Introduction
Rheumatoid arthritis (RA) is a chronic systemic inflammatory disease predominantly affecting joints, leading to tissue destruction and functional disability [
1,
2]. Both genetic and environmental factors are believed to contribute to the dysregulated immune responses seen in this heterogeneous autoimmune disease [
3]. Today, treatment strategies involve traditional disease-modifying antirheumatic drugs as well as biologic agents targeting proinflammatory cytokines (tumour necrosis factor α (TNFα), interleukin (IL)-1 and IL-6), B cells or the activation of T cells [
4]. Despite this arsenal of drugs, at least 30% of the patients are resistant to the available therapies, suggesting that yet other mediators must be important.
The most prominent feature of RA is the progressive destruction of articular cartilage and bone, which is orchestrated by activated RA fibroblast-like synoviocytes (RA-FLSs) [
5,
6]. RA-FLSs not only mediate tissue destruction but also are considered to play a major role in initiating and driving RA in concert with inflammatory cells [
7]. In the healthy synovium, one to three layers of synoviocytes, the macrophage-like type A and the more abundant fibroblast-like type B (also referred to as synovial fibroblast), form the synovial lining layer separating the synovial sublining layer of loose connective tissue from the joint cavity [
8,
9]. The synoviocytes are interconnected with adherens junctions containing cadherin-11 [
10,
11] and E-cadherin [
12,
13] and are embedded in a lattice of extracellular matrix (ECM) resembling an epithelium but lacking a discrete basal membrane as well as gap junctions and desmosomes. Apart from being a marker of FLSs, cadherin-11 has been shown to be essential for the formation of synovial lining structures
in vitro and for the development of inflammatory arthritis in mice [
14,
15].
The morphological hallmarks of RA include activation of FLSs; infiltration of inflammatory cells such as T cells, B cells and macrophages in the sublining; hyperplasia of the synovial lining layer; fibrotic deposition; and subsequent formation of the "pannus" [
16]. This tissue mass expands and attaches to and invades the adjacent cartilage and subchondral bone [
17]. The major cell type accounting for the thickened lining layer as well as for pannus formation is believed to be activated FLSs [
18,
19]. These aggressive cells share many characteristics with tumour cells, with upregulated expression of proto-oncogenes and promigratory adhesion molecules, increased production of proinflammatory cytokines and matrix-degrading enzymes [
7], as well as increased resistance to apoptosis [
20,
21]. There are data indicating that the transformed phenotype of RA-FLS is stable and maintained even in the absence of stimulus from inflammatory cells [
22]. In high-inflammation synovial tissue, RA-FLSs show a gene expression profile characteristic of myofibroblasts, and cells of the synovial lining in RA have been found to express α-smooth muscle actin (α-sma) and type IV collagen [
13,
23]. Thus, it has been suggested that RA-FLSs can undergo a process resembling epithelial-mesenchymal transition (EMT), a phenomenon known from early developmental processes, tissue repair, fibrosis and carcinogenesis [
24,
25]. Recently, it was also suggested that migrating RA-FLSs might be responsible for spreading the disease to distant joints [
26].
Podoplanin (identical to human PA2.26, aggrus and T1α-2), is a small, 38- to 40-kDa, mucin-type transmembrane glycoprotein normally expressed on human lymphatic endothelia, basal epithelial keratinocytes, myoepithelial cells and myofibroblasts of certain glandular tissues, follicular dendritic cells and fibroblastic reticular cells of lymphoid organs and alveolar type I cells [
27,
28]. We demonstrated strong podoplanin expression on subepithelial interstitial cells in human endolymphatic tissue of the inner ear [
29]. The physiologic function of podoplanin is to a large extent unknown, but knockout (KO) studies showed that it is crucial for the development of the lung and deep lymphatics in mice [
28]. The podoplanin-KO mice died at birth as a result of respiratory failure and generalised lymphoedema. Overexpression of this glycoprotein in epithelial cells induced a dentritic cell morphology and increased cell adhesion and migration [
27]. Interestingly, increasing data show that podoplanin is upregulated on the invasive front of human cancers [
27,
30]. The expression of podoplanin is correlated with metastasis and a bad prognosis. In addition, podoplanin (or aggrus) induces platelet aggregation of tumour cells [
31] and has been associated with both EMT-dependent and EMT-independent tumour cell invasion [
32]. There are a few studies indicating increased podoplanin expression in fibroblasts in reactive tissues, such as in chronic pleuritis, in cancer-associated fibroblasts [
33] and in cultured fibroblasts [
34]. However, little is known about the potential role of podoplanin in inflammation and tissue repair. In this study, we were interested to see whether podoplanin is expressed in FLSs in RA and could be associated with the fibrotic transformation of the synovium in this disease.
