Tumor Necrosis Factor � and RANKL Blockade Cannot HaltBony Spur Formation in Experimental Inflammatory Arthritis
Georg Schett,1 Marina Stolina,2 Denise Dwyer,2 Debra Zack,2 Stefan Uderhardt,1
Gerhard Kronke,1 Paul Kostenuik,2 and Ulrich Feige2
Objective. To investigate the kinetics of bony spurformation and the relationship of bony spur formationto synovial inflammation and bone erosion in 2 ratarthritis models, and to address whether bony spurformation depends on the expression of tumor necrosisfactor � (TNF�) or RANKL.
Methods. Analysis of the kinetics of synovialinflammation, bone erosion, osteoclast formation, andgrowth of bony spurs was performed in rat collagen-induced arthritis (CIA) and adjuvant-induced arthritis(AIA). In addition, inhibition experiments were per-formed to assess whether inhibition of TNF� andRANKL by pegylated soluble TNF receptor type I(pegTNFRI) and osteoprotegerin (OPG), respectively,affected bony spur formation.
Results. Bony spurs emerged from the periostealsurface close to joints, and initial proliferation of mes-enchymal cells was noted as early as 3 days and 5 daysafter onset of CIA and AIA, respectively. Initiation ofbony spur formation occurred shortly after the onset ofinflammation and bone erosion. Neither pegTNFRI nor
OPG could significantly halt the osteophytic responsesin CIA and AIA.
Conclusion. These results suggest that bony spurformation is triggered by inflammation and initialstructural damage in these rat models of inflammatoryarthritis. Moreover, emergence of bony spurs dependson periosteal proliferation and is not affected by inhi-bition of either TNF� or RANKL. Bony spur formationcan thus be considered a process that occurs indepen-dent of TNF� and RANKL and is triggered by destruc-tive arthritis.
Arthritis is characterized by a massive influx ofimmune cells into the synovial membrane and its neigh-boring structures such as the tendons, ligaments, and thejoint cavity (1). Chronic joint inflammation leads toprofound changes in the joint architecture, which is thestructural basis for a progressive impairment of function(2). In rheumatoid arthritis (RA), destruction of periar-ticular bone and the articular cartilage is the dominantfeature of structural damage and is radiographicallyreflected by bone erosion and joint space narrowing (3).Conversely, inflammatory joint destruction is sometimesaccompanied by the modeling of bony spurs, also termedosteophytes, which emerge at the joint margins in dis-eases such as psoriatic arthritis (PsA) and ankylosingspondylitis (AS) (4). The reason for the apparentlydivergent bone responses among the various inflamma-tory diseases has not been fully clarified, but appears toinvolve a differential regulation of local bone homeosta-sis in the course of joint inflammation.
Considerable insight into the pathologic mecha-nisms underlying catabolic patterns of joint damage hasbeen obtained in the past few years. Particularly, oste-oclasts have been recognized as the primary bone-resorbing cell in inflamed joints. Cytokines such astumor necrosis factor � (TNF�) and RANKL, both ofwhich are key enhancers of osteoclastogenesis, have
Dr. Schett’s work was supported by the Sonderforschungsbe-reich (SBF) 643, the Interdisziplinares Zentrum fur Klinische For-schung Erlangen, and the Forschergruppe 661 of the Deutsche For-schungsgemeinschaft (DFG), the Austrian Ministry of Sciences(START Program award), the SPIRAL consortium, and the EUprojects Masterswitch, Kinacept, and Adipoa.
1Georg Schett, MD, Stefan Uderhardt, MD, Gerhard Kronke,MD: University of Erlangen–Nuremberg, Erlangen, Germany; 2Ma-rina Stolina, PhD, Denise Dwyer, BS, Debra Zack, MD, PhD, PaulKostenuik, PhD, Ulrich Feige, PhD (current address: EUROCBIGmbH, Benglen, Switzerland): Amgen Inc., Thousand Oaks, Califor-nia.
