Osteoprotegerin expression in synovial tissue from patients with rheumatoid arthritis, spondyloarthropathies and osteoarthritis and normal controls
D. R. Haynes1,
E. Barg7,
T. N. Crotti1,
C. Holding1,
H. Weedon2,
G. J. Atkins1,
A. Zannetino3,
M. J. Ahern4,
M. Coleman5,
P. J. Roberts-Thomson6,
M. Kraan8,
P. P. Tak8 and
M. D. Smith2,4,
Departments of Pathology and
1 Orthopaedics and Trauma, University of Adelaide,
2 Rheumatology Research Unit, Repatriation General Hospital, Daw Park,
3 Department of Haematology, The Hanson Centre for Cancer Research,
4 Department of Medicine, Flinders University of South Australia,
5 SouthPath and
6 Clinical Immunology, Flinders Medical Centre, Adelaide, South Australia,
7 University of Leiden, Leiden and
8 Amsterdam Medical Center, Amsterdam, The Netherlands
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Abstract
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Objectives. To demonstrate the expression of osteoprotegerin (OPG) and receptor activator of nuclear factor
B ligand (RANKL) in synovial tissue from rheumatoid arthritis (RA) patients, establish the cell lineage expressing OPG and compare the expression of OPG in RA, spondyloarthropathies, osteoarthritis and normal synovial tissue.
Methods. Synovial biopsy specimens were obtained at arthroscopy from 16 RA and 12 spondyloarthropathy patients with active synovitis of a knee joint, six RA patients with no evidence of active synovitis, 10 patients with osteoarthritis and 18 normal subjects. Immunohistological analysis was performed using monoclonal antibodies (mAb) to detect OPG and RANKL expression. In addition, dual immunohistochemical evaluation was performed with lineage-specific monoclonal antibodies (macrophages, fibroblasts and endothelial cells) and OPG to determine the cell lineages expressing OPG. The sections were evaluated by computer-assisted image analysis and semiquantitative analysis.
Results. Two patterns of OPG expression were seen, one exclusively in endothelial cells and one expressed predominantly in macrophages in the synovial lining layer. Both patterns of OPG staining could be blocked with excess recombinant OPG. Endothelial and synovial lining expression of OPG was seen in all synovial tissues except those from patients with active RA. In contrast, RANKL expression was seen predominantly in synovial tissue from patients with active disease, mainly in sublining regions, particularly within areas of lymphocyte infiltration.
Conclusions. OPG expression on macrophage type synovial lining cells as well as endothelial cells is deficient in RA patients with active synovitis, in contrast to that seen in spondyloarthropathy patients with active synovitis. This deficiency in OPG expression in the inflamed joint of RA patients may be important in the development of radiologically defined joint erosions.
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Introduction
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Rheumatoid arthritis (RA) is characterized by inflammation of the synovial membrane, leading to invasion of synovial tissue into the adjacent cartilage matrix with degradation of articular cartilage and bone as a consequence. This results in erosion of bone which is often observed as marginal joint erosions radiographically and is predictive of a poorer prognosis [1]. While the pathophysiological mechanisms of cartilage and bone destruction in RA are yet to be completely understood, it is known that matrix metalloproteinases, cathepsins and mast cell proteinases can contribute to cartilage and bone destruction in RA [24]. However, it is now clear that osteoclast formation and activation at the cartilagepannus junction is an essential step in the destruction of bone matrix in RA patients [58]. A number of inflammatory cytokines found in the RA synovial tissue [interleukin (IL)-1
, -1ß and -6, tumour necrosis factor
(TNF-
) and macrophage colony-stimulating factor] have the potential to promote osteoclast formation and bone resorption [911]. However, recent evidence indicates that the interaction between RANKL [also known as osteoclast differentiation factor (ODF), tumour necrosis factor-related activation-induced cytokine (TRANCE) and osteoprotegerin ligand] has an essential role in osteoclastogenesis [5, 6, 12, 13]. RANKL is expressed on osteoblasts, fibroblasts and T cells while receptor activator of nuclear factor
B (RANK) is mainly expressed on pre-osteoclasts, possibly of the macrophage lineage. The end result of the production of inflammatory cytokines, such as IL-1ß and TNF-
, in the inflamed joint is likely to be the up-regulation of RANKL (produced by T cells, fibroblasts and osteoblasts) [8, 13] and RANK (expressed by pre-osteoclasts, T cells and dendritic cells) [13]. Recently it has been suggested that TNF-
may have an additional major role in regulating osteoclast formation, through the TNF receptor type I [14]. In addition to their pivotal role in osteoclast formation, RANKL and RANK have a role in immune cell differentiation and T celldendritic cell interactions [12, 13].
