Background
Idiopathic pulmonary fibrosis (IPF) is the most common form of interstitial lung diseases (ILD) with a worldwide incidence ranging between 14–43 per 100 000 individuals and an average incidence of 7.44 per 100 000 in the UK [
1]. A recent clinical data analysis of UK patient cohorts by Navaratnam
et al. showed a yearly 5 % increase in incidence which was independent of the ageing of the population [
2].
Lung function progressively deteriorates in IPF patients leading to respiratory failure and finally death with a median survival around three years from diagnosis [
3]. Therapeutic options for IPF are limited to date, with pirfenidone and nintedanib being the only medications which seem to slow down disease progression and they have only been tested in patients with mild disease [
4,
5]. These drugs are both expensive and real life usage suggests that toxicity leading to drug discontinuation is higher than reported in the licensing trials [
6].
The pathophysiology of IPF is complex involving fibroproliferation, epithelial cell apoptosis, epithelial-mesenchymal transition (EMT), neo-angiogenesis, and intra-alveolar coagulopathy [
7‐
9]. In fibrotic tissue, effector cells are activated fibroblasts or myofibroblasts residing in large numbers in fibroblastic foci, a characteristic feature of usual interstitial pneumonia. These alpha smooth muscle actin (α-SMA)-expressing cells secrete large amounts of collagen and other extracellular matrix (ECM) components and are thus considered to be responsible for pathological tissue remodelling [
10]. Pro-fibrotic growth factors including transforming growth factor-beta 1 (TGF-β1) and platelet-derived growth factor (PDGF) are believed to drive the fibro-proliferative process in IPF and promote fibroblast-to-myofibroblast differentiation and collagen deposition [
9,
10]. The degree of involvement of EMT in IPF pathogenesis—particularly the number of fibroblasts and myofibroblasts derived from transdifferentiated epithelial cells—is currently debated [
7,
11,
12].
CD248 (also termed Endosialin and TEM-1), is a heavily glycosylated transmembrane protein that was identified initially as a cell surface marker overexpressed in tumour vasculature [
13]. Later it was revealed that in fact CD248 was expressed by pericytes, and not the underlying endothelium [
14]. CD248 is now regarded as a mesenchymal marker, expressed on various fibroblast types, pericytes, mesenchymal stem cells, smooth muscle cells and osteoblasts [
15,
16]. CD248 is also implicated in the regulation of the proliferation of mesenchymal elements [
17,
18].
CD248 was demonstrated to be overexpressed in various mesenchymal cells in pathological conditions including sarcomas [
19], inflammation [
20] and fibrosis [
21]. In renal fibrosis CD248 expression was found to relate to the severity of the disease [
21]. There is no information about CD248 expression in lung disease especially IPF. We hypothesised that CD248 expression would be a marker of severity in IPF. We also investigated its role in fibroproliferation and EMT.
Methods
Patient samples
Lung tissue samples for histology were obtained by diagnostic video assisted thoracoscopic (VATS) lung biopsy and transplant explants. These procedures were performed by surgical teams at Heartlands Hospital and the Queen Elizabeth Hospital, Birmingham between the time-frame January 2005 to December 2012. Lung resections were fixed in formalin, embedded into paraffin and sectioned according to standard procedures. A total of 22 patients’ samples were included in the study. Demographic and clinical data of patients were obtained from electronic and paper records. Patient data is summarized in Table