Drs. Stolina, Zack, Kostenuik, Feige, and Ms Dwyer ownstock or stock options in Amgen Inc.
Address correspondence and reprint requests to GeorgSchett, MD, Department of Internal Medicine III and Institute forClinical Immunology, University of Erlangen–Nuremberg, Kranken-hausstrasse 12, D-91054 Erlangen, Germany. E-mail: [email protected].
Submitted for publication October 15, 2008; accepted inrevised form May 26, 2009.
2644
been recognized to play a central role in animal modelsof arthritis and human RA (5–7). Up-regulation ofRANKL has also been described in the inflamed jointsof patients with osteoarthritis (OA) (8) and those withspondylarthritis (9). The catabolic effects of RANKL areblocked by osteoprotegerin (OPG), a soluble decoyreceptor that prevents osteoclast formation, activation,and survival (10). The relative balance between RANKLand OPG is thought to be an important determinant ofbone resorption (11), and reports of reduced OPG levelsin the inflamed joints of RA patients provide furtherevidence that the RANKL/OPG axis regulates bonedestruction in this condition (12).
Based on these insights, local formation of oste-oclasts and their resorption of bone are considered to bethe primary mechanism for the catabolic pattern ofinflammatory joint damage that is typically seen inarthritis (13). In the context of joint inflammation,TNF� has been implicated in the up-regulation of genesrelated to matrix degradation in chondrocytes (14), andTNF� is present at elevated levels in serum and synovialfluid from RA patients (15,16). The ability of TNF�inhibition to control inflammation in patients with ar-thritis is well established (17).
Much less is known about bony spur formation,which is a prominent feature of inflammatory jointdiseases such as AS and PsA and has been recognized indegenerative joint diseases such as OA or hemochroma-tosis arthropathy. Bony spurs represent spots of newbone formation that emerge from periosteal sites closeto joints (where they are called osteophytes) or interver-tebral spaces (where they are called spondylophytes orsyndesmophytes depending on their pattern of growth)(18). Bony spur formation is considered a process ofendochondral bone formation, requiring the differenti-ation of mesenchymal cells into hypertrophic chondro-cytes and, finally, into osteoblasts, which are cells thatproduce extensive matrix for building up new bone.Although bony spurs can be considered a response-to-stress reaction of the joint, they may also contribute tothe disease burden itself when they lead to fusion of theentire joint and loss of motion (19).
Bony spur formation appears to depend on mol-ecules involved in bone formation, such as transforminggrowth factor � (TGF�), bone morphogenetic proteins,and the Wnt protein family (20–22). Although therelative role of these pathways in bony spur formationand the effects of their mutual interaction are poorlydefined, it is evident that these essential bone-formingmolecular pathways are turned on when joints becomeinflamed or are subjected to mechanical stress. Direct
therapeutic intervention in these pathways is not atreatment strategy that is currently applied, and may alsohave drawbacks because these pathways elicit importantantiinflammatory functions and are required for physi-ologic bone formation and maintenance of bone mass.
More attention, however, is currently beingplaced on the interaction between inflammation, bonemetabolism, and bony spur formation and their rele-vance to current treatment strategies. This is now thefocus for several reasons. 1) Bony spurs are the basis forstructural outcome parameters such as those measuredin AS, and therefore therapeutic modification leading toimprovement is of clinical interest (23,24). 2) TNF�inhibition is of key importance in controlling chronicarthritis, but its effect on bony spur formation is poorlydefined. Recent data in fact suggest that the blockade ofTNF� may not have a major effect on inhibiting theformation of syndesmophytes in the vertebral column ofpatients with AS (25). 3) Bony spur formation is linkedto enhanced bone metabolism, with increased boneformation and bone resorption to shape the newlycreated bone (20–22), and therefore interventions inbone metabolism, such as osteoclast inhibition, mayaffect the formation of bony spurs and also modifystructural joint damage.
