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Rheumatology Advance Access originally published online on February 20, 2006
Rheumatology 2006 45(9):1068-1076; doi:10.1093/rheumatology/kel045
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© The Author 2006. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

RANKL protein is expressed at the pannus–bone interface at sites of articular bone erosion in rheumatoid arthritis

A. R. Pettit, N. C. Walsh1, C. Manning1, S. R. Goldring1 and E. M. Gravallese1,

Institute for Molecular Bioscience, University of Queensland, Brisbane, QLD 4072, Australia and 1Beth Israel Deaconess Medical Center, New England Baptist Bone and Joint Institute, Harvard Institutes of Medicine, 4 Blackfan Circle, Boston, MA 02115, USA.

Correspondence to: E. M. Gravallese, Beth Israel Deaconess Medical Center, Harvard Institutes of Medicine, Room 241, 4 Blackfan Circle, Boston, MA 02115, USA. E-mail: egravall{at}bidmc.harvard.edu


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives. Receptor activator of NF-{kappa}B ligand (RANKL) and osteoprotegerin (OPG) have been demonstrated to be critical regulators of osteoclast generation and activity. In addition, RANKL has been implicated as an important mediator of bone erosion in rheumatoid arthritis (RA). However, the expression of RANKL and OPG at sites of pannus invasion into bone has not been examined. The present study was undertaken to further elucidate the contribution of this cytokine system to osteoclastogenesis and subsequent bone erosion in RA by examining the pattern of protein expression for RANKL, OPG and the receptor activator of NF-{kappa}B (RANK) in RA at sites of articular bone erosion.

Methods. Tissues from 20 surgical procedures from 17 patients with RA were collected as discarded materials. Six samples contained only synovium or tenosynovium remote from bone, four samples contained pannus–bone interface with adjacent synovium and 10 samples contained both synovium remote from bone and pannus–bone interface with adjacent synovium. Immunohistochemistry was used to characterize the cellular pattern of RANKL, RANK and OPG protein expression immediately adjacent to and remote from sites of bone erosion.

Results. Cellular expression of RANKL protein was relatively restricted in the bone microenvironment; staining was focal and confined largely to sites of osteoclast-mediated erosion at the pannus–bone interface and at sites of subchondral bone erosion. RANK-expressing osteoclast precursor cells were also present in these sites. OPG protein expression was observed in numerous cells in synovium remote from bone but was more limited at sites of bone erosion, especially in regions associated with RANKL expression.

Conclusions. The pattern of RANKL and OPG expression and the presence of RANK-expressing osteoclast precursor cells at sites of bone erosion in RA contributes to the generation of a local microenvironment that favours osteoclast differentiation and activity. These data provide further evidence implicating RANKL in the pathogenesis of arthritis-induced joint destruction.

KEY WORDS: Rheumatoid arthritis, Osteoclasts, RANKL, OPG, RANK, Bone erosion.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Articular bone erosions are a characteristic feature of rheumatoid arthritis (RA) and destruction of articular bone is associated with significant morbidity and with a poor prognosis [1]. Recent studies investigating the pathogenic mechanisms of bone destruction in animal models of arthritis have provided substantial evidence that the osteoclast is the cell type primarily responsible for focal bone erosion, and that generation and activity of osteoclasts is dependent upon receptor activator of NF-{kappa}B ligand (RANKL) [2–8]. RANKL is an essential factor for osteoclastogenesis both in vitro and in vivo [9, 10]. This cytokine stimulates precursor cells of the myeloid lineage to differentiate into osteoclasts by binding to its signalling receptor, receptor activator of NF-{kappa}B (RANK) [11, 12]. RANKL also stimulates osteoclast migration, fusion, activation and survival and thus acts at all stages of osteoclast generation and function [9, 13–16]. Osteoprotegerin (OPG) is a naturally occurring soluble decoy receptor for RANKL and effectively blocks the pro-osteoclastogenic action of RANKL at all stages of osteoclast generation and function [17–20]. The current paradigm is that the balance between RANKL and OPG expression determines the level of osteoclast-mediated bone resorption by regulating osteoclast differentiation and activation.

