Drs Bolon and Schett serve as consultants for Amgen Inc., and Dr Bolon owns stock in Amgen Inc. All other authors have no conflict of interest.
RANKL is a Marker and Mediator of Local and Systemic Bone Loss in Two Rat Models of Inflammatory Arthritis
Article first published online: 6 JUN 2005
Copyright © 2005 ASBMR
Journal of Bone and Mineral Research
Volume 20, Issue 10, pages 1756–1765, October 2005
How to Cite
Stolina, M., Adamu, S., Ominsky, M., Dwyer, D., Asuncion, F., Geng, Z., Middleton, S., Brown, H., Pretorius, J., Schett, G., Bolon, B., Feige, U., Zack, D. and Kostenuik, P. J. (2005), RANKL is a Marker and Mediator of Local and Systemic Bone Loss in Two Rat Models of Inflammatory Arthritis. J Bone Miner Res, 20: 1756–1765. doi: 10.1359/JBMR.050601
- Issue published online: 4 DEC 2009
- Article first published online: 6 JUN 2005
- Manuscript Accepted: 2 JUN 2005
- Manuscript Revised: 16 MAY 2005
- Manuscript Received: 20 JAN 2005
- bone erosion;
- adjuvant-induced arthritis;
- collagen-induced arthritis
RANKL is an essential mediator of bone erosions, but the role of RANKL in systemic bone loss had not been studied in arthritis. RANKL protein was increased in rat joint extracts and serum at the earliest stages of arthritis. Osteoprotegerin (OPG) treatment reversed local and systemic bone loss, suggesting that RANKL is both a marker and mediator of bone loss in arthritis.
Introduction: RANKL is well established as an essential mediator of bone erosions in inflammatory arthritis, but the role of RANKL in systemic bone loss in arthritis had not been studied. We hypothesized that serum RANKL could serve as both a mediator and as a novel biomarker for local and systemic bone loss in arthritis. We challenged this hypothesis in two established rat models of inflammatory arthritis. We sought to determine whether serum RANKL was elevated early in disease progression and whether RANKL suppression could prevent both local and systemic bone loss in these models.
Materials and Methods: Detailed time-course studies were conducted in animals with collagen-induced (CIA) or adjuvant-induced (AIA) arthritis to evaluate the onset and progression of inflammation (paw swelling), bone erosions, osteoclast numbers, and RANKL protein levels in arthritic joints and in serum. Additional CIA and AIA rats (n = 8/group) received placebo (PBS) or recombinant OPG (3 mg/kg three times weekly) for 10 days beginning 4 days after disease onset (first macroscopic evidence of hind paw erythema and edema) to assess the role of RANKL in local and systemic bone loss.
Results: RANKL protein was significantly elevated in the joints and serum of CIA and AIA rats within 1–2 days of disease onset. Increased RANKL levels were associated with local (hind paw) and systemic (vertebral) osteopenia in both models. The RANKL inhibitor OPG prevented local and systemic osteopenia in both models of established disease.
Conclusions: RANKL protein is significantly increased both locally and systemically during the earliest stages of inflammatory arthritis in rats, suggesting that serum RANKL might have prognostic value for bone erosions and systemic osteopenia in this condition. RANKL inhibition through OPG prevented local and systemic bone loss in these arthritis models, suggesting that RANKL inhibition is a promising new approach for treating bone loss in arthritis.
FOCAL BONE EROSION is a hallmark of rheumatoid arthritis (RA), a severe immune-mediated disease that progresses over time to yield irreversible skeletal destruction. Bone erosions result from enhanced production of activated osteoclasts in affected joints. (1–5) Increased bone resorption is a general feature of immune-mediated joint diseases, a fact confirmed by the marked increase in osteoclasts that occurs in experimental animal models of polyarthritis. (6–10) Therefore, therapy to control osteoclast activity is a logical strategy for preventing erosive disease in RA.
