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Rheumatology Advance Access originally published online on March 11, 2008
Rheumatology 2008 47(5):605-608; doi:10.1093/rheumatology/ken082
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© The Author 2008. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Inhibition of osteoclasts does not prevent joint ankylosis in a mouse model of spondyloarthritis

R. J. U. Lories, I. Derese and F. P. Luyten

Laboratory for Skeletal Development and Joint Disorders, Division of Rheumatology, Department of Musculoskeletal Sciences, Katholieke Universiteit Leuven, Leuven, Belgium.

Correspondence to: R. Lories, Division of Rheumatology, University Hospitals Leuven, Herestraat 49, B-3000, Leuven, Belgium. E-mail: Rik.Lories{at}uz.kuleuven.be


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
Objectives. The relationship between inflammation, destruction and new tissue formation leading to ankylosis, determines the severity and prognosis of patients with SpA. Recent data in mice and men suggest that new cartilage and bone formation and subsequent ankylosis are uncoupled from chronic inflammation. These data challenge the hypothesis that inflammation and tissue damage trigger an excessive repair response in SpA. We tested whether inhibition of bone erosion by targeting osteoclasts, would prevent or influence joint ankylosis in a mouse model.

Methods. Male DBA/1 mice from different litters were caged together at the age of 8 weeks. Treatment with zoledronic acid (ZA) (100 ng/g) or placebo was started at the age of 10 weeks and administered every 2 weeks. Clinical incidence and severity of arthritis were evaluated twice a week until the age of 26 weeks. At this point, bone density measurements were performed, mice were sacrificed and severity of arthritis was evaluated by histology.

Results. Treatment with ZA did not affect incidence or clinical severity of arthritis in male DBA/1 mice. ZA treatment significantly increased bone mineral density and content as demonstrated by dual X-ray densitometry and peripheral quantitative CT. However, the treatment did not affect histomorphological appearance of arthritis or ankylosis.

Conclusions. These data suggest that bone erosion at the enthesis does not necessarily precede entheseal ankylosis. Therefore, these observations further support the concept that inflammation and new tissue formation in SpA are at least partially uncoupled events and may be different therapeutic targets.

KEY WORDS: Spondyloarthritis, AS, PsA, Ankylosis, Enthesis, Osteoclasts, Bisphosphonates


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 
The human spondyloarthritides (SpAs) are a group of distinct diagnostic entities that share clinical, pathological and genetic features. This disease concept includes AS, PsA, arthritis associated with IBD, reactive arthritis and an undifferentiated and juvenile variant [1, 2]. Entheseal involvement with adjacent bone marrow and synovial inflammation as well as new cartilage and bone formation eventually leading to spine or peripheral joint ankylosis are hallmarks of SpA [3, 4]. The current paradigm suggests that inflammation leads to local injury, thereby eliciting tissue destruction followed by disappearance of inflammation and a subsequent repair response leading to ankylosis [5].

However, emerging data are challenging this concept. The introduction of anti-TNF therapies has profoundly changed the management of SpA as both soluble receptor and antibody-based strategies have an unprecedented effect on signs and symptoms of the disease [6–8]. In contrast to what is seen in the treatment of RA, the limited data available have not provided convincing evidence that anti-TNF prevents or slows down the progression of ankylosis [5, 9–11]. In addition, treatment with soluble TNF receptor etanercept has no effect on ankylosis in a mouse model of enthesitis [12] and mechanistically TNF negatively influences in vitro chondrogenesis [12]. Therefore, inflammation and new tissue formation in SpA may be uncoupled processes.

Osteoclasts have a critical role in physiological and pathological bone remodelling [13]. Bisphosphonates are powerful drugs to inhibit these cells and are widely and successfully used for the treatment of osteoporosis [14]. Inhibition of osteoclasts can also prevent local and systemic bone loss in destructive arthritis [15]. Bisphosphonates, in particular pamidronate, have been used for the treatment of AS and appear to be effective in reducing symptoms and inflammation [16, 17]. The effect of bisphosphonates on structural progression of the disease has not been reported yet.