Another member of the TNF family is TRAIL (TNF-related apoptosis-inducing ligand), which shares homology with RANK and RANKL, which are also members of the TNF family of proteins. TRAIL has also been shown to bind to osteoprotegerin (OPG) [15]. This is a naturally occurring inhibitor of the RANKL interaction with RANK; it binds RANKL with high affinity, preventing RANKL from interacting with RANK [16, 17]. The biological relevance of OPG as a regulator of RANKL/RANK interaction and osteoclast formation and activation is clearly demonstrated by the development of osteopetrosis in OPG transgenic mice and severe osteoporosis in OPG knockout mice [1719].
OPG is an alternative, high-affinity soluble decoy receptor for RANKL which blocks the interaction between RANKL and RANK and significantly inhibits osteoclastogenesis [16, 17]. Like RANK, it is a member of the TNF receptor family but lacks a transmembrane domain and is a secreted protein which is structurally distinct from RANK [13]. Similar to RANK and RANKL, OPG production is stimulated by proinflammatory cytokines, such as IL-1ß and TNF-
[20].
RANK, RANKL, TRAIL and OPG are expressed in tissue of the RA joint [5, 6, 8]. Osteoclasts form from cells isolated from the RA joint [21], large numbers forming rapidly from cells isolated from the pannus region [8]. Furthermore, osteoclast formation can be inhibited by the addition of exogenous OPG. In view of its ability to block RANKLRANK interactions and inhibit osteoclast formation, OPG may have a role in normal homeostasis within the joint and has therapeutic potential in the treatment of conditions such as RA, in which bone destruction is a major sequela of chronic inflammation.
The aim of this study was to demonstrate the expression of OPG and RANKL in synovial tissue from patients with RA, spondyloarthropathies and osteoarthritis (OA) and contrast this with the expression in normal synovial tissue. In addition, we aimed to demonstrate, by dual immunohistochemical labelling techniques, the cell lineages expressing OPG in the synovial tissue.
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Methods
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Materials
Recombinant human OPG (dimeric, amino acids 22401, glycosylated), recombinant human OPG-Fc (dimeric, amino acids 22194 conjugated to human Fc, non-glycosylated) and RANK (amino acids 140317) were generous gifts from Amgen (Thousand Oaks, CA, USA) and recombinant human TRAIL was a gift from Immunex (Seattle, WA, USA).
Patients
Twenty-two RA patients [16] with active synovitis (based on joint pain, swelling and presence of a synovial effusion) in a knee joint and six with no evidence of any synovitis] and 10 patients with a spondyloarthropathy (SpA) and active synovitis of a knee joint were investigated along with 10 patients with OA of the knee joint and 18 normal subjects. The normal subjects were all subjects without any history of any form of arthritis either at the time of or for 5 yr following an arthroscopy for unexplained knee pain performed at a sports medicine facility. At the time of arthroscopy, there was no evidence of any synovial membrane or articular cartilage pathology [22]. All RA patients fulfilled the American College of Rheumatology criteria for RA [23], while the SpA patients fulfilled the European Spondyloarthropathy Study Group (ESSG) criteria for the diagnosis of ankylosing spondylitis and Reiter's disease [24]. Patients with psoriatic arthritis [25] and OA of the knee [26] fulfilled published criteria. All patients gave informed consent, and the study protocol was approved by the institutional medical ethics committee of the Repatriation General Hospital.
