolar lavage (BAL) fluid was obtained from either the lingula or right middle lobe and sent for routine microbiological testing. Bronchial brushings (n = 4–6) were obtained from subsegmental bronchi using a standard single-sheathed nylon cytology brush (5 fr; Wilson-Cook) and dispersed in 5 ml of phosphate-buffered saline (Sigma). Finally, transbronchial specimens were taken from either the right or left lower lobe and sent for histopathological examination to exclude acute vascular rejection based on standard International Society for Heart and Lung Transplantation criteria (57).
PBEC cultures were established from stable lung transplant recipients using a recently described method (14). After bronchoscopy, the suspended brushing samples were centrifuged to form a cell pellet. This was reconstituted in supplemented basal epithelial growth medium [bronchial epithelial cell basal medium (Clonetics) supplemented with bronchial epithelial growth medium Single Quot (Clonetics) and 1% penicillin-streptomycin] and placed in a collagen-coated petri dish (Vitrogen 100; Cohesion). The cells were incubated in a carbon dioxide-enriched incubator (37°C, 5% CO2), and medium was exchanged every 48 h until PBECs reached confluence. In addition, PBECs were grown on eight-chamber slides and stained for cytokeratin using monoclonal mouse anti-human cytokeratin antibodies. PBECs had the appearance of bronchial epithelial cells but without cilia and expressed the epithelial cell marker cytokeratin.
These PBECs were transferred to 24-well plates at a cellular density of 5 × 104 cells/well and expanded in culture to 70% confluence. At this point, they were rested for 24 h by replacing growth medium with resting medium [resting epithelial medium: 500 ml of BEBM, 5 ml of insulin-transferrin-sodium selenite (Sigma), 5 ml of 1% penicillin-streptomycin, and a 500-μl aliquot of gentamycin-amphotericin] before the addition of simvastatin, IL-17, TGF-β alone or in combination.
Simvastatin (Merck) was dissolved in ethanol to give a stock solution that was then frozen to −80°C in aliquots. Subsequent dilutions in resting medium gave concentrations of 5, 2.5, and 0.5 μg/ml for experimentation.
PBECs were incubated with simvastatin at concentrations of 5, 2.5, and 0.5 μg/ml for 48 h (1-ml volume). Additional experiments were carried out with IL-17 alone [recombinant human (rh) IL-17, R&D Systems; 1, 10, and 100 ng/ml], IL-17 (10 ng/ml) and simvastatin (5 μg/ml), TGF-β alone (rhTGF-β1, Biosource; 0.5, 5, and 50 ng/ml), or TGF-β (5 ng/ml) and simvastatin (5 μg/ml). At this time, the cell supernatants were removed and frozen at −80°C for later protein analysis. All experiments were carried out on PBECs derived from patients who were clinically stable, had no evidence of infection on BAL, and had no evidence of acute rejection on histopathological examination of the transbronchial biopsies obtained. In addition, all experiments were carried out on PBECs at passage 2 or 3.
Methylene blue assay.
To adjust for any differences of plating efficiency, variable growth rates of bronchial epithelial cell cultures from different individual patients, or the effect of differing concentrations of simvastatin, IL-17, or TGF-β on cell growth during experiments, we used methylene blue assay. The assay was performed on the remaining cells after removal of supernatants, and cytokine, chemokine, growth factor, and metalloproteinase protein release in individual culture supernatants were normalized and expressed as concentration released per cell number, using a well-described technique (38).
Fluorokine MAP cytokine multiplex kits (R&D Systems) were used in conjunction with a Luminex 100 analyzer, a dual-laser, flow-based sorting and detection platform. This allowed measurement of multiple cytokines simultaneously in a relatively limited sample volume, as in our study. A separate base kit was used for MMP measurement, again in conjunction with a Luminex 100 analyzer. The manufacturer's stated detection ranges and sensitivities were as follows: IL-8, a detection range of 4,250–6 pg/ml with a sensitivity of 0.39 pg/ml; IL-6, a detection range of 4,500–6 pg/ml with a sensitivity of 0.36 pg/ml; VEGF, a detection range of 2,250–3 pg/ml with a sensitivity of 0.81 pg/ml; G-CSF, a detection range of 6,000–8 pg/ml with a sensitivity of 1.04 pg/ml; GM-CSF, a detection range of 1,800–2 pg/ml with a sensitivity of 1.05 pg/ml; MMP-2, a detection range of 61,000–84 pg/ml with a sensitivity of 25.4 pg/ml; and MMP-9, a detection range of 18,000–25 pg/ml with a sensitivity of 7.4 pg/ml.
For IL-17 and TGF-β experiments, the results were expressed as nanograms per 106 cells, whereas for simvastatin and combination experiments, they are expressed as percent changes.
