TWEAK and Fn14 expression in the pathogenesis of joint inflammation and bone erosion in rheumatoid arthritis
- Anak ASSK Dharmapatni1Email author,
- Malcolm D Smith2,
- Tania N Crotti1,
- Christopher A Holding1,
- Cristina Vincent4,
- Helen M Weedon2,
- Andrew CW Zannettino3, 6,
- Timothy S Zheng4,
- David M Findlay5, 6,
- Gerald J Atkins†5, 6 and
- David R Haynes†1
© Dharmapatni et al.; licensee BioMed Central Ltd. 2011
Received: 18 October 2010
Accepted: 24 March 2011
Published: 24 March 2011
TNF-like weak inducer of apoptosis (TWEAK) has been proposed as a mediator of inflammation and bone erosion in rheumatoid arthritis (RA). This study aimed to investigate TWEAK and TWEAK receptor (Fn14) expression in synovial tissue from patients with active and inactive rheumatoid arthritis (RA), osteoarthritis (OA) and normal controls and assess soluble (s)TWEAK levels in the synovial fluids from patients with active RA and OA. Effects of sTWEAK on osteoclasts and osteoblasts were investigated in vitro.
TWEAK and Fn14 expression were detected in synovial tissues by immunohistochemistry (IHC). Selected tissues were dual labelled with antibodies specific for TWEAK and lineage-selective cell surface markers CD68, Tryptase G, CD22 and CD38. TWEAK mRNA expression was examined in human peripheral blood mononuclear cells (PBMC) sorted on the basis of their expression of CD22. sTWEAK was detected in synovial fluid from OA and RA patients by ELISA. The effect of sTWEAK on PBMC and RAW 264.7 osteoclastogenesis was examined. The effect of sTWEAK on cell surface receptor activator of NF Kappa B Ligand (RANKL) expression by human osteoblasts was determined by flow cytometry.
TWEAK and Fn14 expression were significantly higher in synovial tissue from all patient groups compared to the synovial tissue from control subjects (P < 0.05). TWEAK was significantly higher in active compared with inactive RA tissues (P < 0.05). TWEAK expression co-localised with a subset of CD38+ plasma cells and with CD22+ B-lymphocytes in RA tissues. Abundant TWEAK mRNA expression was detected in normal human CD22+ B cells. Higher levels of sTWEAK were observed in synovial fluids isolated from active RA compared with OA patients. sTWEAK did not stimulate osteoclast formation directly from PBMC, however, sTWEAK induced the surface expression of RANKL by human immature, STRO-1+ osteoblasts.
The expression of TWEAK by CD22+ B cells and CD38+ plasma cells in RA synovium represents a novel potential pathogenic pathway. High levels of sTWEAK in active RA synovial fluid and of TWEAK and Fn14 in active RA tissue, together with the effect of TWEAK to induce osteoblastic RANKL expression, is consistent with TWEAK/Fn14 signalling being important in the pathogenesis of inflammation and bone erosion in RA.
TWEAK (TNF-like weak inducer of apoptosis) is a recently described member of the TNF superfamily. It is reported to exert a variety of biological effects through ligation with its receptor, Fn14. The biological effects of TWEAK include induction of pro-inflammatory cytokines, modulation of the immune response and angiogenesis, stimulation of apoptosis and regulation of tissue repair and regeneration [1, 2]. The pro-inflammatory effects of TWEAK/Fn14 signalling are mediated by several signalling cascades, including NF-B and the mitogen-activated protein kinases (MAPK), ERK1/2, JNK1/2 and p38 . TWEAK induces the production of a large number of pro-inflammatory molecules, such as matrix metalloproteinase (MMP1), IL-6, IL-8, MCP-I and Regulated upon Activation Normal T Cell Expressed and Secreted (RANTES) by synoviocytes and fibroblasts, as well as ICAM-1, E-selectin, IL-8, and MCP-1 by endothelial cells . The majority of these cytokines are induced by TWEAK/Fn14 induction of the NF-κβ signalling pathway [3, 5]. The pro-inflammatory effects of TWEAK are seen in various cell types including glomerular mesangial cells , human umbilical vein endothelial cells (HUVEC) , human gingival fibroblasts , human dermal fibroblasts, synoviocytes , chondrocytes, and fibroblasts .