1. No patients received disease modifying treatment (pirfenidone or nintedanib) prior to surgery.
Table 1
Patient demographics and clinical data. We analysed the demographic and clinical data of 22 patients with a histologically confirmed IPF diagnosis. 11 of the patients underwent VATS and had mild-to-moderate fibrotic changes in the lung. Another 11 IPF patients underwent lung transplantation because of severe or end-stage fibrosis. There was no significant difference between the patient groups’ age, smoking status and body mass index (BMI). The lung function parameters Forced Expiratory Volume in 1 s (FEV1), Forced Vital Capacity (FVC), Total Lung Capacity (TLC) and Transfer factor for carbon monoxide in the lung (TLCO) were significantly higher in the patient group with mild-to-moderate fibrotic changes compared to those with severe or end-stage fibrosis (p < 0.01 using Students unequal variance t-test)
Demographic and clinical data |
N | 11 | 11 | - |
Sex | 4 males, 7 females | 9 males, 2 females | - |
Median age (range) | 66 (39–76) | 58 (44–61) | 0.126 |
Smoking in history (percent) | 7 (63 %) | 7 (63 %) | 0.482 |
Pack years (mean ± SEM) | 28.37 ± 11.97 | 21.93 ± 6.61 | 0.784 |
BMI (mean ± SEM) | 28.87 ± 1.37 | 27.67 ± 1.87 | 0.636 |
Lung function |
FEV1 (mean ± SEM) | 2.51 ± 0.19 | 1.59 ± 0.20 | 0.0009 |
FVC (mean ± SEM) | 3.21 ± 0.32 | 1.92 ± 0.23 | 0.0006 |
TLC (mean ± SEM) | 4.77 ± 0.54 | 3.04 ± 0.34 | 0.0014 |
TLCO (mean ± SEM) | 3.69 ± 0.36 | 2.09 ± 0.30 | 0.0099 |
Medication (given only after biopsy) |
Prednisolone | 6 (54.5 %) | 8 (72.7 %) | - |
Azathioprine | 3 (27.2 %) | 4 (36.3 %) | - |
N-acetylcysteine | 4 (36.3 %) | 2 (18.1 %) | - |
Primary human lung fibroblasts were isolated from explanted lung tissue of patients with IPF, acquired at the time of lung transplantation (n = 6) and lung tissue from healthy donors rejected for transplantation as controls (n = 6) at the Institute of Transplantation, Newcastle Upon Tyne Hospitals NHS Trust. All procedures in this study were performed in accordance with approval from the local research ethics committees at the University of Birmingham and Newcastle University, respectively. All patients gave written informed consent for the use of their tissue and clinical data for research purposes. Ethics committee approval number is 07/MRE08/42.
Immunohistochemistry and digital image analysis
Lung tissue samples for immunohistochemistry (IHC) were obtained by VATS from patients with mild-to-moderate fibrosis (
n = 11) or were taken from lungs of patients with end-stage fibrosis undergoing lung transplantation (n = 11). Sections were processed for CD248 immunohistochemical staining using standard methods for diaminobenzidine (DAB) chromogen and haematoxylin for nuclear staining. The B1/35.1 anti-human CD248 antibody [
14] was used for IHC in an automated IHC staining system (Roche Benchmark Ultra). The area staining positive for CD248 was compared between the two patient groups using the ImageJ software for digital image analysis as follows: we took 3–7 high resolution digital images per slide at random locations. Patient identification were blinded during the whole analysis. CD248 signal and haematoxylin signal on the images were digitally separated using the Colour Deconvolution plugin for ImageJ [
22]. Threshold value was set manually at the same levels in both of the channels on all of the images. The area above the threshold was summarised for each image, representing the area stained positive for CD248 or haematoxylin on each image. CD248 positive area was compared to the haematoxylin staining area representing relative CD248 expression in the lung samples of IPF patients. A graphical explanation of this method is shown on Fig.
2a. The relative CD248 staining area were compared between transplant and VATS biopsy patients. We used both Student’s
t-test and Mann–Whitney
U-test for statistical analysis of the data derived from images, where
p < 0.05 denoted statistical significance.
Cell culturing and treatments
Normal human lung fibroblasts (NHLF) were purchased from Promocell (Heidelberg, Germany). At least 3 batches from different patents were used for every experiment. NHLFs were initially cultured and expanded in Fibroblast Growth Medium (Promocell) according to the supplier’s instructions. A549 human lung adenocarcinoma cell line was maintained in DMEM supplemented with 2 mM L-glutamine, HEPES, non-essential amino acids, 100 U/ml penicillin and 100 mg/ml streptomycin and 10 % FCS.
Additional primary fibroblasts were isolated at Newcastle University from the lungs of patients with advanced IPF and the lung tissue from healthy donors rejected for transplantation as controls at the Institute of Transplantation, Newcastle Upon Tyne Hospitals NHS Trust. The isolation method based on a cell outgrowth technique. Briefly, lung tissue pieces (<1 mm3) were cultured in DMEM/F12 (Sigma) supplemented with 10 % FCS, 1 % L-glutamine, 100U/ml penicillin and 100 μg/ml streptomycin to allow cells to migrate out of the tissue. After 7 days the tissue is removed and the cells grown to confluence. Mesenchymal phenotype was confirmed by positive expression of fibronectin, vimentin and α-smooth muscle actin and little/no expression of E-cadherin, ZO-1 and CD45. Fibroblasts were used at the third passage in all experiments.