Materials and methods
Human synovial tissue and cells
Synovial tissue specimens and fluid were obtained from patients with RA (
n = 18) or OA (
n = 9) during joint replacement surgery or therapeutic joint aspiration at Sahlgrenska University Hospital and Spenshult Hospital in Sweden. Both weight-bearing (knee and hip) and non-weight-bearing (shoulder and elbow) joint specimens were included. All RA patients fulfilled the American College of Rheumatology 1987 revised criteria for RA [
35]. Preoperative radiographs were scored according to Larsen index (1 to 5) [
36]: 0 = normal; 1 = slight abnormality, soft tissue swelling, periarticular osteoporosis and slight joint space narrowing; 2 = early abnormality, erosions (obligatory in non-weight-bearing joints) and joint space narrowing; 3 = medium destructive abnormality, erosions and joint space narrowing; 4 = severe destructive abnormality, erosions, joint space narrowing and bone deformation; and 5 = mutilating abnormality. The patient characteristics are outlined in Table
1. All patients gave informed consent, and the procedure was approved by the Ethics Committee of Gothenburg in Sweden. Human primary FLS cultures were established as follows: representative tissue pieces were minced, treated with 1 mg/ml collagenase/dispase (Roche, Mannheim, Germany) for 1 hour at 37°C and passaged through a cell strainer. The cell suspension was rinsed twice in phosphate-buffered saline (PBS), resuspended in Dulbecco's modified Eagle's medium (DMEM) GlutaMAX (Invitrogen, Camarillo, CA, USA) supplemented with 10% heat-inactivated foetal bovine serum (HIFBS) (Sigma, St. Louis, MO, USA), 50 μg/ml gentamicin (Sanofi-Aventis, Paris, France) and 100 μg/ml normocin (Invivogen, San Diego, CA, USA) and incubated at 5% CO
2 at 37°C. Cells in passages 3 through 6 were used.
Table 1
Characteristics of patientsa
Age, mean yr | 61.8 | 68.4 |
Sex, F/M | 13/6 | 6/3 |
Disease duration, mean yr | 21.9 | - |
Seropositiveb, % | 82% | - |
Larsen scorec (mean ± SD) | 2.9 ± 0.6 | - |
DMARDs, % | 72% | - |
Steroids, % | 44% | - |
Biologic drugs, % | 33% | - |
Immunohistochemistry
Paraformaldehyde (PFA)-fixed (Histolab, Göteborg, Sweden), paraffin-embedded (4 μm) or acetone-fixed (Histolab) frozen sections (6 μm) were rehydrated in Tris-buffered saline for 10 minutes. Antigen retrieval was performed when required in a pressure chamber (2100 Retriever; Histolab). Unspecific binding was blocked using serum-free protein block or normal rabbit serum (Dako, Glostrup, Denmark). After incubation with mouse monoclonal antihuman podoplanin (clone D2-40; AbD Serotec, Oxford, UK), mouse monoclonal antihuman cadherin-11 (clone 5B2H5; Invitrogen) or mouse monoclonal antihuman CD90 antibodies (clone AS02; Dianova, Hamburg, Germany), respectively, the specimens were incubated with a biotinylated rabbit antimouse immunoglobulin G F(ab')2 fragment (Dako) followed by streptavidin-conjugated alkaline phosphatase (Dako). Fast Red Naphthol (Sigma) was used as a substrate, and the specimens were counterstained with Mayer's haematoxylin (Histolab) and mounted in Aqua-Mount mounting medium (VWR International Ltd, Leicestershire, UK). The same staining protocol was used for immunocytochemistry of primary FLS seeded onto chamber slides (Lab-Tek; Nunc, Rochester, NY, USA) and fixed in PFA. Normal mouse IgG1 (Dako) was used as a negative control. The podoplanin staining was scored by two independent observers blinded to the procedure according to the following scoring method: 0 = negative staining, 1 = positive staining of single or limited groups of cells in the lining layer, 2 = continuous positive staining of the cells of the synovial lining layer and 3 = same as 2, but with the addition of positive staining of cells in the sublining layer.