Results were analyzed using nonparametric statistical methods (Arcus for Windows), namely, the Wilcoxon signed rank test, with a two-sided P value <0.05 deemed statistically significant.
Effect of IL-17 on PBECs.
After the successful establishment of PBECs from stable lung transplant patients (see methods), experimentation was carried out at passages 2 and 3 only. As a first step, PBECs were incubated with IL-17 at concentrations of 1, 10, and 100 ng/ml for 48 h (n = 10). Cell supernatant levels of IL-8, GM-CSF, G-CSF, IL-6, and VEGF were increased (Table 1). There was no effect on either MMP-2 or -9 (data not shown).
Effect of TGF-β on PBECs.
PBECs were incubated with TGF-β at concentrations of 0.5, 5, and 50 ng/ml for 48 h (n = 8). TGF-β significantly increased IL-6, GM-CSF, MMP-2, and MMP-9. MMP was measured in seven experiments (Table 2). IL-8 and VEGF showed no significant change (results not shown).
Effect of simvastatin on unstimulated PBECs.
PBECs were treated with simvastatin at concentrations of 5, 2.5, and 0.5 μg/ml (n = 9). There was a significant decrease in levels of IL-8, GM-CSF, IL-6, MMP-2, and MMP-9 (Table 3 and Fig. 1, A–E). Basal levels of G-CSF were above the lowest limit of assay detection in only one experiment, where the level did decrease with simvastatin. Basal GM-CSF was below the detection limit in 1/9 experiments, IL-6 in 2/9 experiments, MMP-2 in 1/9 experiments, and MMP-9 in 2/9 experiments (Table 3 and Fig. 1).There was no effect on VEGF (data not shown).
Effect of simvastatin on IL-17-stimulated PBECs.
PBECs were treated with IL-17 at a concentration of 10 ng/ml alone and in combination with simvastatin at a concentration of 5 μg/ml. This was carried out in seven experiments, but stimulated GM-CSF and IL-6 levels remained below the limit of detection in one experiment. IL-8, G-CSF, GM-CSF, and IL-6 levels were all significantly decreased by simvastatin (Fig. 2, A–D).
Effect of simvastatin on TGF-β-stimulated PBECs.
PBECs were treated with TGF-β at a concentration of 5 ng/ml alone and in combination with simvastatin at a concentration of 5 μg/ml. This was carried out for seven experiments. Simvastatin significantly decreased TGF-β-mediated release of IL-6, GM-CSF, MMP-2, and MMP-9 (Fig. 3, A–D).
We have demonstrated the ability of simvastatin to attenuate the ex vivo production of epithelium-derived mediators of neutrophilic airway inflammation, EMT, and airway remodeling both at baseline and following stimulation with physiologically relevant agonists. We propose that this may be a relevant mechanism that underlies the clinical benefits of statin therapy, recently described in OB patients.
There has been a major paradigm shift in the pathogenesis of lung disease, which has placed the epithelium in a critical position, orchestrating airway remodeling and scarring in the development of pulmonary fibrosis (i.e., epithelial origin of fibroblastic foci) and in asthma (epithelial-mesenchymal interactions in the pathogenesis of subepithelial fibrosis and airway remodeling of asthma) (29, 44). Airway neutrophilia is a recognized feature of OB, even in the absence of infection (55). There is evidence of increased epithelial activation in OB, associated with increased expression of MMPs and deposition of subepithelial collagens and other matrix proteins in the lamina reticularis (53, 54). The bronchial epithelium is a well-recognized source of cytokines, chemokines, and growth factors that contribute to inflammatory cell recruitment and activation and to the airway remodeling process of OB (5, 10, 11). IL-8 is a neutrophil chemokine but also is a well-characterized mediator of vascular remodeling in the oncology literature and has been suggested to be involved in the process of EMT in colon carcinoma (2). Enhanced IL-8 levels in BAL of lung transplant patients have been shown to be a predictor of bronchiolitis obliterans syndrome (BOS), and elevated levels of both IL-8 and neutrophils have been demonstrated in the condition (58). IL-6 is secreted by monocytes, macrophages, lymphocytes, and epithelial cells. It plays a role in B-cell differentiation, monocyte proliferation, and neutrophil recruitment to sites of inflammation. It is also involved in neutrophil activation and degranulation (32). Like IL-8, IL-6 has been postulated to play an important role in BOS (3).
GM-CSF can regulate both the accumulation and the activity of neutrophils and is increased in lung disease characterized by neutrophilic infiltration (26). It is produced by airway epithelial cells and fibroblasts and facilitates recruitment of neutrophils to inflammatory sites through increased expression of adhesion molecules on inflammatory cells and by increasing the response to chemotactic stimuli (13, 52). In addition, GM-CSF can prolong neutrophil survival through inhibition of apoptosis and is increased in BAL of lung transplant patients (9). The glycoprotein hormone G-CSF induces proliferation and differentiation of neutrophil progenitor cells, increases neutrophil survival, and is produced by airway fibroblasts and bronchial epithelial cells (56). Increased levels occur in the lungs with stimulation by foreign antigens, allergens, and microbial pathogens (43). It has been demonstrated to cause neutrophil sequestration in rabbit lungs (20).