Recent reports from us  and others  are consistent with TWEAK being a key mediator of joint pathology in murine RA models and in human RA [12, 13]. Specifically, recombinant TWEAK enhanced the production of MCP-1 and MIP-2 by synovial cells from collagen induced arthritis (CIA) mice in vitro, while the addition of TWEAK monoclonal antibody ameliorated paw swelling, synovial proliferation and inflammatory cell accumulation in CIA [10, 11]. A role for TWEAK has been described in human RA, where TWEAK induced the proliferation of synovial fibroblasts and increased the production of inflammatory cytokines and chemokines, as well as the expression of ICAM-1 . High serum levels of TWEAK, TNF-α and IL-6 were seen in RA patients as compared to normal controls . Moreover, serum TWEAK levels correlated with the disease activity score (DAS28) in RA patients and high serum TWEAK levels demonstrated a correlation with short-term response to etanercept treatment . Higher levels of TWEAK were found in RA compared to psoriatic synovium . In the current study we examine TWEAK expression in a larger group of patient-derived samples that encompassed active and inactive RA, osteoarthritic (OA) and normal patients. In addition, levels of soluble (s) TWEAK in the synovial fluids of active RA compared with OA patients were determined.
Pertinent to the pathogenesis of cartilage and bone loss in RA, TWEAK has been demonstrated to promote bone and cartilage destruction through inhibition of chondrogenesis, osteogenesis and the induced production of matrix metalloproteinase (MMP)-3 [10, 15]. We have recently described a role for TWEAK in human osteoblast differentiation  and Polek et al. have proposed its role in osteoclastogenesis . In a more recent study, we demonstrated a significant relationship between serum TWEAK levels and bone erosion markers in patients with bone destructive multiple myeloma . In addition, we have shown that TWEAK, alone and together with TNF-α, induces the expression by osteoblasts and osteocytes, in vitro and in ex vivo bone, of the bone formation inhibitor, sclerostin, via a MAPK-dependent pathway  suggesting a means by which TWEAK may inhibit bone formation during inflammatory bone remodeling. These findings suggest that TWEAK may modulate the bone erosion associated with several diseases, such as rheumatoid arthritis (RA) and multiple myeloma. The present study extends these findings by investigating the effect of sTWEAK on osteoclastogenesis and furthermore the effect of sTWEAK on osteoblasts in vitro.
Materials and methods
Demographic of patient for IHC TWEAK and Fn14 in synovial tissues
Mean Age (years) (range)
40.2 (25 to 58)
69.2 (31 to 86)
72.33 (60 to 79)
69.3 (55 to 77)
Disease duration (months) (range)
3.1 (2 to 6)
21.77 (7 to 36)
C-Reactive protein median (mg/ml) (range)
67.5 (27.0 to 307.0)
6 (2.0 to 26.0)
5 im gold
Synovial fluid samples for ELISA were obtained from 17 active RA (5 female/12 male) and 16 OA (7 female/9 male) subjects. The mean age (± SEM) of the OA group was 66.40 ± 3.18 and of the active RA group was 63.19 ± 3.78.
Immunohistochemical detection of TWEAK
TWEAK was detected using the previously described IHC method [22, 23]. Briefly, tissue sections (5 μm) were deparaffinised, pre-treated with proteinase-K to unmask the antigen and treated with sodium azide in PBS (0.1% w/v) and H2O2 (0.3% v/v) to inhibit endogenous peroxidase. Sections were then incubated overnight with mouse anti-human TWEAK monoclonal antibody (MAb), P2D10 (15 μg/ml) . Secondary antibody (HRP-conjugated goat anti-mouse IgG, DAKO, Botany, NSW, Australia) was then added, followed by HRP-conjugated swine anti-goat IgG (Biosource, Camarillo, CA, USA). The colour reaction was developed using AEC (3,9 aminoethylcarbazole from Sigma, St. Louis, MO, USA). Counterstaining was performed using Harris haematoxylin and lithium carbonate. An isotype-matched, non-binding control antibody (1D4.5, isotype IgG2a) at an identical IgG concentration or omission of the primary antibody, was used as a negative control. Tonsil tissues obtained at surgery were used as controls for lymphocytic staining.
Immunohistochemical detection of Fn14
Fn14 was detected using previously published methods . Briefly, sections were deparaffinised and pre-treated with 10 mM sodium citrate buffer (pH 6.0) at 95°C for 20 minutes. Endogenous peroxidase was then blocked using 0.3% v/v H2O2 in methanol solution. Blocking serum was applied, according to the manufacturer's instructions (Vectastain Universal Elite ABC kit, Vector Laboratories, Burlingame, CA, USA). Sections were then incubated with anti-human Fn14 MAb (ITEM1; Biolegend, San Diego, CA, USA) at 20 μg/ml overnight. Appropriate biotinylated secondary antibody was then added in the presence of 10% normal horse serum. Sections were then treated with avidin-biotin complex reagent before being stained with 3,3' Diaminobenzidine (DAB) (Vector Labs). Counter-staining and isotype-matched negative controls were performed as above for TWEAK staining.