Synovial tissue
A small-bore arthroscopy (2.7 mm arthroscope; Dyonics, Andover, MA, USA) was performed under local anaesthesia as described previously [27]. Biopsies of synovial tissue were obtained from all accessible regions of the knee joint, but mainly from the suprapatellar pouch. The samples were separately snap-frozen in Tissue-Tek OCT (Miles Diagnostics, Elkhart, IN, USA) and stored at -80°C until used. Cryostat sections (6 µm) were mounted on glass slides (Superior, Marienfeld, Germany). The glass slides were boxed and stored at 20°C until immunohistological analysis.
Immunohistochemistry
Serial sections were stained with the following mouse monoclonal antibodies (mAbs): anti-human OPG antibodies (mAb 805 and mAb 8051; R&D Systems, Minneapolis, MN, USA)., anti-human TRANCE (mAb626; R&D Systems), anti-CD68 (EBM11; Dako, Botany, NSW, Australia) to detect macrophages, mAb 67 (Serotec, Oxford, UK), which recognizes CD55, to detect fibroblast-like synoviocytes (FLS), and anti-factor VIII antibody (Dako), to detect endothelial cells.
Endogenous peroxidase activity was inhibited using 0.1% sodium azide and 1% hydrogen peroxide in TrisPBS (phosphate-buffered saline) buffer. Staining for cell markers was performed, as described previously [28]. Following a primary step of incubation with mAbs, bound antibody was detected according to a three-step immunoperoxidase method [2931]. Horseradish peroxidase (HRP) activity was detected using hydrogen peroxide as substrate and amino ethylcarbazole (AEC) as the dye. Slides were counterstained briefly with haematoxylin solution and mounted in Gurr Aquamount (BDH, Poole, UK). Affinity-purified HRP-conjugated goat anti-mouse antibody was obtained from Dako, affinity-purified HRP- conjugated swine anti-goat immunoglobulin from Tago (Burlingame, CA, USA) and AEC from Sigma (St Louis, MO, USA). All antibodies used in this study were from commercial sources with known antigen specificities. All staining was performed at one time with each antibody, using appropriate positive (RA synovial tissue with a previously defined staining pattern, lymph node or tonsil) and negative (omission of primary antibody, control antibody of similar isotype to study antibody) controls. In view of the two different staining patterns for OPG in synovial tissue, absorption studies with recombinant OPG were also performed (see OPG absorption of mAbs 805 and 8051).
Dual immunohistochemistry
Dual immunohistochemical labelling was performed as described previously [31, 32]. In brief, the synovial tissue was incubated with the first primary antibody (mAb 805 or 8051) and subsequent steps in a standard three-step immunoperoxidase method were performed, developing the final colour product using AEC. After washing the tissue and blocking with Tris-glycine, the second primary antibody (anti-CD68, anti-CD55 or anti-factor VIII) was placed on the sections overnight at 4°C, followed by a standard immunoalkaline phosphatase method, developing the colour reaction with Fast Blue. No counterstain was used and the sections were mounted in an aqueous mounting medium.
OPG absorption of mAbs 805 and 8051
The specific activity of the antibodies directed against OPG was blocked by incubation of 0.01 µg/ml mAb 805 or mAb 8051 with 0.1 µg/ml human recombinant OPG overnight at room temperature. A control batch of each antibody was incubated with antibody diluent containing an irrelevant protein at a similar concentration to the recombinant OPG. The following day, both the absorbed and non-absorbed antibodies were centrifuged at 12 000 r.p.m. in a refrigerated centrifuge, diluted in antibody diluent to the usual working dilution and placed on frozen sections of RA synovial tissue. Thereafter, a standard three-step immunoperoxidase method was performed and developed with AEC.