In addition to the evidence provided by studies in animal models of arthritis, several studies in human RA implicate RANKL as an important regulator of osteoclastogenesis. RANKL messenger ribonucleic acid (mRNA) and/or protein expression has been demonstrated in synovial fibroblasts and T cells, including activated CD4+ T cells, in RA synovium remote from bone and in synovial cells cultured ex vivo [2, 21–27]. RANKL protein has also been demonstrated in some macrophages [24] and in macrophage-like cells [23]. In vitro studies have shown that RA synovial fibroblasts and CD4+ T cells can stimulate osteoclastogenesis [22, 23, 25, 28], and OPG inhibits osteoclastogenesis in this setting [22, 25]. However, RA synovial fibroblasts in culture require stimulation with 1,25-dihydroxyvitamin D3 in order to promote the differentiation of osteoclasts from peripheral blood precursors [22, 29, 30]. This stimulation is related to the capacity of 1,25-dihydroxyvitamin D3 to increase the RANKL/OPG ratio in these cells [22]. Additionally, fully mature osteoclasts can be generated in cultures of digested RA synovial tissue, and this osteoclast formation can also be inhibited by the addition of excess OPG [26, 31]. The number of resorption pits formed when these cultures are performed on dentin surfaces correlates with the RANKL/OPG mRNA ratio [26]. Finally, RANKL has been demonstrated to be prominently expressed in tissues or cells collected from RA patients with active disease [27, 32]. These observations indicate that RA synovial tissues contain all of the necessary cells and factors required for osteoclast differentiation and activation and suggest that the RANKL plays a central role in regulating this process.

Cells within RA synovial tissue remote from bone also produce OPG, which can inhibit local RANKL-mediated osteoclastogenesis. Expression of OPG protein in RA synovium has been demonstrated in both fibroblast-like and macrophage-like RA synoviocytes [32, 33] and in endothelial cells [32]. OPG expression is up-regulated in cultured RA synovial fibroblasts by tumour necrosis factor (TNF) [33]. The local balance of RANKL and OPG activity likely determines the role of this cytokine system in the pathogenesis of focal bone erosions. To date RANKL, RANK and OPG protein expression have been examined only in RA synovial tissues remote from bone. Information concerning the topographical expression of RANKL, RANK and OPG protein in the bone microenvironment at sites of erosion is therefore essential for clarifying the contribution of these cytokines to osteoclastogenesis in RA. Additionally, the expression of RANKL, RANK and OPG in RA tissue has not been examined concurrently at the protein level. Therefore, we examined RANKL, RANK and OPG protein expression at sites of articular bone erosion in tissue samples collected from RA patients.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Tissue collection
Tissue samples used in this study were collected as discarded material at the time of surgery from 20 surgical procedures from 17 patients, all of whom fulfilled the American College of Rheumatology (ACR) criteria for RA [34]. Average disease duration in the entire patient group was 16.1 yr (range 4–31 yr) and tissues were collected from surgical sites including nine knees, three wrist joints, five metacarpophalangeal (MCP) joints (arthroplasties) and three tendon sheath (tenosynovium) or carpal tunnel surgeries. Tissue samples collected were from two distinct anatomical locations: synovium remote from bone and the pannus–bone interface. Expression patterns of the proteins of interest were compared between these two anatomical locations. Chart review revealed that 15 of the 17 patients had radiographic documentation of joint destruction in one or more joints pre-operatively: 11 patients had hand erosions, one had hand and knee erosions, one had elbow erosions, one had knee erosions and another had ‘articular destructive changes’ in the knee. One of the two patients without radiographic documentation of erosions underwent tenosynovectomy, and we obtained only synovium remote from bone. Therefore this case was not included in the analysis of sites of bone erosion. Of the 20 samples analysed, six samples contained only synovium or tenosynovium remote from bone, four samples contained bone with adjacent synovium and pannus and 10 samples contained both synovium remote from bone and bone with adjacent synovium and pannus. Written informed consent was obtained from patients, in accordance with the Declaration of Helsinki, and the Beth Israel Deaconess Medical Center Committee on Clinical Investigation approved this study.