Double staining was performed to identify specific cell types expressing TWEAK using a previously published method . Anti-TWEAK antibody was combined with MAbs for human cell surface markers: CD68 (macrophage; clone KP-1, Dako), CD22 (B lymphocyte; MAB1968, R&D Systems, Minneapolis, MN, USA), Tryptase G3 (mast cell; Cell Marque, Rocklin, CA, USA) and CD38 (plasma cells, BD Biosciences, Franklin Lakes, NJ, USA). After the single immunoperoxidase staining described above, the colour reaction was developed using an alkaline phosphatase reaction. Normal donkey serum (20% in PBS) was used to block non-specific binding, followed by incubation with cell surface marker antibodies overnight. Sections were incubated with AP-conjugated donkey anti-mouse IgG (Jackson Immuno Research, West Grove, PA, USA) as secondary antibody followed by incubation with mouse APAAP IgG (Dako, Botany, NSW, Australia). Colour was developed using a Fast Blue substrate. Using this method, immunoperoxidase stained cells were red, immuno-alkaline phosphatase stained cells were blue, while cells with co-expression were purple.
Quantification of immunohistochemical staining
A semi-quantitative (SQA) scoring method was used to assess single immunohistochemical labelling, using a validated scoring system . SQAs were analysed statistically by SPSS 11.5 software (SPSS Inc. Chicago, IL, USA) using non-parametric analysis. A P-value of less than 0.05 was considered to be significant.
Detection of TWEAK mRNA in CD22+ B Cells
Human peripheral blood mononuclear cells (PBMC) were prepared from normal buffy coat blood packs (Australian Red Cross Society, Adelaide, SA) on Ficoll gradients, as previously described . CD22-expressing cells were isolated by fluorescence activated cell sorting (FACS) after staining with a mouse anti-human CD22 monoclonal antibody directly conjugated to phycoerythrin (anti-human CD22RPE, Clone 4KB128, Dako, Golstrup, Denmark), by a method essentially described previously . CD22+ or CD22- cells were pelleted by centrifugation and resuspended in Trizol reagent. Total RNA and complementary DNA (cDNA) were prepared, and real-time reverse transcription polymerase chain reaction (RT-PCR) performed for the expression of TWEAK mRNA, as previously described .
Detection of sTWEAK in synovial fluids
sTWEAK levels were measured following the manufacturer's instructions using a TWEAK instant ELISA kit (Bender MedSystem, Burlingame, CA, USA). Briefly, samples were added in duplicate to a 96-microwell plate, which had been pre-coated with antibody, capture antibody and secondary antibody and colour was developed using tetramethylbenzidine (TMB) substrate. The absorbance was read at 450 nm using Multiskan Ascent plate reader (Thermo Labsystems, Helsinki, Finland). A standard curve was generated from the control sera provided with the kit. The difference between the mean levels of sTWEAK in the two groups was analysed using an independent sample t test. A P-value of less than 0.05 was considered to be significant.
Osteoclastogenesis assays in human PBMC and RAW 264.7 cells
Human CD14+ PBMC were isolated by FACS, as described previously in detail . The isolated cells were plated at 2 × 105 cells/well into a 96-microwell plate for tartrate resistant acid phosphatase (TRAP) staining or on whale dentine slices for resorption assays. Cells were cultured in αMEM medium containing 10% foetal calf serum (FCS) and 10 nM dexamethasone (Fauldings, Adelaide, SA, Australia) and rhM-CSF (25 ng/ml; Millipore, Temecula, CA, USA). Cultures received additional rhTWEAK and/or rhRANKL as indicated and were fed every three days. TRAP activity was assessed at 9 days and resorption was assessed by scanning electron microscopy (SEM) after 14 days, as described previously .
Murine RAW 264.7 cells were plated at 1 × 105 cells/well into 96-well plates, in the presence or absence of recombinant human (rh) Receptor Activator of Nuclear factor Kappa-B Ligand (RANKL) (100 ng/ml) (Millipore) and/or rhTWEAK (10 to 800 ng/ml). Cells were cultured for up to seven days in α-MEM medium containing 10% FCS and 10 nM 1α,25-dihydroxyvitamin D3 (1,25 D)  then stained for TRAP using a commercial kit (Sigma) at Day 7.