Immunofluorescence and confocal microscopy
Paraffin-embedded synovial sections were subjected to a double-staining procedure: incubation with rabbit antihuman cadherin-11 (Invitrogen), rabbit anti-matrix metalloproteinase (MMP)-9 (AB805; Millipore, Billerica, MA, USA), rabbit antihuman E-cadherin (clone H-108; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or rabbit anti-α-sma (PA1-37024; Thermo Scientific, Rockford, IL, USA) antibodies followed by addition of Alexa Fluor 555-conjugated goat antirabbit IgG (Invitrogen) or, in one step, Alexa Fluor 647-conjugated mouse antihuman CD68 (clone KP1; Santa Cruz Biotechnology). Second, mouse antihuman podoplanin (clone D2-40) incubation was followed by Alexa Fluor 488-conjugated goat antimouse IgG (Invitrogen). Alternatively, biotinylated mouse antihuman podoplanin (Acris Antibodies GmbH, Herford, Germany) and Alexa Fluor 488-conjugated streptavidin were added prior to mouse antihuman cadherin-11 (clone 5B2H5) and Alexa Fluor 555-conjugated goat antimouse IgG (Invitrogen). Slides were placed in ProLong Gold antifade reagent mounting medium with 4',6-diamidino-2-phenylindole (Invitrogen). Normal mouse IgG1 or normal rabbit serum (Dako) was used as negative controls. Images were collected using a confocal microscope (LSM700; Zeiss, Oberkochen, Germany). The background fluorescence level was set with the negative controls, and images were analysed using Zen image analysis software 2009 (Zeiss).
Western blot analysis
Membrane proteins from tissue and cell pellets were prepared by sodium carbonate treatment [
37]. In brief, lyophilized material was resuspended in 0.1 M sodium carbonate before sonication. After removal of cell debris, the membrane fraction was collected by ultracentrifugation at 115,000
g for 75 minutes. The membrane proteins were solubilised with 7 M urea, 2 M thiourea, 40 mM Tris, 1% C7 detergent (wt/vol) and 4% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate buffer (wt/vol) and kept at -80°C before use.
Samples, together with recombinant unglycosylated human podoplanin core protein (ProSpec, Ness-Ziona, Israel), were separated by 20% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions with 10 mM dithiothreitol. After being transferred onto polyvinylidene fluoride membrane, the blots were probed with mouse antihuman podoplanin (1:50; D2-40) and detected with a horseradish peroxidise-conjugated rabbit antimouse antibody (1:2,000; DakoCytomation) and chemiluminescence (SuperSignal West Femto Maximum Sensitivity Substrate; Thermo Scientific).
Flow cytometry
Primary synovial cell cultures from patients with RA (n = 6) and patients with OA (n = 5) were trypsinised, resuspended in fluorescence-activated cell sorting buffer (5% HIFBS, 0.09% sodium azide and 0.5% ethylenediaminetetraacetic acid in PBS) and transferred onto a 96-well plate. For intracellular staining (CD68; α-sma), cells were PFA-fixed and permeabilised with 0.1% Triton X-100 in PBS. Unspecific binding was blocked using 1% HIFBS in PBS or Beriglobin P (human IgG; Apoteket, Sweden). Staining was performed with allophycocyanin (APC)-conjugated mouse antihuman CD90, phycoerythrin (PE)-conjugated mouse antihuman CD68, PE-conjugated mouse antihuman CD29 (BD Biosciences, San Jose, CA, USA), mouse antihuman podoplanin (clone D2-40), mouse antihuman cadherin-11 (clone 5B2H5), rabbit antihuman α-sma (PA1-37024) and isotype controls (BD Biosciences). The unconjugated antibodies were incubated with secondary PE-conjugated rat antimouse IgG1 (BD Biosciences) or APC-conjugated goat antirabbit IgG (Santa Cruz Biotechnology) in a second step. Fluorescence was measured using the FACSCanto II system (BD Biosciences) equipped with DIVA 6.2 software (BD Biosciences), and data were analyzed using FlowJo 8.7.3 software (Tree Star Inc., Ashland, OR, USA). The isotype controls were used to set the gates for positive and negative populations.