Tissue processing and histology
Tissues were fixed for 48 h in 4% paraformaldehyde and specimens containing bone were subsequently decalcified for at least 2 weeks in 14% EDTA. Specimens were processed for paraffin embedding (Citadel 1000, Shandon, Pittsburgh, PA, USA) and 5 micron serial sections were cut for histology or immunohistochemical staining. Tartrate resistant acid phosphatase (TRAP) staining was performed by a modification of a previously described method [35]. Briefly, sections were incubated for 15 min at 37°C in freshly prepared 0.1 mol/l Tris buffer, pH 5.0, 1.35 mmol/l naphthol AS-MX phosphate (Sigma, St Louis, MO, USA), 0.362 mol/l N, N-dimethylformamide, 3.88 mmol/l Violet LB salt (Sigma) and 25 mmol/l sodium tartrate. Slides were rinsed for 10 min and counterstained with haematoxylin. Additionally, at least one section from each sample was stained with haematoxylin and eosin (H&E) for histological assessment.

Antibodies
Primary antibodies used in this study were as follows: rabbit polyclonal anti-human soluble RANKL antibody (PeproTech Inc., Rocky Hill, NJ, USA); rabbit polyclonal anti-human RANK (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); mouse monoclonal anti-human OPG (IMGENEX, San Diego, CA, USA); rabbit polyclonal anti-human CD3 (DakoCytomation, Carpinteria, CA, USA); mouse anti-human CD68 antibody (PM-G1, DakoCytomation); mouse anti-human CD20 (DakoCytomation); and isotype matched control antibodies (polyclonal rabbit IgG, Santa Cruz Biotechnology Inc. and PeproTech Inc., mouse IgG1 and mouse IgG2a, DakoCytomation and mouse IgG3k, BD Pharmingen). The recombinant soluble human RANKL protein used for blocking experiments was purchased from PeproTech Inc.

Immunohistochemistry
Immunohistochemistry was performed using an immunoperoxidase technique with diaminobenzidine (DAB) as the chromogen. Expression of RANKL, RANK, OPG and CD3 were examined in serial sections. Briefly, sections were deparaffinized and rehydrated followed by microwave antigen retrieval in 10 mM EDTA pH 7.5 at 93°C for 10 min and allowed to cool for at least 2 h. Sections were washed in Tris buffered saline (TBS) and endogenous peroxidase activity was blocked by incubating the sections in 3% H2O2 (diluted in TBS) for 30 min. Sections were incubated for 60 min in serum block [10% fetal calf serum (FCS) plus 10% normal swine serum or 10% normal rabbit serum diluted in TBS] and then incubated with the primary antibody for 60 min. Sections were subsequently incubated for 30 min with a biotinylated F(ab')2 fragment of swine anti-rabbit or rabbit anti-mouse immunoglobulin (DakoCytomation), followed by horseradish peroxidase (HRP)-conjugated streptavidin (DakoCytomation) and developed with DAB (DakoCytomation) chromogen to the manufacturer's specifications. The specificity of the staining was confirmed by using matched isotype control antibodies. The specificity of the anti-RANKL antibody was further tested by a pre-incubation of this antibody with recombinant soluble human RANKL in blocking experiments, as well as by obtaining the expected pattern of RANKL staining in the positive control tissues, tonsil and lymph node (DakoCytomation). All sections were counterstained with haematoxylin. All incubations were carried out at 25°C sections were washed between each step with TBS. Slides were examined and photographed using a transmitted light microscope (Nikon, Tokyo, Japan) and AxioCam digital camera (Carl Zeiss International) using Improvision imaging software.