Human primary osteoblasts were cultured from cancellous bone samples obtained from patients undergoing total hip replacement surgery, as described previously . Cells (105/well) were seeded into wells of a six-well plate and cultured overnight in αMEM medium containing 10% v/v FCS. Cells were then cultured untreated or treated with recombinant human TWEAK (50 ng/ml) for three days . Cells were enzymically removed and stained for STRO-1  as previously described , and RANKL (MAB6261, R&D Systems) using an anti-mouse IgM-PE and anti-mouse IgG-FITC conjugate, respectively. Isotype-matched negative control antibodies 1A6.12 (IgM) and 1B5 (IgG1) were used to determine the level of background fluorescence for each fluorochrome and the compensation settings. Stained cells were analysed on a FACStarPLUS flow cytometer (Becton Dickinson, Sunnyvale, CA, USA). The percentages of cells positive for either or both STRO-1 and RANKL were calculated.
Fn14 was highly expressed in synovia from active RA patients (Figure 1E) compared to other groups (Figure 1F-H). There was a statistically significant difference between Fn14 expression in normal tissue compared to diseased groups (Figure 2), but not between active RA and inactive RA groups. There was a strong correlation between the relative expression of TWEAK and Fn14 in synovial tissues (Kendall tau_ b test, r = 0.443, P = 0.001).
Cell types expressing TWEAK and Fn14
TWEAK in synovial fluids from RA and OA
Effect of soluble TWEAK on osteoclastogenesis in vitro
Effects of soluble TWEAK on cell surface RANKL expression
The findings of this study are consistent with the growing evidence that TWEAK is a mediator of the joint destruction both in animal models of RA [10, 11] and in human RA . Recently, a number of cell types involved in the pathogenesis of RA have been reported to express TWEAK and its receptor, Fn14. While synovial fibroblasts from human patients with active RA have been reported to express TWEAK and Fn14  another report demonstrated TWEAK expression by an unidentified CD45-positive haemopoietic cell population and Fn14 expression by both CD45-positive and -negative cells . In contrast, van Kuijk and co-workers  reported TWEAK/Fn14 expression by macrophages but not by lymphocytes. The current study demonstrates that several cell types in RA synovial tissue express TWEAK, including CD68-positive macrophages, confirming the results of van Kuijk and co-workers . Interestingly and in contrast to van Kuijk and co-workers, we observed that a sub-population of CD22-positive B lymphocytes and CD-38 positive plasma cells found in lymphocyte aggregates in the sub-lining synovial tissues expressed TWEAK. Our findings are consistent with those of Kraan and coworkers who found that the numbers of CD38+ plasma cells and CD22+ B cells in RA were the best discriminating markers when comparing RA to non-RA inflammatory synovial samples .
In the current study, TWEAK expression by plasma cells is in agreement with our recent finding that demonstrated the expression of TWEAK in bone marrow plasma cells in patients with multiple myeloma . In that study, serum levels of TWEAK correlated with the serum bone resorption marker, β-crosslaps, and levels of the osteoclast recruitment chemokine, CXCL12, strongly implying a pathological role for plasma cell-derived TWEAK in bone erosion associated with multiple myeloma . In the current study, we found that the TWEAK-positive B-Lymphocytes in the germinal centres of normal tonsil sections were rare, consistent with the infrequency of CD22+ B cells at this site . While the CD22+TWEAK+ expressing B cells may be a specific population associated with the chronic inflammation seen in RA, CD22-positive B lymphocytes isolated from human normal peripheral blood also expressed abundant levels of TWEAK mRNA, suggesting that B-lineage cells are a source of TWEAK. Based on our findings in multiple myeloma  these cells may possibly also be associated with the bone erosion characteristic of RA. Interestingly, an immunotherapeutic approach that targets CD22, epratuzumab, has proved efficacious in the treatment of non-Hodgkin lymphoma, and in the autoimmune diseases, systemic lupus erythematosus and primary Sjögren's syndrome . Our findings suggest that CD22 and CD38 are also potential therapeutic targets in RA.
We were unable to verify the cell types expressing Fn14 using dual label IHC for technical reasons; however, many of these cells also had the morphology of lymphocytes and monocytes and importantly our observation indicates that Fn14 was strongly expressed by multinucleated cells within the synovial tissue. We have previously demonstrated that these multinucleated cells were preosteoclasts . It is well known that the active rheumatic joint contains an influx of osteoclast precursors . This is also consistent with a recent study by our group demonstrating the expression of TWEAK and Fn14 by multinucleated cells in gingival tissues from chronic periodontitis patients . This would suggest that TWEAK-Fn14 signalling may stimulate osteoclastogenesis and hence could contribute to the bone loss associated with chronic inflammatory diseases [10, 11].