Bone resorption is regulated primarily by the relative balance between RANKL and osteoprotegerin (OPG). RANKL is the primary mediator of osteoclast formation, function, and survival. OPG is a soluble decoy receptor that neutralizes RANKL and thereby prevents osteoclast formation, function, and survival. (11, 12) In arthritic joints, RANKL is generated by activated synovial fibroblasts(13–15) and activated T lymphocytes. (7, 16, 17) Several lines of evidence convincingly show that RANKL is required for bone erosions in arthritic joints. The most direct demonstration is the absence of osteoclasts and greatly reduced joint destruction observed in RANKL knockout mice with arthritis. (18) Additional support is provided by studies showing that therapy with the RANKL inhibitor OPG prevents bone erosion in two rodent models of aggressive, immune-mediated joint destruction: adjuvant-induced arthritis (AIA)(7, 19, 20) and collagen-induced arthritis (CIA). (21)
Whereas much attention has been focused on local bone erosion, RA is also associated with systemic bone loss(22) that can be further exacerbated by glucocorticoid therapy. (23) RANKL inhibition through OPG can prevent systemic bone loss in rats treated with the glucocorticoid prednisolone, (24) but the effects of RANKL inhibition on systemic bone loss have not been studied in inflammatory arthritis models. Risk factors for systemic bone loss in RA patients include immobility and persistently active disease. (22) Unfortunately, there are no convenient biochemical markers that identify those RA patients at greatest risk for systemic bone loss. It was recently reported that serum RANKL levels are positively correlated with disease progression in RA patients, (25) suggesting that RANKL might serve as a useful biomarker for local bone loss. Serum RANKL was also reported to correlate positively with bone resorption markers and with generalized bone loss in arthritis patients. (26) Thus, serum RANKL may represent a novel marker of systemic bone loss in arthritis. If serum RANKL was increased during the very early stages of disease progression, it could provide an early indication for intervention with antiresorptives to prevent both local and systemic bone loss. To explore these questions, we used two well-characterized rat models of inflammatory arthritis (collagen-induced and adjuvant-induced arthritis), to examine the kinetics of inflammation, bone erosions, systemic bone loss, and RANKL protein concentrations in arthritic joints and in serum. We then examined the efficacy of the RANKL inhibitor OPG for preventing local and systemic bone loss when treatment was initiated within 2 days of the first significant increase in serum RANKL.
MATERIALS AND METHODS
Lewis rats (Charles River, Wilmington, MA, USA) of both sexes (males, 180–200 g; females, 150–175 g) were acclimated for 1 week and randomly assigned to treatment groups (n = 8/group). Animals were given tap water and fed ad libitum with pelleted rodent chow (8640; Harlan Teklad, Madison, WI, USA) containing 1.2% calcium and 1.0% phosphorus. For necropsy, all animals were killed by carbon dioxide. These studies were conducted in accordance with federal animal care guidelines and were preapproved by Amgen's Institutional Animal Care and Use Committee.
Induction of CIA
Porcine type II collagen (10 mg; Chondrex, Redmond, WA, USA) was dissolved in 0.1N acetic acid (5 ml) 2 days before use on a rotating plate in the refrigerator. Subsequently, collagen was emulsified 1:1 with incomplete Freund's adjuvant (IFA; Difco, Detroit, MI, USA), yielding a final concentration of 1 mg/ml, using an emulsification needle and glass syringes (Popper and Sons, New Hyde Park, NY, USA). CIA was induced in each animal by intradermal injection of emulsified collagen in IFA at 10 different sites (100 μl/site) over the back.
Induction of AIA
AIA was induced as described(27) by a single intradermal injection at the base of the tail of heat-killed Mycobacterium tuberculosis H37Ra (0.5 mg; Difco) suspended in 0.05 ml of paraffin oil (Crescent Chemical Co., Hauppauge, NY, USA).