OPG expression was determined by one observer using the following grading system based on the number of cells expressing OPG throughout a section: low, fewer than 10 cells per x10 microscope field of view expressed OPG; moderate, more than 10 cells but less than 50% of total cells within a x10 field of view expressed OPG; high, more than 50% of cells within a x10 field of view expressed OPG. Two independent observers confirmed the anatomical distribution of OPG expression.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
RANKL is expressed at sites of bone erosion in RA
We examined RANKL protein expression in samples containing synovium remote from bone (available in 16 samples, mean disease duration 14.7 yr, range 4–25 yr). Typical synovial inflammation was observed in these samples (including lining layer hyperplasia and accumulation of leucocytes: CD3+ T cells, CD20+ B cells and/or CD68+ macrophages), indicating ongoing disease in these patients. RANKL protein was observed in many cells within the synovial lining layer, including scattered cells that were intensely stained (Fig. 1A). In addition, isolated cells within the sublining, located most commonly within and bordering perivascular mononuclear cell aggregates, expressed RANKL protein, as has been previously described [23–25, 32]. Specificity of staining was confirmed in serial sections after blockade of the RANKL antibody by pre-incubation with recombinant human RANKL protein (Fig. 1B) and staining with isotype-matched control antibody. This general expression pattern was observed in all 16 samples.


Figure 1
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  		FIG. 1. RANKL is expressed at sites of bone erosion in RA. The figure shows immunohistochemical staining for RANKL protein expression (brown) in synovium and bone samples collected from RA patients. All sections were counterstained with haematoxylin. (A) RANKL protein in the synovial lining layer demonstrating expression in lining layer cells with intense staining (arrows) in a small subset of these cells. (B) To confirm specificity of staining, a serial section to (A) was stained with anti-RANKL antibody pre-incubated with a 10-fold excess of soluble RANKL protein. The majority of staining was eliminated, demonstrating specificity. (C) RANKL staining in the bone microenvironment demonstrating focal expression of RANKL in cells at a site of pannus invasion into subchondral bone. The boxed area indicates RANKL expression adjacent to multinucleated osteoclast-like cells in multiple resorption lacunae at a pannus–bone interface. The arrow indicates a nearby bone–pannus interface that has no evidence of active bone erosion and no adjacent RANKL-expressing cells. The boxed area is shown at higher magnification in (D). (D) RANKL-expressing cells near and immediately adjacent to multinucleated osteoclast-like cells in resorption lacunae (arrowheads). (E) and (F) RANKL expression in cells within pannus tissue invading bone at low (E) and high (F) magnification. In this example RANKL-expressing cells are at the interface of the invading pannus and bone and are near a multinucleated osteoclast-like cell at a site of erosion (the arrowhead indicates the same cell in both images). (G) RANKL expression in cells at a site of pannus invasion into subchondral bone. In this example, RANKL is expressed in cells within one region of a large area of erosive activity containing numerous multinucleated osteoclast-like cells. (H) RANKL expression in a lymphocyte aggregate within bone marrow at a site remote from bone erosion. Original magnification: x50 (C and E) and x100 (A, B, D and F–H).

 
To determine if RANKL is expressed at sites of bone erosion, we investigated the expression of RANKL protein in samples collected from RA patients that contained the pannus–bone interface and adjacent synovium (available in 14 samples). These samples were collected from patients with long-standing disease (mean duration 17.9 yr, range 10–31 yr) from both large (six total knee replacements) and small (five MCP arthroplasties and three wrist fusions) joint surgeries. A striking observation was that RANKL protein expression was not widespread at the pannus–bone interface, including sites of pannus invasion into bone. RANKL expression was restricted to isolated areas within many, but not all, sites of osteoclast-mediated bone erosion and was expressed by cells of varying morphology (Fig. 1C–G). RANKL protein was identified most commonly in focal areas where pannus tissue invaded through cartilage and into subchondral bone (Fig. 1E–G). We also identified RANKL protein on cells in areas of osteoclast-mediated resorption in regions where inflamed synovial tissues invaded into the bone marrow space (Fig. 1C and D). These patterns of RANKL staining were observed in 12 of the 14 patient samples studied that contained the pannus–bone interface. Two samples had no RANKL expression. Notably, these samples had minimal to no apparent osteoclast-like cells associated with bone surfaces as assessed in H&E- or TRAP-stained sections.