Most of the animal models of inflammatoryarthritis display bony spur formation. Adjuvant-inducedarthritis (AIA), collagen-induced arthritis (CIA), andalso the K/BxN serum transfer model of arthritis are allcharacterized by formation of bony spurs along thejoints. The kinetics of bony spur formation and therelationship of bony spur formation to inflammation andbone resorption, however, are poorly understood. More-over, it is not known whether inhibition of inflammationand/or inhibition of bone resorption in these modelscould affect bony spur formation. The introduction ofTNF� blockade was not effective in blocking bony spurformation in the male DBA/1 mouse arthritis model,which is an experimental model characterized by mini-mal inflammation and extensive bone growth, suggestingthat these mechanisms may be uncoupled (26).
We were therefore interested in studying whethertherapeutic interventions to block inflammation or boneresorption would be effective in modifying the formationof bony spurs in 2 standard models of inflammatoryarthritis, AIA and CIA. To accomplish this, we firstdefined the kinetics of bony spur formation in AIA andCIA. In addition, we investigated whether inhibition ofTNF�, as a strategy to decrease inflammation, or inhi-bition of RANKL, as a strategy to inhibit bone resorp-
TNF� AND RANKL BLOCKADE IN EXPERIMENTAL INFLAMMATORY ARTHRITIS 2645
tion, could affect the formation of bony spurs in these 2forms of inflammatory arthritis.
MATERIALS AND METHODS
Animals and induction of arthritis. Young adult Lewisrats (54 males and 54 females), weighing 80–100 grams, werepurchased from Charles River (Wilmington, MA). Animalswere acclimatized for 1 week under normal environmentalconditions and fed a pelleted rodent chow (no. 8640; HarlanTeklad, Madison, WI) with tap water ad libitum. Initially, atotal of 60 rats was assigned to a time-course experiment. AIAwas induced in male rats (n � 30) by a single intradermalinjection of 0.5 mg heat-killed mycobacteria H37Ra (Difco,Detroit, MI), suspended in paraffin oil, into the tail base. CIAwas induced in female rats (n � 30) by multiple intradermalinjections with a total of 1 mg porcine type II collagen(Chondrex, Redmond, WA), emulsified 1:1 in Freund’s incom-plete adjuvant (Difco), into the skin of the back. Rats subjectedto these inductions for AIA or CIA were killed at disease onset(day 0) or on days 1, 2, 3, 4, 5, 10, 14, 20, or 27 after diseaseonset.
In addition, AIA (n � 24) or CIA (n � 24) was inducedin another group of 48 rats, and these animals were randomlyassigned to 1 of the following 3 treatment groups (n �8/group): pegylated soluble TNF receptor type I (pegTNFRI,or pegsunercept; 4 mg/kg/day by daily subcutaneous [SC]bolus), OPG (consisting of the RANKL-binding portion ofnative OPG fused with the constant [Fc] domain of IgG; 3mg/kg/day given every other day by SC bolus), or vehiclecontrol. Treatments were started 4 days after the onset ofclinical disease and continued for 10 days. Moreover, in a totalnumber of 24 mice, we performed a preventive treatment with3 different doses of OPG (0.1, 1, and 10 mg/kg; each n �8/group), in comparison with a vehicle control group (n �8/group). These treatments were started at the onset of clinicaldisease (day 0) and continued for 10 days.
This study was conducted in accordance with federalanimal care guidelines. Approval for the study was provided bythe Amgen Institutional Animal Care and Use Committee.
Assessment of paw swelling. Swelling of the hind pawswas assessed daily from disease onset to day 20 after diseaseonset. In AIA, paw swelling was measured by water plethys-mography, as previously described (27). In CIA, paw swellingwas quantified using calipers (Fowler Sylvac Ultra-Cal MarkIII; Sylvac, Crissier, Switzerland) to measure the ankle diam-eter of the hind paws.