Microscopic analysis
After immunohistochemical staining, sections stained for OPG (mAbs 805 and 8051) and RANKL were analysed in a random order by computer-assisted image analysis, analysing six high-power fields for each section, as described previously [28, 3133]. Area of staining, integrated optical density [IOD, equivalent to area of staining x mean optical density (MOD)] and MOD were all measured using computer-assisted image analysis. In addition, these sections were also scored by a semiquantitative method on a 4-point scale by two independent observers in a random order, as described previously [28, 30].
Western blot and ELISA assays of OPG monoclonal antibodies
The immunospecificity of the two monoclonal antibodies against OPG was investigated using western blotting and an enzyme-linked immunoassay (ELISA). Western blot analysis was carried out using unreduced (dimeric) and reduced (monomeric) recombinant human OPG. Two forms of OPG were used, dimeric OPG (110 kDa) and dimeric OPG-Fc (90 kDa). Reduced (dithiothreitol) and unreduced OPG were run on gels and immunoprecipitated with mAb 805 and mAb 8051 antibodies using standard methods (Fig. 1
). In another study, primary cultures of synovial cells, obtained from tissue explants, were obtained from two inactive RA patients. The supernatants and cell lysates were absorbed to mAb 8051 linked to magnetic beads (Dynabeads M-450; Dynal Biotech, Oslo, Norway) and the bound protein was eluted to concentrate the antigens recognized by mAb 8051. These eluates were then run on a western blot and probed with mAb 8051.

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FIG. 1. The specificity of binding of mAb 805 and mAb 8051 is shown using western blot analysis. Lane A, recombinant human OPG (110 kDa dimer); lane B, reduced recombinant human OPG (55 kDa monomer); lane C, recombinant human OPG-Fc (90 kDa dimer); lane D, reduced recombinant human OPG-Fc (45 kDa monomer).
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ELISAs were carried out using the OPG antibodies as recommended by the commercial supplier (R&D Systems). Briefly, mAb 805 or mAb 8051 was used as the primary (capture) antibody and a biotinylated goat polyclonal antibody (BAF805; R&D Systems) was used as the secondary antibody. As it is reported that mAb 805 can block the binding of TRAIL to OPG (mAb 805 specification sheet; R&D Systems), the effects of RANKL and TRAIL on the binding of OPG to both antibodies were compared (Fig. 1
). OPG, at various concentrations (0.0053.33 ng/ml), was incubated with recombinant human RANKL or TRAIL for 1 h at 37°C then tested in the ELISA assay using either mAb 805 or mAb 8051 as the primary antibody (Fig. 2
).

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FIG. 2. The ability of human recombinant RANKL and TRAIL to inhibit the binding of mAb 805 and mAb 8051 was tested as described in the text (Methods). All OPG dilution curves were carried out in triplicate. (A) RANKL strongly inhibits OPG binding to mAb 805. (B) TRAIL inhibition of OPG binding to mAb 805 is weaker than RANKL inhibition. (C) Both RANKL and TRAIL are weak inhibitors of OPG binding to mAb 8051 even at concentrations of 100 ng/ml.
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Statistical analysis
Non-parametric statistics were used to analyse the mean ranks of the semiquantitative scores, the number of vessels and percentage staining for the five groups of patients, using KruskalWallis one-way analysis of variance. As the data for area, MOD and IOD, measured by digital image analysis, were not normally distributed, the data were log-transformed and analysed using one-way analysis of variance. Post hoc discrepancies between groups were analysed using Tukey's HSD (honestly significant difference) and Bonferroni tests. Statistical significance was accepted when P < 0.05.
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Results
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Clinical and demographic features
Clinical and demographic data of the RA and SpA patients included in the study are presented in Table 1
. Only one patient in the active RA group and no patients in the inactive RA group were on immunosuppressive agents or corticosteroids, which have been reported to alter OPG and/or RANKL production. The 10 OA patients included five males and five females, five of whom were taking NSAIDs at the time of synovial tissue removal at knee replacement surgery, with a mean age of 69.4 yr (range 5678). The normal subjects included 11 males and seven females with a mean age of 33.3 yr (range 1854).