Human Primary Lung epithelial cells were isolated from tissue samples from lobectomy patients with normal lung function. Cells were isolated and cultured as described elsewhere [
23]. TGF-β1 was obtained from R&D Systems (Abingdon, UK). Final concentration of TGF-β1 was 10 ng/ml.
siRNA transfection
CD248 knock-down (KD) in NHLFs was carried out using specific siRNA (Life Technologies). We used Lipofectamine 2000 reagent and Opti-MEM (both from Life Technologies) according to the manufacturer’s instructions. Cells were cultured after transfection for 48 h. Efficiency of KD was determined using PCR and flow cytometry (Additional file
1: Figure S4A and B, respectively). Cell viability was >95 % after 48 h of siRNA transfection as determined by the trypan-blue exclusion test.
Proliferation assay
A commercial ELISA-based bromodeoxyuridine (BrdU) proliferation assay kit was used from Calbiochem (Watford, UK) according to the manufacturer’s instructions. Briefly, previously serum-starved cells were seeded into a 96-well tissue culture plate in DMEM + 0.1 % FCS; BrdU and stimulants were added afterwards. Cells were incubated for 24 h to incorporate BrdU, then the proliferation was stopped using the fixative agent supplemented with the kit. The development and colorimetric measurements were performed according to the manufacturer’s instructions. Proliferation experiments were repeated for at least 3 times with cells from 3 different donors.
Flow cytometry
Cultured cells were detached using a non-enzymatic cell dissociation solution (Sigma Aldrich) to preserve the trypsin-sensitive CD248 epitope. Labelling was carried out using an anti-CD248-FITC monoclonal antibody [
24]. Samples were analysed on a CyanADP Analyzer flow cytometer and Summit v4.3 software. CD248 protein expression is presented as median fluorescent intensity (MFI).
Real time qPCR
Total RNA was isolated from cultured cells using the NucleoSpin RNA isolation kit with on-column DNase digestion (Machery-Nagel), cDNA synthesis was performed using a High Capacity RNA-to-cDNA kit (Applied Biosystems) following manufacturer’s protocols. For real-time qPCR experiments, master mixes with or without SYBR Green were used (Roche). The list of primers is available in the online supplement. (Additional file
1: Table S1) PCR experiments were performed on a Light Cycler 480 Instrument (Roche). In the plots reverse ΔCt values versus GAPDH expression are presented; the calculation formula was reverse ΔCt = Ct(GAPDH) - Ct(Target) [
25]. The mean Ct values were calculated for 3 or more independent experiments.
Statistics
Data were analysed on using SPSS for Windows 16.0 (SPSS, Inc., Chicago, IL). Data were tested for normality using Spearman’s chi squared test and analysed by non-equal variance t test or Mann–Whitney U test. Data are expressed as mean ± SE unless otherwise indicated. Correlations were calculated using least squares linear regression test.
Discussion
In this study we have evaluated CD248 expression in the lungs of patients with mild early stage IPF and lung transplant explants. Immunohistochemistry demonstrated intense staining of established fibrotic areas of the lung and expression related to the severity of lung function abnormalities. We confirmed increased expression of CD248 in IPF derived fibroblasts compared to normal primary pulmonary fibroblasts.
Formation of fibroblastic foci is a key feature reflecting sites of active on-going fibrogenesis. Increased numbers of fibroblastic foci have been associated with disease activity and a more rapid disease progression in IPF patients [
2,
10]. Fibroblastic foci are often strong sources of TGF-β1 but expression is variable [
26]. One unexpected finding in our study was that fibroblastic foci had less CD248 staining than the areas of established fibrosis. One explanation for this could be our finding that TGF-beta suppressed expression of CD248 in IPF derived fibroblasts in contrast to its effects on normal lung fiborblasts. (Fig.
3 and Additional file
1: Figure S4) A recent article by Babu et al. [
27] suggests that the TGF-β1-mediated suppression of CD248 is present in normal murine embryonic fibroblasts but not murine lymphoma cell lines or in cancer-associated fibroblasts suggesting perhaps that the local cellular environment influences CD248 expression.
The fact, that TGF-β1 treatment leaves CD248 expression levels unchanged in normal lung fibroblasts but decreases the expression in IPF-derived cells (Fig.