Conventional histology and detection of osteoclasts. Atnecropsy, the right hind paws of rats with AIA or CIA wereremoved at the fur line just proximal to the tibiotarsal (hock)joint. The paws were then fixed in zinc formalin for 2 days,decalcified with a 1:4 mixture of 8N formic acid and 1N sodiumformate, and then divided longitudinally along the median axis,processed into paraffin, and cut serially at 4 �m. One sectionwas stained with hematoxylin and eosin (H&E) to allow forconventional histopathologic assessment. The other sectionwas studied immunohistochemically to visualize osteoclasts,using an indirect immunoperoxidase procedure for the detec-tion of cathepsin K, an osteoclast-specific protease.
Immunohistochemistry was performed on an auto-mated tissue stainer (Model Mark 5; DPC, Flanders, NJ)
according to a standard method (28). Briefly, sections werepretreated with 0.1% trypsin in 1% CaCl2 (Sigma, St. Louis,MO) for 15 minutes, blocked with CAS Block (Zymed Labo-ratories, San Francisco, CA) for 10 minutes, and incubatedwith a proprietary rabbit polyclonal anti–cathepsin K antibody(1 �g/ml; Amgen, Thousand Oaks, CA) for 60 minutes. Theprimary antibody was localized using sequential 30-minuteincubations with biotin-conjugated goat anti-rabbit polyclonalsecondary antibody (Vector, Burlingame, CA) (used at 1:200),peroxidase-blocking solution (Dako, Carpinteria, CA) for 25minutes, and avidin–biotin complex and peroxidase reagents(ABC Elite Kit; Vector). The reaction was visualized usingdiaminobenzidine (DAB�Substrate Chromagen System;Dako) for 3 minutes. The osteophytic proliferative responsewas assessed using an antibody against the proliferation anti-gen Ki-67 (Novocastra, Newcastle-upon-Tyne, UK) accordingto the protocol described above. Immunostaining for type Xcollagen was done by incubating sections with a specificantibody against type X collagen (29) (kindly provided byKlaus Von der Mark, Erlangen, Germany) overnight at 4°C.The sections were then incubated with alkaline phosphatase–streptavidin for 30 minutes at room temperature before detec-tion with fast red Texas Red/naphthol solution (Sigma), result-ing in red staining.
Semiquantitative lesion scoring. Synovial inflamma-tion and bone erosion were assessed in H&E-stained sectionsusing a semiquantitative scoring system as previously described(30). Inflammation was scored according to the followingcriteria: 0 � normal, 1 � presence of a few inflammatory cellsin perisynovial tissue, 2 � mild inflammation, with a few smallfocal aggregates and modest buildup in perisynovial tissue, 3 �moderate inflammation, with many small aggregates and ex-tensive buildup in perisynovial tissue, 4 � marked inflamma-tion, with large aggregates and extensive buildup in perisyno-vial tissue. Bone erosion in AIA was scored as follows: 0 �normal, 1 � minimal, with a few erosion sites in tarsal bones,2 � mild, with a modest number of erosion sites in tarsalbones, 3 � moderate, with many erosion sites in tarsal bones,4 � marked, with partial destruction of the tibia and extensivedestruction of tarsal bones, 5 � extensive, with fragmentationof tarsal bones and full-thickness cortical penetration of thetibia. Bone erosion in CIA was quantified as follows: 0 �normal, 1 � minimal, with 1–2 small, shallow erosion sites, 2 �mild, with 1–4 erosion sites of medium size and depth, 3 �moderate, with �5 erosion sites partially extending throughthe cortical bone, 4 � marked, with multiple foci partially orcompletely extending through the cortical bone, 5 � extensive,with cortical penetration at �25% of the bone length. Analysisincluded the tibiotarsal articulation and all intertarsal joints.Osteoclasts in AIA and CIA were quantified according to thefollowing scores: 0 � normal (no osteoclasts), 1 � presence ofa few osteoclasts (lining fewer than 5% of most affected bonesurfaces), 2 � some osteoclasts (lining 5–25% of most affectedbone surfaces), 3 � many osteoclasts (lining 30–50% of mostaffected bone surfaces), 4 � abundant osteoclasts (lining�50% of most affected bone surfaces).