Stimulation experiments
Primary FLSs from one OA patient were seeded into complete DMEM in triplicates in six-well plates (100,000 cells/well) and incubated until confluence. The cells were serum-starved in DMEM supplemented with 2% heat-inactivated foetal calf serum for 6 hours before the different human recombinant cytokines were added: 10 ng/ml TNFα (Sigma), 1 ng/ml IL-1β and 1 ng/ml TGF-β1 (R&D Systems, Minneapolis, MN, USA). The cells were harvested by trypsinisation after 12, 24 and 48 hours, and podoplanin expression was measured using flow cytometry with antipodoplanin antibody (clone D2-40). The experiment was repeated four times with different primary cell cultures, including RA-FLSs, with similar results.
Statistical analysis
Differences in protein expression between the patient groups detected by immunohistochemistry (IHC) and flow cytometry were evaluated using the Mann-Whitney nonparametric test.
Discussion
In this study, we have shown that the tumour-associated proinvasive glycoprotein podoplanin is highly expressed in synovial lining layer cells in RA but is rarely found in OA synovial specimens. The expression of podoplanin was most pronounced in areas with signs of inflammation (that is, the presence of leukocyte infiltrates and ectopic lymphoid structures) and synovial transformation (indicated by lining layer hyperplasia, MMP-9 expression and upregulation of cadherin-11 and α-sma). Furthermore, the podoplanin-expressing lining layer cells expressed cadherin-11 but not the macrophage marker CD68, suggesting that these synoviocytes were FLSs rather than synovial macrophages.
All included RA patients had progressed to erosive disease (Larsen index score >1), and all except one had a high podoplanin expression score (IHC score >1) of the synovial tissue from the replaced joint (Table
1). However, without the rarely available tissue specimens from nonerosive and early RA joints to compare these tissues with, we could not analyze whether there is a correlation between erosive disease and podoplanin expression.
The function of podoplanin is far from elucidated. On one hand, this small glycoprotein is constitutively expressed on the apical surface of lymph endothelia as well as on specialised epithelia (for example, podocytes) facing fluid compartments [
28,
42,
43]. On the other hand, podoplanin is crucial for processes involving cell migration, such as the specific embryologic development of deep lymphatics [
28] and the invasion and metastasis of certain tumour cells or tissues [
32]. Podoplanin has been shown to bind ezrin, an actin filament membrane linker protein, on the inside of the cell
in vitro
[
30,
44]. It has therefore been suggested that podoplanin is involved in directing actin polymerisation, thereby forming the cellular protrusions needed for migration.
In our study, the marked and widespread expression of podoplanin in lining layer cells in RA was not restricted to the apical cell surface. Instead, it resembled the strong whole cell surface-staining pattern of podoplanin in tumour tissues [
30]. It has been shown that RA-FLSs of highly inflammatory synovial tissue show a gene expression profile characteristic of myofibroblasts [
23]. We detected coexpression of podoplanin and α-sma of FLSs in areas of synovial transformation and found that the expression of E-cadherin was low or absent in the podoplanin-expressing lining layer cells. We know from earlier studies that podoplanin can promote EMT of epithelial Madin-Darby canine kidney cells
in vitro
[
44]. EMT is a biologic process in which polarised epithelial cells undergo sequential changes into a mesenchymal cell phenotype with increased migratory potential and the production of ECM components [
24]. Loss of E-cadherin and gain of α-sma expression constitute examples of such changes. We therefore hypothesise that podoplanin is involved in an EMT-like transdifferentiation of RA-FLSs into myofibroblasts.
Podoplanin has been observed in interstitial fibroblasts in different inflammatory environments
in vivo and
in vitro
[
33,
34]. In agreement with this observation, we found a locally increased expression of podoplanin in interstitial cells of the sublining connective tissue in specimens from patients with RA. However, it is difficult to determine whether upregulated podoplanin expression in the sublining in some RA specimens was a result of general inflammation or whether this phenomenon was part of a specific activation and transdifferentiation of FLSs in RA.