Here, we demonstrate that inhibition of osteoclasts with the bisphosphonate zoledronic acid (ZA) does not affect ankylosis originating from the entheses in mice. This study therefore provides further evidence that new tissue formation, inflammation and bone erosion in SpA are uncoupled events.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Acknowledgements
 References
 
Animal experiments
DBA/1 mice were from Janvier (Le Genest St Isle, France). All experiments were approved by the Ethics Committee for Animal Research (KU Leuven, Belgium). Male mice from different litters were mixed and caged in groups of six mice at the age of 8 weeks. From week 10 (day 70) to 24 (day 168) mice were treated every 2 weeks with ZA (Novartis, Basel, Switzerland) (100 ng/g) or phosphate buffered saline (PBS) by intraperitoneal injection (n = 9 animals per group). Mice were scored twice a week for clinical signs of arthritis as previously described [12]: 0 (no symptoms), 1 (redness and swelling in one toe), 2 (redness and swelling in more than one toe), 3 (toe stiffness) and 4 (deformity or ankle involvement). At week 26, animals were sacrificed and hind paw forefeet were processed for histology. Histomorphological severity was determined as described [12]: 0 (normal toe), 1 (acute inflammation including dactylitis), 2 (entheseal cell proliferation), 3 (cartilage formation), 4 (bone formation) and 5 (joint ankylosis). A cumulative score from all toes was calculated per mouse.

Bone density measurements
Total body (without head) bone density was determined using dual-energy X-ray absorptiometry (DEXA) (Piximus densitometer, Lunar, GE Healthcare, Diegem, Belgium). Trabecular and cortical bone mineral content and cortical thickness were assessed by peripheral quantitative CT (pQCT) (XCT Research M+, Norland Medical Systems, Coopersurgical, Trumbull, CT, USA) using software version 5.40. Slices of 0.2 mm thickness were scanned using a voxel size of 0.07 mm. Three scans 2.4–2.6 mm were taken from the distal end of the femur. An additional scan was taken 4 mm from the distal end of the femur to determine cortical bone parameters.

Statistical analysis
Data were compared using two-tailed independent group tests. When normal distribution could be assumed, Student's t-test was used. In all other cases, the non-parametric Mann–Whitney U-test was used. In all tests, a P-value <0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Acknowledgements
 References
 
ZA does not inhibit clinical arthritis
Preventive treatment with bi-weekly administration of ZA (100 ng/g) did not have an effect on clinical incidence (eight out of nine animals in the PBS control group vs seven out of nine animals in the ZA group) or severity of arthritis [severity score 1.67 ± 0.5 (mean ± S.E.) in the PBS control group vs 1.89 ± 0.8 in the ZA group (n = 9 per group)] in male DBA/1 mice (Fig. 1). Onset of arthritis and pattern of joints involved (mainly the 4th and 5th digits) were similar in both groups. Of note, similar dosages in the human TNF-transgenic mouse model of destructive arthritis prevented not only local and systemic bone loss but also had no effect on synovial inflammation [15]. In contrast, pamidronate treatment also reduced symptoms and decreased bone marrow oedema in patients with SpA [17].


Figure 1
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  		FIG. 1. ZA treatment does not affect clinical incidence (A) or severity (B) of spontaneous arthritis in male DBA/1 mice. Mice were treated with ZA (100 ng/g) or placebo from the age of 10 weeks onwards and scored twice per week (n = 9 per group; for clinical severity, data are shown as mean ± standard error).

 
ZA does not affect histomorphology of ankylosing enthesitis
As expected, intensive ZA treatment from week 10 to week 24 significantly increased both trabecular and cortical bone density in male DBA/1 mice at 26 weeks (Table 1). In contrast, ZA treatment did not influence histomorphological severity of ankylosing enthesitis as compared with controls [median score 2 (quartiles 0–6) in the PBS groups vs 3 (quartiles 0–6) in the ZA group, n = 9 per group] (Fig. 2A). Detailed analysis of different stages in the process of ankylosing enthesitis demonstrated a similar pattern in the ZA and the control group (Fig. 2B).


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  		TABLE 1. Bone density measurements in 26-week-old male DBA/1 mice

 

Figure 2
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  		FIG. 2. ZA treatment does not affect the histomorphology of spontaneous arthritis in male DBA/1 mice. (A) Histomorphological severity scores (n = 9 per group). (B) Percentage of affected mice showing either proliferative changes, new cartilage formation or new bone formation at the enthesis.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Acknowledgements
 References
 
ZA does not inhibit the onset and progression of arthritis in the spontaneous DBA/1 mouse model of SpA. This suggests that in this animal model bone erosion by osteoclasts is not required to trigger the process of entheseal new cartilage and bone formation that is leading to ankylosis. These data provide further support for the hypothesis that inflammation, joint destruction and joint remodelling are uncoupled and mostly independent processes in this disease model. The relationship between these is of critical importance to improve our understanding of the pathology of SpA and a number of important questions related to mechanisms of disease remain to be solved.