Histomorphometric analysis of bony spurs. In addi-tion, the size of the entire bony spur of the navicular bone aswell as its bony fraction was analyzed quantitatively by histo-morphometry. Previous studies have validated the navicularbone as a sensitive indicator for the extent of arthritic changes
2646 SCHETT ET AL
in experimental arthritis in rats (28,31). Bony spurs are mostprominent at the navicular bone as well. In addition, the areaof the periosteal surface of the navicular bone covered by thebony spur and the numbers of cathepsin K–labeled osteoclasts(multinucleated cells attached to bone) were analyzed quanti-tatively by histomorphometry. All parameters were analyzedusing commercial image-analysis software (OsteoMeasure pro-gram 2.2; Osteometrics, Atlanta, GA) as previously described(32).
Radiographs. The left hind paws were placed in posi-tion on Kodac X-OMAT TL high-resolution specimen-imagingfilm (Eastman Kodak, Rochester, NY) and radiographed witha Faxitron X-ray system (Model 43855A; Faxitron X-ray,Buffalo Grove, IL). Images were shot at 26 kV for 10 seconds.
Statistical analysis. All results are expressed as themean � SEM. Groups were compared by nonparametricKruskal-Wallis test using GraphPad Prism software (version 4;GraphPad Software, San Diego, CA). P values less than orequal to 0.05 were used to delineate significant differencesbetween groups.
RESULTS
Emergence of bony spur formation followingsynovial inflammation and bone destruction. To assessthe kinetics of bony spur formation in arthritis, we first
performed a sequential analysis of the development ofbony spurs in the AIA model, as well as in CIA. In eachrat model of arthritis, massive synovial infiltration in thehind paws occurred in conjunction with the onset ofclinical symptoms of arthritis (day 0) (results for AIA areshown in Figures 1A–D, while those for CIA are avail-able from the corresponding author upon request).Onset of periosteal proliferation was observed as earlyas 5 days after the onset of disease in the AIA model(Figure 1A) and as early as 3 days after the onset ofdisease in the CIA model (results not shown). Moreover,initial formation of small bone erosions could be de-tected before the bony spurs had emerged (Figure 1B),suggesting that spur formation in AIA and CIA maydepend on an initial resorptive phase of arthritis.
Increased osteoclast counts also preceded theappearance of bony spurs (Figure 1C), suggesting thatan initial resorptive stimulus paves the way for growth ofbony spurs. In the late stages of disease, when growth ofbony spurs was most pronounced (Figure 1D), osteoclastcounts gradually decreased, suggesting a switch of bone
Figure 1. Relationship of bony spur formation to inflammation and bone erosions in rat adjuvant-inducedarthritis (AIA). A, Sequential assessment of hind paw inflammation was performed by measurement of pawdiameter in rats with AIA from day 0 to day 27 after disease onset. B and C, Semiquantitative sequentialassessment of bone erosion (B) and osteoclast counts (C) was performed in the same mice. D, Quantitativemeasurement of bony spur size was carried out by histomorphometry. Bars show the mean and SEM. � � P �0.05 versus day 0. Results for rats with collagen-induced arthritis are available from the corresponding authorupon request.
TNF� AND RANKL BLOCKADE IN EXPERIMENTAL INFLAMMATORY ARTHRITIS 2647
metabolism to more bone formation and less resorption.Interestingly, the pattern was almost identical betweenAIA (Figure 1) and CIA (results not shown).