The observation that Fn14 is expressed by cells associated with blood vessels is consistent with the report that TWEAK regulates human endothelial cell proliferation, migration and survival in vitro [33, 34]. Both TWEAK and Fn14 expression was associated with synovial tissue blood vessels suggesting that TWEAK has an autocrine effect on the angiogenesis seen in RA synovial tissue. Furthermore, this is consistent with the perivascular expression of TWEAK and Fn14 expression in RA and psoriatic synovial tissue .
A significant decrease in serum TWEAK has been reported in RA patients responding to treatment with etanercept [13, 14] while persistent TWEAK/Fn14 expression was seen in a RA patient cohort receiving infliximab treatment  suggesting a role for TWEAK in modulation of disease activity. Our data demonstrated significantly lower expression of TWEAK in the inactive RA synovial tissues. In the majority of patients this followed successful non-TNF targeted DMARD treatment, suggesting that DMARDs may reduce TWEAK expression as seen in the etanercept-treated patients. Higher TWEAK expression in active RA was also reflected in the increased synovial fluid levels of sTWEAK compared with those in OA in our study. Based on this, we suggest that monitoring the sTWEAK levels in synovial fluids could possibly be another marker of disease progression.
It was previously reported that TWEAK stimulates the differentiation of RAW 264.7 monocyte/macrophage cells into functional osteoclasts, and did so in the absence of Fn14 expression . However, we could not reproduce these findings in vitro using recombinant TWEAK to directly stimulate osteoclast formation from RAW 264.7 cells, or from human PBMC, either unfractionated or sorted on the basis of CD14 expression, a fraction shown previously to contain the osteoclast precursor [25, 35]. Although the present study demonstrated that multinucleated cells expressed Fn14, our data indicate that the stimulation of osteoclast formation by TWEAK may not be due to a direct interaction with osteoclast precursors. However, we have demonstrated that stromal osteoblasts express Fn14 and are sensitive to the effects of TWEAK [10, 16], including the induced expression of RANKL and CXCL12 mRNAs . This supports an indirect role for TWEAK in recruiting and generating osteoclasts, via the osteoblast. In the current study we confirmed that TWEAK induced the expression of cell surface RANKL protein and did so in a population of human osteoblasts expressing the immature osteoblast cell surface marker, STRO-1, cells we have previously demonstrated are capable of supporting osteoclastogenesis . This is reminiscent of our earlier study, in which RANKL mRNA expression was preferentially induced in STRO-1-positive osteoblasts in response to 1α,25-dihydroxyvitaminD3 . In addition, we recently reported a novel role for TWEAK in the regulation of osteoblast differentiation  and showed that TWEAK, and the combination of TWEAK and TNFα, induced the expression of sclerostin, a negative regulator of bone formation , and as we recently demonstrated , human osteoblast differentiation and matrix mineralization. Thus, the elevated TWEAK expression we see in RA may not only stimulate bone resorption by osteoclasts by induction of RANKL on osteoblasts but also inhibit new bone formation, contributing to the reduction in bone mass observed in inflammatory bone disease .
The current study found high levels of TWEAK expression in synovial tissues and synovial fluids from patients with active RA. Together with its ability to induce cell surface expression of RANKL by osteoblasts and possibly induce activation of B cells into plasma cells we postulate that TWEAK in this context would be pro-osteoclastogenic and would thus contribute to the bone loss associated with this chronic inflammatory disease. These findings support the concept that TWEAK should be considered as a therapeutic target in RA.
American College of Rheumatology
collagen induced arthritis
disease activity score
disease modifying anti-rheumatic drug
fluorescence activated cell sorting
foetal calf serum
horse raddish peroxidase
human umbilical vein endothelial cells
mitogen-activated protein kinases
peripheral blood mononuclear cells
phosphate buffered saline
Receptor activator of NF Kappa B Ligand
Tumor necrosis factor
tartrate resistant acid phosphatase
TNF-like weak inducer of apoptosis.
This work has been supported in part by grants from the Australian National Health and Medical Research Council [ID 453568 to D.R.H]; C.J Martin Fellowship [ID299078 to T.C] and NHMRC R. Douglas Wright Fellowship to G.J.A.
The authors thank Dr. Peter Diamond, Myeloma Research Laboratory, for performing CD22 cell sorts, and Renee Ormsby, Bone Cell Biology Group, for performing real-time RT-PCR. We express our gratitude to Dale Caville, Discipline of Anatomy and Pathology, The University of Adelaide for his photographic expertise.
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