RANKL expression was also observed in occasional scattered cells within the bone marrow space in inflamed tissues and in areas resembling more normal bone marrow. CD3+ T-cell infiltration in the bone marrow microenvironment was not a prominent feature in the majority of samples studied from these patients with long-standing RA. Small numbers of CD3+ cells were observed in the bone microenvironment in most samples, although these were not usually immediately associated with sites of obvious bone erosion. However, three of the samples studied contained large lymphocytic aggregates within the bone marrow and RANKL expression was observed in some cells scattered within and bordering these aggregates (Fig. 1H). Staining in serial sections with the T-cell marker CD3 demonstrated some overlap of the staining pattern for RANKL and CD3 in these areas (data not shown). Finally, 10 samples contained intact cartilage and in three of these samples RANKL expression was observed in a small number of chondrocytes.

RANKL protein is expressed adjacent to RANK+ osteoclast-like cells
Staining in serial sections (Fig. 2A–E) demonstrated that RANKL was often expressed by cells located near to TRAP+ or RANK+ multinucleated and mononuclear cells located on or adjacent to bone surfaces. No staining was noted with an isotype-matched antibody (Fig. 2E) or the additional control of antibody blockade by pre-treatment with recombinant soluble RANKL protein. The majority of multinucleated cells on bone expressed RANK, as did numerous mononuclear cells in the bone microenvironment. The presence of RANK expression on multinucleated cells within a sample varied, suggesting that the expression of this receptor may be regulated in later stages of osteoclast activation. RANK expression was observed in cells that had morphological features of osteoblasts in six of the 14 samples examined. Additionally, RANK expression was observed in the synovial lining layer and in cells within and bordering perivascular mononuclear cell infiltrates in the sublining area in all samples containing synovium remote from bone (data not shown).


Figure 2
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  		FIG. 2. RANKL-expressing cells are adjacent to TRAP+ and RANK+ multinucleated cells at sites of bone erosion. The figure shows histochemical and immunohistochemical staining in serial sections at a site of subchondral bone erosion from an RA patient sample. All sections were counterstained with haematoxylin and in all images ‘Bone’ indicates a similar anatomical location in the same bone fragment. (A) Staining for TRAP enzyme activity (red) demonstrating TRAP+ multinucleated and mononuclear cells. (B) RANKL protein expression (brown) demonstrating RANKL+ cells adjacent to the location of TRAP+ osteoclast-like cells. (C) RANK expression in multinucleated osteoclast-like cells and numerous mononuclear cells within and near this site of subchondral bone erosion. RANK+ multinucleated cells are adjacent to the location of RANKL+ cells in (B) and correlate with TRAP+ multinucleated cells in the near serial section in (A). (D) OPG is expressed in few cells in tissue adjacent to bone at sites of erosion. A low level of OPG staining was noted in some multinucleated osteoclast-like cells and also in mononuclear cells. (E) Staining with isotype-matched control antibody for anti-RANK and anti-RANKL staining. Original magnification for all images: x100.