Initial time-course studies were performed in CIA and AIA rats to monitor the kinetics of disease onset and progression. Groups of normal healthy control rats (n = 2/time-point) and arthritic rats (n = 8/time-point) were killed before, during, and after initial disease onset. Disease onset was defined as the first macroscopic evidence of hind paw erythema and edema. Treatment studies were conducted to evaluate the impact of OPG therapy on RANKL-mediated bone erosion and systemic osteopenia. CIA or AIA rats were injected subcutaneously with vehicle (PBS) or with human recombinant OPG-Fc, a RANKL inhibitor, at 3 mg/kg every 3 days for 10 days. (28) Nonimmunized healthy control rats were treated with vehicle (PBS). In both arthritis models, treatment was initiated 4 days after disease onset, when bone erosions were established. (19) Rats were necropsied 10 days later (onset + 14 days).
Assessment of arthritis
Arthritis progression was assessed in vivo by noninvasive measurement of either hind paw volume by water plethysmography(27) or hind paw diameter using precision calipers (Cole Palmer, Vernon Hills, IL, USA).
At necropsy, blood was collected by intracardiac puncture, and serum was obtained by conventional methods. The tibiotarsal region (ankle) of one hind paw (from time-course and treatment studies) and one lumbar vertebra (from treatment studies) were frozen in liquid nitrogen and pulverized, after which protein was extracted with a standard digestion buffer (50 mM Tris buffer, pH 7.4, containing 0.1 M sodium chloride and 0.1% Triton X-100). To monitor bone resorption, TRACP-5b levels were measured by a commercial ELISA kit that used a mouse monoclonal anti-rat TRACP-5b primary antibody, with 0.1 U/liter as the lower limit of sensitivity (IDS, Phoenix, AZ, USA). Values were normalized to the total protein concentration, which was determined using a standard kit (BCA Protein Assay; Pierce Co., Rockford, IL, USA). RANKL protein was assayed using a commercial ELISA kit that used a polyclonal anti-mouse RANKL primary antibody (R&D Systems). Mouse and rat forms of RANKL share 96% amino acid identity, (29) and we determined that the RANKL antibodies provided in the R&D mouse RANKL kit cross-reacted with recombinant rat RANKL (Amgen, Thousand Oaks, CA, USA). The mouse RANKL ELISA dose-dependently detected both rat and mouse recombinant RANKL standards, although cross-reactivity for rat RANKL was only about 34% relative to mouse RANKL (data not shown).
Histopathology and histomorphometry
One hind paw from each rat was processed and embedded in paraffin, and osteoclasts were identified in tissue slices using an indirect immunoperoxidase method directed against the osteoclast marker cathepsin K, as previously described. (30) H&E was used as counterstain. Bone erosion and intralesional osteoclasts were scored using tiered, semiquantitative grading criteria (Table 1) by a veterinary pathologist who was blinded to treatment conditions.
Bone histomorphometry was performed on decalcified sections of the lumbar vertebrae, using an Osteomeasure workstation (Osteometrics, Decatur, GA, USA). Sections were stained for TRACP and counterstained with H&E. Three fields of cancellous bone were measured at x20 magnification, providing a measurement area of 0.365 mm2 per section. Data were generated for cancellous bone volume/total volume (BV/TV), number of osteoclasts/bone perimeter (NOc/BPm), and number of osteoblasts/bone perimeter (NOb/BPm).
For selected paws, a serial section was labeled by standard isotopic in situ hybridization techniques(31) to follow the pattern of RANKL expression in arthritic joints over time. Tissue samples were immersion fixed in zinc formalin, decalcified, and embedded in paraffin. The tissue blocks were sectioned at 5 μm onto charged slides and processed using in situ hybridization(32) to localize RANKL. The33P-labeled probe was transcribed from nucleotides 395–640 of the rat RANKL gene (Genbank AF187319.1), amplified by RT-PCR, and cloned into the pGEM-T vector (Promega). The probe was hybridized to sections overnight at 60°C, followed by RNase digestion, and rinsed in a series of SSC washes with highest stringency of 0.1x SSC at 55°C for 30 minutes. Slides were coated with NTB2 emulsion (Kodak) and exposed for 3 weeks in the dark at 0–4°C, developed, and counterstained with H&E.