ZA does not appear to affect clinical signs of arthritis in different mouse models of both RA and SpA ([15] and data presented here). This is in contrast with clinical and radiological observations in SpA patients treated with another bisphosphonate, pamidronate [16, 17]. This suggests that different bisphosphonates may not have the same effect in a clinical setting. Also, in this study, we focused on bone destruction and ankylosis. Both inflammation and tissue remodelling contribute to the clinical appearance of arthritis in this specific model, but histomorphological analysis suggests that the former may be short lived or even a secondary phenomenon. From this perspective, other models and further patient data appear necessary to analyse the effect of ZA on inflammation in SpA.

New tissue formation, in particular bone formation in the presence of chronic inflammation, is a rare event and SpA appears to be quite unique from this perspective. This suggests the existence of a permissive environment that would stimulate rather than inhibit new tissue formation. Despite intensive research in the field, few molecular differences between SpA and RA, which typically presents with the opposite phenotype, have been convincingly demonstrated. The work of Baeten et al. [18] suggested a number of distinguishing features in the synovium of the knee between both patient groups. Recent data on Dickkopf-1 (DKK1), a WNT antagonist, suggest a role for canonical WNT signalling in phenotypical differences between RA and SpA [19]. DKK1 appears to suppress tissue repair responses in the human TNF transgenic mouse model. In addition, serum levels of DKK1 are low in patients with AS, whereas they are high in patients suffering from RA. DKK1 expression is induced by TNF. Inhibition of DKK1 not only stimulates new bone formation by osteoblasts but also inhibits osteoclasts.

TNF also inhibits in vitro chondrogenesis from periosteal cells by TGF-β superfamily members [12]. In contrast, this critical cytokine up-regulates different bone morphogenetic proteins [20], which are likely to play a role in entheseal ankylosis [18, 21]. This suggests that local balances between cytokines and growth factors and the presence or absence of distinct progenitor cell populations in the tissue may be critical factors that determine within the context of a specific microenvironment whether erosion or remodelling ultimately dominates the outcome of the disease. For instance, DKK1 has been demonstrated at the sites of joint erosion in different mouse models [19, 22], whereas BMPs have been demonstrated in enthesophytes [21]. Of interest, recent data in human SpA suggest that erosion and new tissue formation occur at anatomically different sites in the enthesis organ [23].

Ankylosis in SpA is often considered an exaggerated repair response secondary to inflammation and damage [5]. Our observations in the DBA/1 model in this and in a previous study demonstrating that etanercept treatment does not affect ankylosis [12], are not in line with this hypothesis and provide support for an alternative paradigm in which common triggers lead to both the development of chronic inflammation and to joint remodelling and ankylosis. The nature of such triggers is unknown at this point but increasing evidence suggests that they may not be limited to activators of the innate and adaptive immune system, but that other triggers such as biomechanical factors may also play a role [4]. However, biomechanical signals do not explain the relationship between SpA and gut inflammation, SpA and bacteria such as Klebsiella and obviously the strong association with HLA-B27 [2]. Nevertheless, it is not excluded that some of these factors rather play a role in the chronicity of inflammation. From this point of view, early changes at the enthesis may be a lot more common than generally thought and may only lead to a chronic disease in a number of genetically predisposed individuals in which a chronic inflammatory reaction fully develops. In others, entheseal stress may pass clinically unnoticed and not lead to chronic disease.

In summary, this study in a mouse model of ankylosing enthesitis and SpA does not support the hypothesis that bone erosion by osteoclasts necessarily precedes joint remodelling and ankylosis, but rather provides further evidence that new tissue formation can occur independently from inflammation and injury.

Formula


   Acknowledgements
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
References
 
The authors would like to thank Mrs Jenny Peeters for technical assistance with the DEXA and pQCT measurements.

Funding: This work was supported by grant G.0213.07 from the Scientific Research Foundation Flanders (FWO Vlaanderen) and a GOA grant from the KU Leuven. R.J.U.L. is the recipient of a post-doctoral fellowship from FWO Vlaanderen.

Disclosure statement: R.J.U.L. has filed a patent on the use of BMP inhibitors in the treatment of spondyloarthritis. R.J.U.L. has received speaker and consultancy fees from Wyeth, Merck Sharp & Dohme, Centocor and Schering-Plough. F.P.L. has filed a patent on the use of BMP inhibitors in the treatment of spondyloarthritis. The other author has declared no conflicts of interest.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Acknowledgements
 References
 

  1. Wright V. Seronegative polyarthritis: a unified concept. Arthritis Rheum (1978) 21:619–33.[Web of Science][Medline]
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Submitted 14 December 2007; revised version accepted 1 February 2008.
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This Article
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