Specificity of binding of OPG antibodies
Figure 1
shows that mAb 805 only recognized the unreduced (dimeric) form of both OPG molecules, whereas mAb 8051 detected the unreduced (dimeric) and reduced (monomeric) forms of both OPG molecules. In addition, western blots of supernatants and cell lysates from synovial tissue cell cultures showed that the only protein bound by mAb 8051 was monomeric and dimeric OPG (results not shown). The supernatants and cell lysates of primary cultures of synovial cells demonstrated that mAb 8051 specifically detected only two bands of protein that corresponded in molecular weight to the native monomeric and dimeric OPG (results not shown) which is also seen with recombinant human OPG (Fig. 1
).
Further information on the specificity of binding of the two monoclonal antibodies was demonstrated using an ELISA system. Figure 2
shows the standard dilution curve for measurement of recombinant OPG and the ability of RANKL and TRAIL to block the detection of OPG. Binding of mAb 805 to OPG was blocked by both RANKL and TRAIL. RANKL was more than 10-fold more effective at blocking OPG binding, possibly indicating that RANKL bound to OPG with higher affinity than TRAIL. mAb 8051 was only affected slightly by RANKL and TRAIL, even at a concentration of 100 ng/ml. These results show that mAb 805 recognizes an epitope associated with RANKL and TRAIL binding and that mAb 8051 recognizes an epitope that is not closely associated with RANKL and TRAIL binding.
Immunohistochemistry
Two distinct patterns of staining for OPG in synovial tissue were seen. As demonstrated in Fig. 3
, mAb 805 stained exclusively endothelial cells, while mAb 8051 stained mainly the lining layer of the synovial membrane. However, at higher concentrations of mAb 8051 weak staining of endothelial cells was also seen (results not shown). The synovial tissue staining with both mAbs could be blocked with excess of recombinant OPG (Fig. 4
). Dual immunohistochemical labelling with lineage-specific mAbs for macrophages (CD68), FLS (mAb67) and endothelial cells (factor VIII) clearly demonstrated that mAb 805 was staining endothelial cells while mAb 8051 was staining the intimal macrophages rather than the FLS (Fig. 5
). The computer-assisted image analysis results and semiquantitative scores for mAbs 805 and 8051 are displayed in Table 2
and Fig. 6
. Expression of OPG, as detected by mAbs 805 and 8051, was seen in all patients with the notable exception of RA patients with active synovitis at the time of synovial biopsy, whether measured by semiquantitative scoring (P < 0.0005) or image analysis (P < 0.0005 for area and IOD, P < 0.005 for MOD). There was no significant difference in OPG expression, as detected by staining with mAb 805 or mAb 8051, between inactive RA, SpA, OA and normal groups.

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FIG. 3. OPG staining in synovial tissue from an RA patient with active disease (A and B), an RA patient with psoriatic arthritis (C and D), an RA patient with OA (E and F) and a normal subject (G and H) with mAb 805 (A, C, E, G) and 8051 (B, D, F, H). A colour reproduction of this figure can be viewed on the following website: http://www.repat.com.au/topic.asp?Page_ID=202.
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FIG. 4. Recombinant OPG blocks the staining of OPG using mAb 805 (A and B) and 8051 (C and D) on RA synovial tissue from a patient with inactive disease. (A and C) Immunoperoxidase staining with non-absorbed mAb. (B and D) Immunoperoxidase staining after absorption of mAb with recombinant OPG. A colour reproduction of this figure can be viewed on the following website: http://www.repat.com.au/topic.asp?Page_ID=202.