μCT analysis of arthritic joints
Hind paws were examined with an eXplore MS MicroCT System (GE Healthcare, Waukesha, WI, USA). Each paw was placed in the scanner in a 60-mm-diameter acrylic tube with a density phantom, filled with PBS, and stabilized with gauze. Using Volumetric Conebeam technology, whole paws were scanned at 0.5° rotations for 200° (80 kVp, 80 μA) and reconstructed to yield images with a voxel size of 33 × 33 × 33 μm. Rat paw scans, from distal tibia/fibula to midmetatarsal bones, were examined by 3-D surface rendering with a common threshold, optimized using histomorphometric techniques (GEMS MicroView). Single sagittal slices, bisecting the calcaneus, were also generated with common contrast settings to qualitatively assess erosion within the bones.
For time-course studies in CIA and AIA rats, two normal healthy control rats were killed at each time-point. These rats were age and sex matched to the arthritic groups, and none of the endpoints from these healthy rats showed any significant changes over the 32-day time-course. We therefore pooled data from all healthy control rats for each disease model, yielding a total of 28 healthy control rats per model. In OPG treatment studies, each experimental and control group consisted of eight age- and sex-matched animals. Statistical comparisons were made at the end of the OPG treatment period. All results were expressed as the group mean ± SE. Clinical (continuous) data were assessed using a paired t-test, whereas histopathologic scores (ordinal data) were analyzed using the χ2 test. A p value of 0.05 was used to delineate significant differences between groups.
Arthritis time-course studies
Disease onset in CIA and AIA models was defined as the first day of hind paw erythema and edema, as indicated by arrows on the x-axes of Figs. 1, 2, 3, and 4. Increases in hind paw volume (Figs. 1A and 1B) and protein content (Figs. 1C and 1D) were evident at disease onset (day 0) and increased rapidly thereafter. Paw volumes and protein content peaked between days 5 and 10 after disease onset and remained elevated in both models for the duration of the 27-day follow-up. Hind paw swelling and total protein was somewhat greater in the AIA model compared with the CIA model.
For both models, bone erosion in the hind paw was significantly enhanced by 2 days after disease onset and peaked at onset + 10 days (Figs. 2A and 2B). Osteoclast numbers were also significantly increased in hind paws of both models starting 2 days after onset (Figs. 2C and 2D). Erosions and increased osteoclast counts remained evident for the duration of follow-up.
Serum RANKL protein concentrations increased rapidly (within 2 days) in both CIA and AIA rats (Figs. 3A and 3B). In both models, serum RANKL remained significantly elevated for the duration of follow-up. RANKL protein was also significantly increased in joint extracts of rats with either CIA or AIA (Figs. 4A and 4B). These local increases in RANKL protein were apparent within 1–3 days of the onset of paw swelling, and RANKL protein remained elevated for 20–27 days. TRACP-5b, an osteoclast-specific marker of bone resorption, was increased locally (in joint extracts; Figs. 4C and 4D) but not systemically (in serum; Figs. 3C and 3D) in both CIA and AIA rats. These local increases in TRACP-5b occurred concurrent with or slightly after the observed increases in local RANKL concentrations.
In both CIA and AIA rats, RANKL mRNA transcripts were expressed in the bone marrow of the tarsal bones and distal tibia as well as in adjacent soft tissues starting at disease onset (Fig. 5). This expression was spatially and temporally coordinated with the influx of leukocytes into these regions, as determined by histopathology (Fig. 5 H&E inset,). RANKL mRNA levels increased over time in proportion to the expanding inflammatory infiltrate. The most intense RANKL expression was localized to the inflamed synovium in both models and was typically localized adjacent to bone erosions.
OPG treatment studies
RANKL is specifically inhibited by OPG, so we treated CIA and AIA rats with OPG to examine the potential contribution of RANKL to local and systemic bone loss. OPG therapy was initiated 4 days after initial disease onset (3 mg/kg, SC, on days 4, 7, 10, and 13), and rats were killed on day 14. Consistent with the time-course studies, serum RANKL was significantly elevated in both CIA and AIA rats when measured 14 days after disease onset (p < 0.05 versus normal controls; data not shown). The systemic antiresorptive effect of OPG was shown by a 90–95% reduction in serum TRACP-5b in both CIA and AIA rats (p < 0.001 versus PBS controls; Fig. 6).