 
OPG protein is expressed in synovium remote from bone but expression is restricted at sites of bone erosion
The balance between RANKL and OPG expression determines the outcome for osteoclastogenesis. We therefore examined and compared the expression pattern of OPG in RA samples containing synovium remote from bone and samples containing the pannus–bone interface. In the six samples that contained only synovium (three samples) or tenosynovium (three samples) remote from bone, OPG expression was moderate to high in all samples. In contrast, OPG expression in the bone microenvironment, particularly at sites of bone erosion and RANKL expression, was more limited (Fig. 2B and D). In the four samples that contained only pannus/synovium adjacent to bone, OPG expression was generally limited (low to moderate grading). In eight of the 10 samples from which we had both pannus/synovium adjacent to bone and synovium remote from bone, OPG expression levels were clearly higher in tissue remote from bone than in tissue located near bone (Fig. 3A and B). This difference in the localization of OPG expression was also present in the remaining two samples that included tissue from both anatomical locations, but the difference in OPG expression patterns was not as striking in these two samples. These data indicate that in RA there is a gradient of OPG protein expression, with lower OPG expression in the local RA bone microenvironment compared with synovial tissues remote from bone.


Figure 3
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  		FIG. 3. OPG staining is more prominent in synovial tissue remote from bone than in the bone microenvironment, particularly near sites of erosion. The figure shows immunohistochemical staining for OPG in RA samples containing either synovium remote from bone or sites of pannus invasion into bone with adjacent synovium. All samples were counterstained with haematoxylin. (A) OPG staining (brown) in cells within synovium remote from bone demonstrating strong expression of OPG in numerous cells within the synovial lining layer (top and bottom edges of the tissue) and some expression in a moderate number of cells scattered in sublining tissue. (B) Staining for OPG in the same patient sample as shown in (A) in an area that contains bone with adjacent pannus. Minimal OPG staining is present at the bone–pannus interface. Staining is noted in multinucleated osteoclast-like cells (arrow), as well as in superficial cells within pannus. RANKL staining was observed in a small number of cells at this erosion site (data not shown). (C) Multinucleated osteoclast-like cells in the bone microenvironment demonstrating staining for OPG. (D) Isotype-matched control antibody staining in a serial section to (C). Arrows indicate the same multinucleated osteoclast-like cells in (C) and (D). (E) OPG staining in osteoblast-like cells (arrows) was observed in some samples in areas of normal subchondral bone and bone marrow in which there was no evidence of osteoclast-mediated bone erosion. (F) Isotype-matched control antibody staining in a serial section to (E). Original magnification; x50 (A and B) and x100 (C–F).

 
The cellular pattern of OPG expression differed between the RA bone microenvironment and synovium remote from bone. In tissue adjacent to bone, multinucleated cells on the bone surface were the common cell type positive for OPG staining (Figs 2D and 3C). The presence of OPG staining on osteoclast-like cells was variable, suggesting that expression of OPG, or a cell surface protein or receptor that can bind OPG, is differentially regulated during osteoclast maturation and/or function. Occasional OPG expression was observed in osteoblast-like cells (Fig. 3E), as well as in some chondrocytes (data not shown) and in occasional scattered cells within bone marrow and invading pannus tissue (Figs 2D and 3B). In synovial tissue remote from bone the most common cells stained for OPG were cells in the synovial lining layer and in endothelium, as has been previously shown [32]. In addition, scattered cells expressing OPG were noted within the sublining (Fig. 3A).


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
Studies in animal models of arthritis have provided strong evidence suggesting a central role for RANKL in the generation of osteoclasts and in subsequent bone erosion in inflammatory arthritis [2–4, 6, 7]. RANKL and RANK protein expression have been demonstrated in inflamed synovium near sites of bone erosion in animal models of RA [36–42]. Similarly, RANKL and OPG protein expression have been previously demonstrated in RA synovial tissues collected from sites remote from bone erosions [23–25, 32, 43]. However, the expression of RANKL, RANK and OPG in RA tissue at the bone–pannus interface has not been reported. Since bone erosion occurs at this interface and in subchondral bone adjacent to invading synovial tissues, determination of RANKL protein expression at these sites in RA joints is critical for definitively establishing the contribution of this cytokine to the process of bone erosion.