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FIG. 5. Cell lineages expressing OPG were determined using dual immunohistochemical labelling on serial sections of synovial biopsy from an RA patient with inactive disease. (A) mAb 805immunoperoxidase with AEC (red). (B) Anti-factor VIII, using immunoalkaline phosphatase and Fast Blue (blue). (C) Dual labelling with mAb 805 (red), anti-factor VIII (blue) and both (purple). (D) mAb 8051immunoperoxidase with AEC (red). (E) Immunoalkaline phosphatase with anti-CD55Fast Blue (blue) (F) Immunoalkaline phosphatase with anti-CD68Fast Blue (blue). (G) Dual labelling with mAb 8051immunoperoxidase (red) and anti-CD55 (blue). (H) Dual labelling with mAb 8051immunoperoxidase (red), anti-CD68 (blue); dual-labelled cells are purple. Magnification x200 except G and H, which are x400. A colour reproduction of this figure can be viewed on the following website: http://www.repat.com.au/topic.asp?Page_ID=202.
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TABLE 2. Digital image analysis results (IOD and MOD) and mean semiquantitative scores for the expression of OPG using mAbs 805 and 8051 in synovial tissue from RA, OA and SpA patients and normal subjects
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FIG. 6. mAb 805 (A and B) and mAb 8051 (C and D) staining measuring IOD (A and C) and area (D) using image analysis and manual counting of positive blood vessels (B). Horizontal bars represent means.
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RANKL expression in the same patient group was detected by immunohistochemistry (Fig. 7
) and quantitated by digital image analysis and semiquantitative analyses (Table 3
). As can be seen in Fig. 7
, RANKL expression was predominantly seen in T-cell-rich regions of the RA synovial membranes, with no expression of RANKL in regions of the RA synovial membranes stained with mAbs 805 or 8051 for OPG. There was an inverse relationship between OPG and RANKL expression in RA synovial membranes, active RA patients having high RANKL and low OPG expression, resulting in a high RANKL:OPG ratio (Table 3
). There was no statistically significant difference in synovial membrane RANKL expression between active and inactive RA patients, although there was a trend to increased RANKL expression in patients with active RA, as measured by both image analysis and a semiquantitative scoring system.

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FIG. 7. Immunohistochemical labelling of the synovial membrane from a patient with active RA (A, C, E, G) and inactive RA (B, D, F, H) with anti-CD3 (A, B), anti-TRANCE (RANKL) (C, D), mAb 8051 (OPG: E, F) and mAb 805 (OPG: G, H). Magnification x200 (inserts x400). A colour reproduction of this figure can be viewed on the following website: http://www.repat.com.au/topic.asp?Page_ID=202.
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TABLE 3. Digital image analysis results (IOD and MOD) and mean semiquantitative scores for the expression of RANKL using mAb626 in synovial tissue from active and inactive RA
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Discussion
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Osteoclasts are responsible for the resorption of bone during normal bone metabolism and the destruction of bone seen in a variety of pathologies, such as RA. Over the past 5 yr, several important factors have been identified that regulate osteoclast formation [6, 12, 13]. The cartilagepannus junction in RA contains many types of cells which produce inflammatory cytokines reported to stimulate osteoclast differentiation and bone resorption, including IL-1
, IL-1ß, IL-6, IL-11 and TNF-
[9]. OPG production is stimulated in vitro by proinflammatory cytokines, such as IL-1ß and TNF-
[20]. This study has demonstrated that OPG is present in the synovial tissue lining normal joints and may have an important role in regulating osteoclast formation within the normal joint. We have also demonstrated that there are two different staining patterns for OPG within the joint, one of which is predominantly found in the intimal macrophages found in the synovial lining, while the other is exclusively endothelial. It is unclear why two separate staining patterns for OPG were seen in the same tissue with two different monoclonal antibodies, particularly as both staining patterns could be blocked with excess recombinant OPG.