The systemic arthritis-induced increase in RANKL and the antiresorptive effect of OPG were consistent with changes in BMD in both the hindpaws and lumbar vertebrae. In the hind paw (ankle), DXA analysis revealed that CIA and AIA rats had significantly lower BMD compared with normal controls (Figs. 7A and 7B). OPG treatment of CIA or AIA rats led to a significant increase in ankle BMD (p < 0.001 versus CIA or AIA rats), resulting in normalized BMD values. TRACP-5b protein concentrations were markedly elevated in protein extracts from both CIA and AIA rat hind paws (p < 0.05 compared with their normal controls; Figs. 7C and 7D). OPG treatment greatly reduced TRACP-5b concentrations in hind paws from both arthritis models (p < 0.05 versus arthritic controls).
DXA analysis of the lumbar vertebrae also showed significant reductions in BMD in AIA and in CIA animals compared with normal controls (p < 0.01; Figs. 8A and 9A). OPG treatment of AIA rats caused a significant increase in lumbar BMD (p < 0.05 versus arthritic controls). OPG treatment of CIA rats led to a nonsignificant increase in BMD, such that the reduction in BMD in these arthritic animals was no longer significantly different from normal controls. Histomorphometry of lumbar vertebrae revealed changes in cancellous bone volume (BV/TV) that were largely consistent with BMD changes. BV/TV was significantly reduced in the lumbar vertebrae of both CIA and AIA rats (p < 0.001 versus normal controls), and OPG treatment significantly increased BV/TV in both CIA and AIA rats to levels that were similar to normal controls (Figs. 8B and 9B). Loss of cancellous bone volume in both arthritis models was associated with significant increases in osteoclast numbers (p < 0.05 versus normal controls), suggesting that increased bone resorption contributed to systemic bone loss (Figs. 8C and 9C). Osteoblast numbers were not significantly different in CIA rats, whereas AIA rats had significantly reduced osteoblasts (p < 0.01 versus normal controls; Figs. 8D and 9D).
Representative μCT images of normal and arthritic rat paws are shown in Figs. 10 and 11. These images were generated 14 days after disease onset. In both disease models, arthritis presented as a dramatic deterioration of joint integrity associated with focal erosions, generalized osteopenia, and osteophyte formation. A 10-day course of therapy with recombinant OPG, starting 4 days after disease onset, largely preserved bone mass and joint integrity in both models.
RANKL has been implicated as a pathogenetic factor for bone loss in patients with rheumatoid(16, 25, 33, 34) or psoriatic(35) arthritis. In animal models of arthritis, RANKL plays a causal role in the initiation and progression of local bone erosions. The essential role of RANKL in bone erosions has been clearly established in animals with AIA, (7) CIA, (21) serum-transfer arthritis, (18) and arthritis associated with overexpression of TNF-α. (36) The current studies were performed to further our understanding of two important questions: is RANKL also involved in systemic bone loss in arthritis models, and is RANKL protein elevated early enough in disease progression to have potential prognostic value for local and/or systemic bone loss?
The results from two distinct preclinical models of inflammatory arthritis suggest that the answer to both questions is yes. Regarding the potential role of RANKL in systemic bone loss, the lumbar vertebrae of both CIA and AIA rats had significantly increased serum RANKL, significantly lower BMD, reduced cancellous bone volume, and increased osteoclast numbers compared with normal healthy control rats. The RANKL inhibitor OPG effectively reversed all of these changes, which suggests that RANKL played a role in bone loss despite the lack of an arthritis-related inflammatory response at this skeletal site. These results are consistent with the reported association between serum RANKL levels and vertebral bone loss in rheumatoid arthritis patients. (26) Regarding the potential use for RANKL as a biomarker of bone loss, serum RANKL levels were significantly increased in both CIA and AIA rats within 2 days after the first signs of disease onset (paw swelling). The increase in serum RANKL protein occurred before, or concurrent with, the induction of bone erosions, osteoclasts and TRACP-5b within arthritic paws. This early increase in serum RANKL could provide an indication for therapeutic intervention before significant bone loss has occurred. The benefits of early intervention were shown by the effective control of bone erosions in arthritic rats when OPG treatment was initiated 4 days after disease onset (2 days after the earliest increase in serum RANKL).