In this study we examined RANKL protein expression at sites of pannus invasion into bone and at sites of subchondral bone erosion. We have demonstrated the expression of RANKL protein by cells at sites of osteoclast-mediated bone resorption within the bone microenvironment in RA at both of these anatomical sites. RANKL protein was focally expressed in these locations and expression was limited to a relatively small number of cells. Expression of RANKL was typically observed in cells that were adjacent to multinucleated osteoclast-like cells and RANK-expressing mononuclear osteoclast precursor cells. RANKL expression in the bone microenvironment was not abundant, but was focused at sites of bone resorption, suggesting specific expression of RANKL at these sites during the erosive process. RANKL protein was not detected at all sites of osteoclast-mediated erosion, underscoring the focal nature of RANKL expression. This limited pattern of expression suggests that RANKL acts locally in osteoclast-mediated bone resorption in this disease and that the disease process does not result in dramatic generalized up-regulation of RANKL in the bone microenvironment. The pro-inflammatory cytokines TNF and interleukin (IL)-1 can regulate osteoclast differentiation and function in the presence of RANKL in vitro [44–49]. It is possible that in RA the expression of RANKL is an essential and limiting step, and that ‘permissive’ levels of RANKL cooperate with TNF, IL-1 and possibly other locally expressed cytokines to drive osteoclast differentiation and function. It should be noted that the samples studied were from patients with long-standing disease. RANKL protein expression in synovium remote from bone was reported by Haynes et al. [32] to be more prevalent in tissues collected from RA patients with active disease. Therefore, RANKL expression in the bone microenvironment in early and/or more active RA may be more prevalent than that noted in our study.

It is also likely that the RANKL protein detected in this study under-represents the total RANKL protein in the bone microenvironment in RA. RANKL protein is expressed as a membrane-associated form, which requires cell–cell contact for its biological action. Soluble forms of RANKL have also been described, resulting either from enzymatic cleavage of the membrane-bound protein or from expression of alternative soluble forms [50–54]. The fixation and immunohistochemistry protocols used in the current study are unlikely to allow detection of soluble RANKL, as soluble proteins can easily dissipate from tissues. Additionally, the anti-RANKL antibody used may be unable to detect RANKL bound by OPG or RANK. Therefore, the RANKL protein observed in this study may reflect only the level of unbound and presumably active membrane-associated RANKL.

Since the prevalence of OPG expression is a critical determinant of the degree to which RANKL can stimulate osteoclast generation and activation, we also examined the expression of OPG protein in RA synovium remote from bone and at the pannus–bone interface. There was a striking difference observed in the expression of OPG protein at these sites, with prominent expression observed in synovium remote from bone and more restricted expression detected within the bone microenvironment, particularly at sites of bone erosion where RANKL expression was noted. These observations suggest that OPG expression in the bone microenvironment is limited in RA, particularly at sites of focal bone erosion, which may contribute to a local microenvironment that favours osteoclast differentiation and activation. As noted, our observations may only represent intracellular OPG protein expression and OPG bound to other proteins on the cell surface.

We observed that some multinucleated osteoclast-like cells were detected with the anti-OPG antibody in the bone microenvironment. Osteoclast expression of OPG has been previously reported [55–57]. However, we did not observe a significant overlap between RANKL and OPG expression patterns, as was described in these prior studies. It is possible that the osteoclast-associated OPG staining is due to detection of OPG bound to RANKL–RANK complexes or to TNF-related apoptosis-inducing ligand (TRAIL) on the surface of osteoclasts, although the majority of OPG staining does appear to be cytoplasmic. Interestingly, Colucci et al. [58] recently demonstrated that osteoclastogenesis can still occur in vitro in the presence of high OPG levels due to the formation of OPG/TRAIL complexes, possibly leading to the sequestration of OPG by TRAIL, which subsequently prevents its binding to RANKL. Additionally, it has been suggested that a tertiary OPG–RANKL–RANK surface complex can induce protease expression in the RAW264.7 pre-osteoclast monocytic cell line [59]. Therefore, the localization of OPG to osteoclasts in the RA bone microenvironment could be contributing to the erosive process, rather than being inhibitory.