Microarchitecture of growth of bony spurs in AIAand CIA. Initial lesions in AIA and CIA were charac-terized by a proliferation of mesenchymal cells at peri-
Figure 3. Bone formation and osteoclast accumulation in bony spurs in rat AIA and CIA. A and B, Newly formed bonewithin bony spurs (A) and osteoclasts within bony spurs (B) were assessed quantitatively in rat AIA and CIA byhistomorphometry. Bars show the mean and SEM. C, Photomicrographs of bony spurs show results of hematoxylin andeosin staining of hind paws in AIA and CIA (first and third panels, respectively) and staining with an antibody againstcathepsin K in AIA and CIA (second and fourth panels, respectively). Osteoclasts are indicated in brown within the bony spur(broken arrows; the margin of the lesion is indicated with solid arrows) (original magnification � 10). S � synovium; C �cartilage-like tissue; B � bone; P � periosteum (see Figure 2 for other definitions).
Figure 2. Sequence of bony spur formation in rat antigen-induced arthritis (AIA) (top) and collagen-induced arthritis (CIA)(bottom). Photomicrographs of hematoxylin and eosin–stained sections of hind paws of rats with AIA or CIA show the periosteum ofthe navicular bone (original magnification � 5 in top; � 10 in bottom). Sections were obtained 5, 10, 20, and 27 days after diseaseonset. Arrows indicate the proliferation front. Note that bony spurs in AIA are much larger than in CIA.
2648 SCHETT ET AL
osteal sites in the vicinity of the joint space (Figure 2).Bony spurs showed a rapid and consistent growth in bothmodels over time, peaking in their size at the finalobservation time point, 27 days after the onset ofarthritis. The size of the lesions was much more pro-nounced in AIA as compared with CIA, but microarchi-tectural changes were very similar between the 2 models.The surface of the lesions contained dense accumula-tions of mesenchymal cells that showed high prolifera-tive activity, with almost all of the cells in both modelspositively staining for the proliferation marker Ki-67(results available from the corresponding author uponrequest). Mesenchymal cells are known to form adensely packed multilayer comprising the outer surfaceof the bony spur, which determines the growth of thebony spur. Underneath this dense and proliferatingmesenchymal lining layer of the spur, we observedhypertrophic chondrocytes producing an extensiveamount of matrix and expressing type X collagen (re-sults available from the corresponding author uponrequest). Finally, the inner regions of the spur, whichwere closest to the former periosteal surface, appearedto be remodeled into bone.
The periosteal surface of the navicular bone is anarea that reproducibly shows marked growth of bonyspurs. Assessment of the periosteal surface revealed thatthis process affected a major part of the navicularsurface early on and affected virtually the entire navic-ular bone at later stages (results not shown). In fact, theperiosteum appears to be essential to allow the bonygrowth in these 2 inflammatory arthritis models. Disrup-tion of the integrity of the periosteum and cortical bonecompletely prevented the proliferative response. Thiswas evident at sites where osteoclasts had penetratedcortical bone (results available from the correspondingauthor upon request). At these sites, no proliferativeresponse was found, whereas the neighboring sites withintact periosteal bone interphase showed a massiveosteophytic proliferation. This suggests that bony spursrequire an intact periosteum covering cortical bone fortheir formation.
Simultaneous occurrence of bone deposition andosteoclast influx into bony spurs. We next studiedwhether the formation of new bone within the bony spuris linked to the emergence of osteoclasts, which aregenerally required for the remodeling of mineralized
Figure 4. Effects of tumor necrosis factor � (TNF�) and RANKL blockade on bony spur formation in ratadjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA). Hind paws of rats were treated withvehicle (blue line), pegylated soluble tumor necrosis factor receptor type I (PEG sTNFRI) (green line), orosteoprotegerin (OPG) (orange line). A, Clinical assessment of joint swelling in AIA and CIA. Arrow indicatesinitiation of treatment in each group. B, Quantitative assessment of bony spur formation in AIA and CIA byhistomorphometry. Bars show the mean and SEM.