3) allows us to speculate about the changes in the regulation of gene and protein expression occurring in IPF pathogenesis. Our results suggest that CD248 expression becomes newly regulated by TGF-beta signalling in IPF. Interestingly our experiments showed CD248 expression changes only on the protein expression but not on the mRNA levels (Fig.
3). This phenomenon is typical of post-transcriptional protein regulation, so we hypothesize that TGF-beta regulates CD248 protein levels by influencing the post-transcriptional mechanisms rather than the on the level of gene transcription. However, the identification of the exact regulatory mechanism is beyond the scope of this project. Higher CD248 expression has been associated in tumour-associated fibroblasts and pericytes while in normal stroma CD248 is hardly detectable [
28]. In pulmonary fibrosis we observed elevated CD248 expression. Similarly Smith et al. described elevated CD248 expression in kidney fibrosis [
21]. Since it is known that CD248 binds to ECM components, it is interesting to speculate that profound changes can be observed in ECM modelling/remodelling in both fibrotic and malignant diseases and how these changes might regulate CD248 expression levels and ECM-derived cues for cellular functions, like proliferation, adhesion and migration.
We found that CD248 is not expressed on pulmonary epithelial cells during TGF-β1-induced epithelial-mesenchymal transition, nor on a lung carcinoma cell line (Fig.
5). This is in concordance with the findings of Rouleau et al. [
29] The authors describe high levels of CD248 expression in the stromal components but not on the malignant cells of carcinomas. Interestingly, the malignant cells of sarcomas proved to be highly positive for CD248 expression [
19,
29]. These findings strongly suggest that CD248 is a genuine mesenchymal marker which is not induced by epithelial-mesenchymal transition in contrast to other mesenchymal proteins like S100A4 or vimentin [
7,
30,
31]. Presently there is an ongoing debate on whether mesenchymal elements in fibrotic organs derive in large numbers from epithelial cells [
7]. Although CD248 is not a fibroblast-specific marker [
14,
24] these findings might highlight the usefulness of CD248 as a marker for separating mesenchymal-like cells originating from epithelial cells from genuine mesenchymal cells. This could aid appropriate flow sorting protocols in studies looking at epithelial mesenchymal interactions.
This research has its limitations. Firstly, immunohistochemistry is not a truly quantitative technique as DAB staining intensity shows no linear correlation with antigen expression levels [
22]. That is why we chose to measure CD248 staining area instead of staining intensity. By using the area of extent of CD248 staining compared to the area stained by non-immune nuclear stain haematoxylin we did show significant correlations with disease severity and lung function. Further we demonstrated that IPF derived fibroblasts do express greater levels of CD248 than normal lung fibroblasts.
Secondly, whilst we demonstrated an inhibitory effect of CD248 siRNA upon proliferation, this was a relatively small effect but that may have been due to only 40-50 % efficiency of CD248 knockdown. We were unable to test the CD248 siRNA in IPF derived fibroblasts due to their limited availability. Finally, CD248 siRNA did not affect myofibroblast differentiation or collagen expression so the exact role CD248 plays in fibrogenesis is still unclear.
Conclusion
In conclusion, CD248 appears to be a specific marker of mesenchymal cells that is elevated in the lungs of patients with IPF. It is noteworthy, that epithelial cells do not express CD248 when undergoing EMT, in contrast to other commonly used EMT biomarkers. CD248 expression correlated with markers of disease severity. CD248 was functionally important in terms of proliferation of primary pulmonary fibroblasts but does not appear to alter myofibroblast differentiation. CD248 may represent a novel therapeutic target in IPF to reduce fibro-proliferation.
Ethical statement
All procedures in this study were performed in accordance with approval from the local research ethics committees at the University of Birmingham and Newcastle University. All patients included in this study gave written informed consent for the use of their tissue and clinical data for research purposes. Ethics committee approval number 07/MRE08/42.
Consent for publication
Not applicable for this research.
Availability of data and materials
All relevant data and materials are published in the manuscript and supplementary materials.
Competing interests
All authors have declared no competing interests related to this publication.
Author contributions
Concept and design: DRT, AJF, CDB, DB, APC, JEP; Cell preparation, laboratory work and data analysis: LEC, DB, GL, LB, VKD; Patient recruitment: DT, AJF, LB, LEC, RT, DB; IHC and digital image analysis: DB, GL, LEC; Preparation of manuscript & figures: DB, DRT, LEC, CDB, APC, JEP. All authors have read and approved the manuscript.