Background
Sarcoidosis is a multi-organ inflammatory disorder characterised by tissue infiltration of mononuclear phagocytes and lymphocytes as well as noncaseating granuloma formation. Although sarcoidosis can affect any organ, more than 90 % of all patients exhibit pulmonary involvement [
1]. Especially common in the northern part of Europe is the distinct subgroup of patients with Löfgren’s syndrome (LS), clinically characterised by an acute disease onset with bilateral ankle arthritis and/or erythema nodosum, fever and bilateral hilar lymphadenopathy with or without parenchymal infiltration [
2]. While the cause behind sarcoidosis remains uncertain, antigen exposure in genetically susceptible individuals is believed to be a triggering factor [
1]. A subgroup of patients develop chronic disease with fibrosis and eventually 5 % die of respiratory failure [
3,
4]. Moreover, even without developing fibrosis, sarcoidosis can have a substantial impact on quality of life [
5]. There is no specific treatment for sarcoidosis and no clinically established disease markers are available for diagnosis, monitoring of disease activity or prognosis, although genetic associations with the disease course have been described.
The diagnostic procedure in patients with suspected sarcoidosis in Sweden generally includes bronchoscopy for biopsies and BAL fluid retrieval, which presents an excellent opportunity for collecting and studying cells and soluble components such as proteins present at the affected site. Previous studies have investigated serum [
6,
7], BAL fluid as well as BAL cells (macrophages) [
8] in the search for potential markers of disease. However, comparative analysis of BAL fluid and serum from the same individuals has so far not been extensively analysed.
In this study, we performed protein profiling of BALF and serum using the antibody suspension bead array technology with the aim to find proteins associated to sarcoidosis. These bead arrays currently allow profiling of up to 384 analytes in 384 samples simultaneously, only using a few microliters of sample. Through the antibodies generated within the Human Protein Atlas, an initiative aiming to produce antibodies to all human proteins (
www.proteinatlas.org), the arrays can be customized depending on the context and they have previously been applied in studies on various diseases and sample materials [
9,
10].
Discussion
Here we present a proteomic profiling approach using antibodies and bead arrays to investigate protein abundance in BAL and serum in order to find proteins associated to sarcoidosis. As a systemic inflammatory disease sarcoidosis can affect many body compartments, however the lungs are engaged in more than 90 % of patients. Hence BAL fluid is probably most suitable for identifying protein patterns of sarcoidosis. This study focused on proteins previously linked to sarcoidosis, lung function and inflammation resulting in a discovery phase screening 94 protein targets in 68 BAL samples from sarcoidosis patients, asthmatics and healthy controls. All targets were furthermore screened in 249 additional BAL samples from sarcoidosis patients and healthy controls, including additional antibodies towards three targets selected for further investigation.
Several proteins were identified with higher abundance in BAL of sarcoidosis patients, including cadherin 5 (CDH5), transferrin (TF), C-C motif chemokine 24 (CCL24), interleukin 15 (IL15) apolipoprotein A (LPA) and mitochondrial superoxide dismutase (SOD2). However, in the final analysis we focused on Fibronectin 1 (FN1) and Chemokine (C-C motif) ligand 2 (CCL2) that had the strongest associations to sarcoidosis in both sample sets. Fibronectin is a glycoprotein with a soluble form (plasma fibronectin) and an insoluble form (cellular fibronectin) found in plasma, extracellular matrix as well as on the cell surface [
19]. It is involved in cell adhesion and migration processes, including wound healing, coagulation and host defence. Increased levels of fibronectin in BAL fluid have previously been associated with sarcoidosis and other interstitial lung diseases [
20‐
23]. The concordance between our data and these studies, where FN1 was measured by ELISA, confirm the suitability of our assay for protein analysis in BAL. Furthermore, our results showing no FN1 differences in serum of sarcoidosis patients compared to healthy controls mirror previous results in plasma [
21]. Currently, elevated BAL levels of fibronectin is suggested as a marker of lung inflammation rather than reflecting sarcoidosis specific processes [
20,
24,
25]. Nevertheless, the small set of asthma patients included in this study displayed lower levels of FN1 compared to the sarcoidosis groups and were in line with the healthy controls.
Chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein 1 (MCP-1), is a cytokine recruiting monocytes, memory T cells and NK cells to sites of active inflammation and has been implicated in the pathogenesis of other inflammatory diseases such as atherosclerosis, RA and multiple sclerosis [
26]. We found increased levels of CCL2 in BAL fluid of sarcoidosis patients compared to controls, which is in line with previous studies [
27,
28] although there are contradictory reports as well [
29]. Palchevskiy et al. found elevated BAL levels of CCL2 and CCL5 throughout all stages of sarcoidosis [
27], but while Christophi et al. found increased mRNA levels of CCL2 in tissue samples from sarcoid granulomas, the CCL2 levels could not differentiate sarcoid granulomas from other granulomatous inflammation [
30]. In our study, we could not find altered levels of CCL2 in serum, which is in line with recent results [
7].