TNF� AND RANKL BLOCKADE IN EXPERIMENTAL INFLAMMATORY ARTHRITIS 2649
tissue. Assessment of bone growth in bony spurs in AIAand CIA showed a kinetic pattern that was similar to thesize of the expansion of the entire lesion (Figures3A–C). The bony part of the spur, however, was consis-tently smaller than the entire lesion, which indicated aconsistent growth of the lesions as well as consistentremodeling of fibrous and cartilage-like tissue into bone.Small deposits of bone were found in lesions even in theearly phase of disease (day 5). Emergence of bone withinthe osteophytic lesion triggered the appearance of oste-oclasts in the lesions. The marked increase in bone sizewithin bony spurs from day 5 to day 14 was then ac-companied by a dramatic accumulation of osteoclasts inthese lesions, with levels peaking on day 20 in AIA andday 14 in CIA, before gradually decreasing thereafter.
Lack of effect of TNF� and RANKL inhibition ongrowth of bony spurs. To address whether growth ofbony spurs depends on inflammation or osteoclast gen-eration, we used a potent antiinflammatory approach(TNF� inhibition) as well as an effective method toblock osteoclasts (RANKL inhibition) in the AIA andCIA models. Inhibitory treatments in both AIA and CIAwere started in the early phase of arthritis (day 3), when
initial bone erosions had started to form. Blockade ofTNF� using pegTNFRI significantly reduced inflamma-tion but did not affect the formation of bony spurs(Figures 4A and B), suggesting that inhibition of inflam-mation does not inhibit the periosteal bone response.This finding was identical in both AIA and CIA.
OPG treatment of rats with AIA or rats with CIAresulted in marked (�95%) reductions in osteoclastnumbers in the hind paws (results not shown), as de-scribed in a previous report related to this same study(30). This level of reduction of osteoclasts was notassociated with changes in the size of the bony spurs, asevident on radiographs, in either AIA or CIA (Figures5A and B). Moreover, preventive treatment with OPGwas not effective in blocking bony spur formation.
Bony overgrowth was found after treatment withall 3 doses of OPG, ranging from 0.1 mg/kg to 10 mg/kg,and was as pronounced as that in vehicle-treated mice(Figures 6A–D). Even at higher doses, when OPGcompletely blocked the formation of osteoclasts andbone resorption, fully formed bony spurs were observed,suggesting that the presence of osteoclasts is not essen-tial for the formation of bony spurs.
Figure 5. Radiographic evidence of bony spurs after TNF� and RANKL blockade. The hind paws of rats with AIA (A) andrats with CIA (B) were treated with vehicle, OPG, or pegylated TNFRI, and radiographs were obtained at the end of the study.Representative results are shown. See Figure 4 for definitions.
2650 SCHETT ET AL
DISCUSSION
Bony spurs are a frequently observed pathologicfeature of both degenerative and inflammatory jointdiseases and usually grow at the edges of the synovialjoint or at insertion sites of tendons (entheses). Currentconcepts suggest that bony spur formation may repre-sent a response-to-stress mechanism of the joints (4,19).Both mechanical and inflammatory triggers can lead toformation of bony spurs, and mechanical triggers seemto be particularly important in the formation of bonyspurs along the entheses (18,33). Bone turnover in bonyspurs is enhanced and reflects the type of spur typicallyseen in subchondral bone, where the rate of boneturnover exceeds the levels observed in epiphyseal andmetaphyseal cancellous bone compartments (34). It is asyet unclear why bony spur formation is particularlyprominent in certain forms of human joint disease suchas AS and PsA, and why it is virtually absent in otherforms of joint disease such as RA. Novel concepts,however, indicate molecular differences related to theexpression of proteins involved in osteoblast differenti-ation as an underlying principle. The relationship of
inflammation, bone erosion, and bony spur formationhas not been completely elucidated.