It is possible that this was due to different forms of OPG being recognized by the two monoclonal antibodies. Our western blot analysis of the specificity of the binding of the antibodies indicates that OPG associated with endothelial cells is likely to be native dimeric OPG, whereas the OPG predominantly found in the synovial lining is not a native dimer but possibly a breakdown product or monomer of OPG. In addition, further information about the different forms of OPG present in the tissues is revealed by our immunobinding studies. OPG protein expressed by the endothelial cells is identified by monoclonal antibodies that detect a conformational epitope involved in the binding of OPG to RANKL, whereas the monoclonal antibody that detects both endothelial and intimal macrophage OPG binds to a linear epitope in a region of OPG unrelated to binding to RANKL. This means it is possible that OPG produced by endothelial cells is able to block RANKLRANK interactions but the OPG produced by synovial cells is unable to block RANKL activity. Alternatively, the failure of mAb 805 to detect OPG on the synovial lining layer may be due to binding of RANKL to OPG on the intimal macrophage, blocking binding of mAb 805 to the conformational epitope. It is also possible that mAb 8051 detected lining layer cells because of the detection of RANKLOPG complexes on macrophage lineage cells in the synovial lining layer. The latter explanations appear unlikely as RANKL cannot be detected at either the mRNA [21] or the protein level on CD68-positive synoviocytes, and RANKL expression in RA synovial membranes is seen in T-cell-rich areas of the synovial membrane and not on the synovial lining layer [34].
OPG knockout mice have been shown to develop arterial calcification [18, 35] in addition to severe osteoporosis, suggesting that vascular endothelial expression of OPG may have a role in homeostasis [35]. This may be caused by a normal basal level of extracellular matrix calcification that requires active inhibition to avoid pathological calcification [36]. This raises the possibility that alterations in OPG expression on endothelial cells in RA patients may play a role in the excess cardiovascular morbidity and mortality in RA [37]. OPG expression by vascular endothelial cells has been described recently and shown to be up-regulated by the inflammatory cytokines IL-1ß and TNF-
[20]. If this is also the case in vivo, we might expect that OPG protein expression would increase during active RA, when elevated levels of these cytokines are reported. However, this was not observed in the present study, indicating that other mechanisms regulate OPG protein expression in RA. Low levels of OPG in synovial fluid from RA patients, compared with OA, trauma and gout patients, has been reported in a recent abstract [38] and subsequent paper [34], which would support the results found in synovial tissue in this study.
We demonstrated that endothelial OPG expression is present in the synovial tissue of normal subjects and both inflammatory and degenerative arthritides, with the notable exception of active RA patients. We also demonstrated the presence of OPG [both endothelial (mAb 805) and synovial lining (mAb 8051)] in the synovial membrane of RA patients with no active synovitis at the time of synovial biopsy, suggesting that successful treatment of the disease state in RA may up-regulate OPG expression and possibly inhibit bone erosion at the joint margins. This hypothesis needs to be tested by studying sequential synovial biopsies from patients undergoing treatment with disease-modifying anti-rheumatic drugs (DMARDs) for RA and correlating the expression of OPG with radiological outcome measures in hand and feet X-rays. There is, at present, no published study which demonstrates the effects of currently used DMARD treatment in RA on the production of biological regulators of osteoclast formation, such as RANKL, RANK and OPG. The results of this study, however, raise the possibility that a deficiency in OPG expression may have a role in the pathogenesis of the bone erosions which characterize RA and suggest that OPG may well have a therapeutic role in the future management of RA.
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Acknowledgments
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This study was supported by grants from the National Health and Medical Research Council, Department of Veteran Affairs, the Clive and Vera Ramaciotti Foundation, the J.H. and J.D. Gunn Foundation and the Rebecca L. Cooper Medical Research Foundation. The authors wish to acknowledge the technical assistance of Mr Martin Hutchens.
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Notes
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Correspondence to: M. D. Smith, Rheumatology Research Unit, Repatriation General Hospital, Daws Road, Daw Park, South Australia 5041, Australia. E-mail: malcolm.smith{at}rgh.sa.gov.au 
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Submitted 14 January 2002;
Accepted 10 June 2002