The availability of extensive clinical information is vital for this kind of study as it allows for correlation of protein patterns beyond healthy/disease. In this study, cadherin 5 (CDH5) was found to correlate positively with BAL lymphocyte counts of both patient cohorts. However, no association was found to the other available parameters including smoking. As sarcoidosis is considered to be a Th1-driven disease characterized by an accumulation of lymphocytes in BAL fluid, CDH5 levels as a measure of lymphocyte infiltration could potentially be used to monitor disease progression. Furthermore, ROC analysis separating patients from healthy controls with AUC values of 0.91 and 0.71 for FN1 and CCL2 respectively, indicated applicability for clinicians in ambiguous patient cases. To that end, future studies evaluating these proteins in relation to other interstitial lung diseases are needed.
Interestingly, none of the proteins analysed revealed concordant differences in the two sample sets between the subtypes of sarcoidosis, namely Löfgren’s syndrome (LS) and non-Löfgren’s syndrome (Non-LS). These groups of patients differ not only in their clinical symptoms and disease progression but have also been found to display differential genetic profiles [
31]. Even so, they also share common characteristics, including granuloma formation, which could associate with similar protein expression patterns in the local environment of the lung. The patient population included in this study consisted mainly of patients with early stages of disease as indicated by the high representation of subjects classified with chest radiographic stage I and II. However, we do not see this as a major limitation of the study as the intended aims included whether protein profiles could provide potential markers to support diagnosis and monitoring of disease progression or treatment response – all of which focus on the earlier stages of disease. Therefore, a clinically useful marker would either predict disease course already at disease onset or help set the diagnose, rather than reflect protein expression at a later stage of the disease. In addition, patients at later stages are usually on treatment, potentially influencing several aspects of the pulmonary inflammation most likely including protein expression. Nonetheless, further insight into overall disease pathogenesis would have benefited from more patients in stages III and IV.
In this study, we observed higher levels of total protein concentration in BAL from sarcoidosis patients compared to healthy controls as well as asthma patients. Such difference could potentially influence protein analysis, thus it is important to elucidate whether this has a technical/processing or biological origin. Since the BAL samples were concentrated prior to analysis, we chose to complement our initial analysis and also include 74 of the samples in an unprocessed state. Although all proteins could not be detected in crude sample material, which in turn supports the use of concentrated material, the high correlations of FN1 and CDH5 indicates that the differences in total protein concentration are not due to variability in the concentration procedure but rather of biological origin. Furthermore, sarcoidosis patients have previously been reported with elevated BAL levels of albumin, suggested to be related to the chronic inflammation and not the lavage procedure itself. This explains the differences in total protein concentration as of biological origin and importantly shows that normalizing based on measured total protein or albumin concentrations might mask disease related differences in the analysis phase [
28,
29].
The most abundant proteins in BAL fluid are plasma proteins (albumin, immunoglobulins, etc.) thought to be derived from diffusion across the blood-air barrier [
32]. As implied earlier, interstitial lung diseases such as sarciodosis, lead to increased BAL fluid levels of plasma proteins, however there is no consensus on the underlying mechanisms (altered barrier permeability, active transport or local production) [
32‐
34]. In our recent evaluation of autoimmune IgG specificities in sarcoidosis, results were highly concordant between BAL and serum with correlation coefficients above 0.81 [
35]. A bi-directional transcytosis mediated by the Fc-gamma receptor was the suggested mechanism for transport across the epithelium. Thus IgG produced locally in the lung is spread systemically and circulating IgG will be present in the lung environment. Looking at the total set of 94 proteins investigated in this study, individual protein levels in BAL compared to serum had weak to moderate correlations (up to Rho 0.48). This implies a minor role for a general increased barrier permeability causing the increased plasma protein content of BAL from sarcoidosis patients. The fact that serum levels of the 94 investigated proteins could not differentiate sarcoidosis patients from controls further indicate that differences seen in BAL are the results of local processes in the lung or active transport rather than reflecting a systemic state.
Abbreviations
BAL, bronchoalveolar lavage; BSA, bovine serum albumin; CCL2, C-C motif chemokine 2; CCL24, C-C motif chemokine 24; CDH5, cadherin 5; FN1, fibronectin; HPA, human protein atlas; IL15, interleukin 15; LPA, apolipoprotein A; LS, Löfgren syndrome; MFI, median fluorescence intensity; SOD2, mitochondrial superoxide dismutase; TF, transferrin.
Acknowledgements
This study was supported by grants from Science for Life Laboratory Stockholm, the ProNova VINN Excellence Centre for Protein Technology (VINNOVA, Swedish Governmental Agency for Innovation Systems), the Knut and Alice Wallenberg Foundation, The Center for Inflammatory Diseases, Karolinska Institutet and KTH Center for Applied Proteomics financed through the Erling-Persson Family Foundation. The authors wish to acknowledge Mun-Gwan Hong for fruitful discussions and the entire staff of the Human Protein Atlas for their efforts. Financial support in the form of research grants was also received from The Swedish Heart Lung Foundation, The Swedish Research Council, The Mats Kleberg Foundation, The King Oscar II Jubilee Foundation, The Stockholm County Council, and The Swedish Association for Chest Physicians.