VEGF has been demonstrated to be a mediator of vascular and airway remodeling and inflammation (18, 40). It enhances antigen sensitization and has a key role in the adaptive airway inflammatory response. It has been shown to increase the number of activated dendritic cells in a murine model of asthma (27). In a bronchial epithelial cell monolayer, VEGF causes increased permeability, thereby facilitating inflammatory cell influx (16).VEGF release from epithelial cells has been stimulated by both respiratory syncytial virus and rhinovirus (16, 23). In a heterotrophic rat tracheal model of transplantation, VEGF has been shown to potentially enhance airway luminal occlusion by increasing recruitment of mononuclear inflammatory cells (24), and a role for VEGF in lung transplantation has been postulated (24, 25, 33).
It is increasingly recognized that statins are more than simply lipid-lowering agents. In particular, statins have been reported to possess wide-ranging anti-inflammatory effects, including the lowering of serum C-reactive protein in coronary disease and chronic obstructive pulmonary disease (COPD), accompanied by a lowering of mortality in observational studies (8, 15, 30, 31, 37, 42, 47). A recent study suggested that statin use exerts a protective effect on lung function in nonsmokers, ex-smokers, and current smokers, although the benefit is greatest in ex-smokers (1).
Although other authors have demonstrated various potential anti-inflammatory effects of statins by using several different ex vivo cell models (28, 34, 35, 50), we have shown for the first time that simvastatin modulates the epithelial release of IL-8, GM-CSF, MMP-2, and MMP-9 in ex vivo cultures obtained from lung transplant patients. In addition, we have demonstrated inhibition of mediators of inflammation and remodeling by simvastatin in cultures stimulated with physiologically relevant agonists. Our experiments showed that simvastatin reduced levels of mediators in epithelial cells stimulated with IL-17. Previous studies suggest a role for IL-17 in regulating the airway inflammatory response, and intratracheal installation of IL-17 in an in vivo rat model resulted in neutrophil recruitment into the airways and increased neutrophil activity as measured by the release of myeloperoxidase and elastase (19). In addition, mice with a homozygous deletion of the IL-17 receptor have markedly diminished recruitment of neutrophils into the lung in response to a challenge with a gram-negative pathogen. It is proposed that IL-17 plays a major role in neutrophilic airway inflammation and might represent a link between T-cell alloimmune injury and the subsequent neutrophilic pathophysiology of OB (48, 58). A recent study has shown increased levels of IL-17 in BAL during acute lung allograft rejection (49).
The other stimulus used in our PBEC experiments was TGF-β. TGF-β plays a major role in airway remodeling (39, 41, 51). Bronchial epithelial cells are known to be capable of releasing TGF-β, and TGF-β has been shown to be involved in the epithelial cell regulation of fibroblast proliferation (17, 36). One of the key actions of TGF-β is the induction of matrix proteins and inhibition of collagen breakdown, either by reducing matrix-degrading proteases such as MMPs or by inducing protease inhibitors and tissue inhibitors of metalloproteinases (TIMPs). In patients with OB, TGF-β has been shown to be increased in BAL (10, 11), and TGF-β is also the recognized prototypical driver of EMT, which was recently described in human lung allografts (54).
Our demonstration that simvastatin blocked a range of mediators from patient-derived PBECs both at baseline and following stimulation with two physiologically relevant stimulants is consistent with a positive effect on a broad range of factors effecting neutrophilic airway inflammation, EMT, angiogenesis and fibrosis. Our work did not include studies on the possible intracellular pathways involved. It is known, however, that statins block mevalonate synthesis, which has an indirect effect on Ras proteins, which in turn have critical roles in signal transduction and effect cell differentiation, proliferation, and apoptosis (22). Therefore, statins may indirectly have an influence on multiple cellular mechanisms, and our data are consistent with a broad spectrum of activity that may underlie the clinical benefits recently described in OB. Further mechanistic studies and controlled trials of statin therapy would be appropriate in lung transplantation, and such research may be relevant to other airways disease such as COPD.
This research was funded by the Newcastle upon Tyne Hospitals Special Trustees, European Respiratory Society Fellowships (to D. M. Murphy and C. Ward), a Medical Research Council (MRC) Fellowship (to I. A. Forrest), the McPhail Trust (to J. L. Lordan), and an MRC project grant (to C. Ward and P. A. Corris). Simvastatin was kindly donated by Merck & Company, Incorporated.
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