We were unable to definitively identify the cellular source(s) of RANKL in the RA bone microenvironment, despite performing staining in serial sections with markers of fibroblasts, osteoblasts, B cells and macrophages. Several previous studies have reported RANKL expression in RA synovium remote from bone in a subset of fibroblast-like cells and in infiltrating mononuclear cells, including CD3+, CD4+ and CD45RO+ T cells [2, 21–27]. RANKL expression has also been reported in synovial macrophages [23, 24]. The three most likely sources of RANKL in the bone microenvironment appear to include invading synovial fibroblasts, lymphocytes, especially CD4+ T cells, and mesenchymal cells of the osteoblast lineage. Synovial fibroblasts represent a potential source of RANKL expression at sites of bone erosion as previous studies have demonstrated RANKL protein expression in RA synovial fibroblasts cultured in vitro [21, 22]. Additionally, in a recent publication synovial fibroblasts were implicated as an important source of RANKL in an erosive animal model of arthritis [60]. Similarly, in a previous study we identified cells expressing parathyroid hormone receptor adjacent to sites of bone erosion and speculated that these cells could represent osteoblast-lineage cells [61]. Cells of the osteoblast lineage can be induced to up-regulate RANKL production and down-regulate OPG production by pro-inflammatory cytokines known to be expressed in pannus tissues, including IL-1, TNF, parathyroid hormone-related peptide and others (reviewed in [62, 63]). Therefore in this setting, mesenchymal cells of the osteoblast lineage may be an important source of local RANKL expression.

T cells are also a potential source of RANKL at sites of RA bone erosion. However, in this study of tissues representing late disease, only three of 14 samples had a prominent T-cell component within the inflammatory infiltrate in the bone microenvironment, and only one of these samples had significant numbers of T cells within sites of bone erosion. In most of the samples studied, a small number of scattered T cells were observed in the bone microenvironment and occasionally at sites of bone erosion. We did observe some overlap in the pattern of RANKL and CD3 staining in the bone microenvironment, suggesting that T cells are a potential, if minor, source of RANKL near erosion sites. It is possible that T cells in the bone microenvironment express soluble forms of RANKL that may not have been detected by the technique used in this study [64], and therefore T cells would not be required to be located directly within the erosive site to influence osteoclast generation and function. This argument also applies to other cellular sources of RANKL. To fully elucidate the potential contribution of RANKL-expressing T cells to osteoclastogenesis in the RA bone microenvironment it will be important to examine samples from patients with shorter disease duration and/or more active disease, as the composition of the cellular infiltrate may well be influenced by the stage of disease.

In conclusion, we have demonstrated the expression pattern of RANKL, RANK and OPG protein in the RA bone microenvironment. Other studies of RA tissues have not specifically examined expression patterns at this site. These observations support the hypothesis that the local RA bone microenvironment enables osteoclastogenesis. This is achieved at least in part by the induction of RANKL expression at sites of erosion, with a concomitant relatively restricted expression of OPG at this site. This study provides additional evidence supporting the hypothesis that RANKL plays a critical role in the differentiation of osteoclast precursor cells to osteoclasts at the erosive site in RA. Effective inhibition of the biological activity of RANKL in RA is therefore likely to restrict the process of focal bone erosion.

Formula


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
The authors wish to thank Dr Zhenxin Shen for his assistance in obtaining and processing surgical samples.

This research was supported by a NHMRC C.J. Martin Fellowship (ARP) and by NIH RO1-AR-47665 (EMG).

The authors have declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

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Submitted 12 October 2005; revised version accepted 13 January 2006.
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A. E. Kearns, S. Khosla, and P. J. Kostenuik
Receptor Activator of Nuclear Factor {kappa}B Ligand and Osteoprotegerin Regulation of Bone Remodeling in Health and Disease
Endocr. Rev., April 1, 2008; 29(2): 155 - 192.
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