To confirm the specificity of the D2-40 antibody and the expression of podoplanin in RA synovial tissue, we performed SDS-PAGE and Western blot analysis of protein extracts showing a distinct band of about 45 kDa. The mature glycosylated form of podoplanin has been estimated to be about 38 to 40 kDa [
27]. The difference in approximated molecular weight could be explained by the reported heterogeneity of podoplanin in SDS-PAGE, which arises as a result of heavily
O-linked glycosylation of the core protein [
27] as well as a slightly unspecific migration of the used molecular weight markers.
Characteristics of RA are the phenotypic changes and hyperplasia of FLSs of the lining layer. Conventional isolation of FLSs from synovial tissue yields homogeneous fibroblast cultures [
45], but the interindividual morphological variation is large, and cultures presumably arise from both the synovial lining and sublining layers.
We established primary cultures of FLSs from human synovial tissues by enzyme digestion and found that the cells had typical fibroblast morphology. Nearly all of the primary FLSs stained positive for the fibroblast marker CD90/Thy-1 and most expressed β1 integrins. However, the IHC staining of human synovia using the anti-CD90 antibody revealed positive expression in the sublining, but not in the lining layer cells (Figures
3A and
3B). Fibroblasts possess a remarkable phenotypic plasticity [
41] as well as a positional identity [
46]. The synovium (lining layer versus sublining layer) of both healthy and RA patients harbour phenotypically different (by morphology and expression of surface markers) populations of fibroblasts. CD90 might therefore be a good marker for interstitial tissue fibroblasts, but not for the FLSs forming the epithelium-like lining of the synovium. In addition, fibroblasts change the expression of several surface molecules
in vitro and acquire an "active" phenotype with prominent stress fibres and focal adhesions [
47] when cultured on plastic. We therefore concluded that most of the established primary FLS cultures in this study originated from the sublining connective tissue or acquired a sublining fibroblast phenotype (with respect to CD90 expression) in culture. Using IHC, we found that cadherin-11 was expressed both in the lining layer and in cells of the sublining tissue in reactive areas, but when using flow cytometry, we found it on average in only 10% of the isolated primary FLSs. These data support the assumption that the isolated primary FLSs in these experiments originated from the sublining rather than from the lining layer.
Fibroblasts have been shown to upregulate podoplanin in culture [
34]. In this study, we did not observe any significant difference in mean podoplanin expression between the RA-FLS and OA-FLS cultures. Only one culture, derived from an RA patient, was growing without contact inhibition, a characteristic of activated FLSs in RA. All cells of this culture were expressing podoplanin. Taken together, our results suggest that cultures of the lining layer FLS phenotype are hard to establish by using this technique and that primary FLSs, like other fibroblasts, probably upregulate podoplanin in culture. The observed upregulated expression of α-sma of the primary FLSs constitutes another example of an acquired feature of cultured fibroblasts.
Finally, we found a more than twofold increase in podoplanin expression in primary FLSs after stimulation with IL-1β and TNFα compared with controls. Interestingly, we also detected an increase in podoplanin expression in response to TGF-β1 stimulation. TGF-β1 is a key mediator of EMT and promotes the differentiation of fibroblasts into myofibroblasts in wound healing and fibrosis. Furthermore, TGF-β1-induced podoplanin in human fibrosarcomas [
48] was found to be increased in arthritic joints in RA [
49] and promoted EMT of FLS
in vitro
[
13]. The fact that proinflammatory cytokines and growth factors, known to be present in high concentrations in the RA joint, stimulate podoplanin expression in primary FLSs
in vitro supports our finding that podoplanin is upregulated in the synovium of RA patients and might be involved in the transdifferentiation of FLSs in RA.
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Competing interests
MIB has received consulting fees from Schering-Plough, UCB, Pfizer, Roche and GlaxoSmithKline (less than US$10,000 each). NK was working for Bristol-Myers Squibb from 2006 to 2009 on an unrelated project. The authors declare no other competing interests.
Authors' contributions
AKHE did the major design of the study and the coordination and establishment of the biobank, carried out the experiments, performed the imaging and FACS analysis and drafted the manuscript. TE participated in the design of the study and performed the Larsen scoring of radiographs. TE and CA included the patients in the study and collected patient data and tissue samples. CJ performed the SDS-PAGE and Western blot analyses. MB participated in the analysis of the FACS data and helped to draft the manuscript. MIB participated in the design of the study and analysis of the data and helped to draft the manuscript. All authors read and approved the final manuscript.