Generalized osteopenia and osteoporosis is common in arthritis patients, (22) but the etiology is not well understood. Systemic bone loss in arthritis is likely to be multifactorial, with contributing factors including immobility, glucocorticoid therapy, (23) and increased bone resorption. (37, 38) The role of bone formation in arthritic bone loss is complex, with some clinical studies of arthritis patients showing reduced bone formation markers(39) and others showing increases. (40–43) The suppression of bone formation may be an early and transient response to arthritis, with subsequent increases with later-stage disease. Whether suppressed bone formation contributes to systemic bone loss in preclinical arthritis models is also unclear. Systemic bone loss has been previously described in a murine model of CIA, but bone formation rates were not reduced in the femur of these mice. (44) Previous studies in AIA rats have shown increased bone resorption and decreased bone formation. (45) In our studies, histomorphometry data from the lumbar vertebrae were largely consistent with these prior observations. Osteoblast numbers were not significantly reduced in the vertebrae of CIA rats but were significantly reduced in AIA rats. It is therefore likely that reduced bone formation may have contributed to systemic bone loss in AIA rats. In both CIA and AIA rats, cancellous bone volume was reduced and osteoclast numbers were increased in the lumbar vertebrae. Inhibition of RANKL with OPG significantly reduced osteoclast numbers and fully restored bone volume, which suggests that RANKL probably contributed to systemic bone loss in both models.
Clinical and preclinical studies have shown that systemic bone loss is relatively well controlled with antiresorptive therapy, whereas focal bone erosions tend to be more resistant to therapy. For example, the bisphosphonate pamidronate improved systemic BMD in rheumatoid arthritis patients but without any reduction in radiographic measures of focal bone erosions. (46) Recent preclinical studies suggest that more potent antiresorptives may be required to address focal bone erosions. Zoledronic acid (ZOL) was recently reported to reduce bone erosions in arthritic CIA rats and in arthritic TNF-α-transgenic mice when therapy was started at the first signs of joint inflammation. (47, 48) In the CIA rat study, ZOL also increased paw swelling and raised arthritis scores. (48) In TNF-α transgenic mice, ZOL was administered at a high dose (100 μg/kg), 5 days per week for 6 weeks. Whereas this regimen was effective at reducing bone erosions, it is unclear whether such aggressive dosing would be feasible in patients because of the potential for renal toxicity with ZOL. (49, 50) We recently compared the effects of OPG versus ZOL on bone erosions and renal toxicity in the more aggressive AIA rat model. OPG reduced focal bone loss in AIA rats by 80–90%, without any effects on renal histopathology. ZOL was capable of achieving similar levels of bone protection, but only at doses that caused significant renal histopathology in 100% of animals. (51) These results suggest that potent antiresorptive agents are promising for controlling bone erosions if renal toxicity can be avoided.
These data are, to our knowledge, the first that describe the local and systemic kinetics of RANKL protein induction in a quantitative manner in arthritis models. The results suggest that serum RANKL might be a convenient marker of disease progression in rheumatoid arthritis patients. We also described systemic bone loss in both arthritis models that was reversed by the RANKL inhibitor OPG, even when treatment was initiated after disease onset. If the role of RANKL in either of these arthritis models is consistent with its role in human disease, it is possible that serum RANKL may become a useful marker to identify arthritic patients that are most likely to suffer from local and systemic bone loss. RANKL inhibitors are a promising new class of therapeutics with the potential to control systemic bone loss and local bone erosions in rheumatoid arthritis.
The authors thank Janet Buys, Yan Cheng, Chris De La Torre, Diane Duryea, Darlene Kratavil, Ruiyuan A Luo, Efrain Pacheco, and Li Zhu for technical assistance. Holly Zoog assisted with preparation of manuscript.
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