For understanding the process of bony spur for-mation, it is of particular interest to characterizewhether an initial erosive phase is necessary to promotethe growth of these bony spurs. Herein we show thatbony spur formation clearly follows inflammation, initialosteoclast formation, and bone erosion in 2 typicalmodels of inflammatory arthritis, suggesting that bonyspur formation can indeed be regarded as a response ofbone to joint inflammation. The induction of bony spursis preceded by an erosive stimulus, which shares similar-ities with fracture healing. Both processes are initiated inresponse to bone damage and involve the production ofnew bone through endochondral ossification (35). Infracture healing, new bone formation occurs on perios-teal surfaces that are adjacent to, but not directly within,the damaged bone areas, and this formation typicallyresults in bridging of the fracture site (36). We observedthat bony spurs formed only above intact periosteum,which seems to be analogous to the periosteal responseobserved in fracture healing. The periosteal surface is
Figure 6. Preventive RANKL blockade does not affect bony spur formation in AIA. Rats with AIA were treated withvehicle or different doses of OPG starting at the onset of disease. Hind paws were scored for A, inflammation (synovitis),B, bone erosion, C, osteoclasts, and D, histomorphometrically assessed area of bony spur formation. Bars show the meanand SEM. � � P � 0.05 versus vehicle. See Figure 4 for definitions.
TNF� AND RANKL BLOCKADE IN EXPERIMENTAL INFLAMMATORY ARTHRITIS 2651
considered to be one of the most active sites of bonemodeling (37), and bony spur formation is a stepwiseprocess originating from the periosteum.
Another similarity between bony spur formationand fracture repair is that the bone formation responsesin these models were not inhibited by OPG (38). It istherefore reasonable to suggest that osteoclasts are notessential for these bone formation responses to occur,even though osteoclasts are prominent histologic fea-tures in both models. Osteoclasts are clearly importantfor the remodeling of fracture calluses, since osteoclastinhibition typically results in a larger fracture callus(39,40). The lack of effect of osteoclast inhibition onbony spur formation that was observed in the presentstudy suggests that these lesions do not remodel, whichcould explain their persistent nature, in contrast to thetransient nature of fracture calluses.
Animal models of arthritis vary in their potentialto form bony spurs, as do human inflammatory jointdiseases. Whereas TNF�-transgenic mice do not formbony spurs and the histologic pattern is purely erosiveunless bony spur formation is stimulated by pharmaco-logic interventions, other arthritides in rodents, such asthat in the male DBA/1 mouse model, are dominated byosteoproliferation and formation of bony spurs is thehallmark of the disease, with little inflammation (41,42).In fact, Lories and colleagues have demonstrated thatosteoproliferation in male DBA/1 mice cannot beblocked by inhibition of TNF�, indicating that, after theinitial inflammatory trigger, bone formation might pro-ceed uncoupled from inflammation (42). This wouldindeed support clinical data obtained in AS, showingthat the formation of syndesmophytes (bone spurs alongthe vertebral column) are not affected by TNF�-blocking therapy.
Interestingly, standard models of inflammatoryarthritis, such as AIA, CIA, or the serum transfer modelof arthritis, also display formation of bony spurs, andthus these models do not exactly mimic the diseaseprocesses of RA. In fact, bony spur formation has notbeen rigorously studied in these models, because it hasnot been the focus of attention for therapeutic interven-tions targeting joint inflammation and structural damagesuch as bone erosion and cartilage degradation.Scharstuhl and colleagues performed an elegant analysisof bony spurs in murine CIA, and their results showedthat TGF� is an important mediator of growth of theselesions, suggesting that the release of growth factorsfrom mesenchymal cells is indeed a key prerequisite forbony spur formation in CIA (21). Moreover, the inhib-itory effect of nonsteroidal antiinflammatory drugs on
new bone formation, by blocking the synthesis of pros-taglandin E2, has long been known (43).
Although it has been proven clinically effective,TNF� inhibition in the present study was not able toblock bony spur formation in either AIA or CIA. Thisreinforces the current concept that bone formation iscrucial for the development of bony spurs and thatTNF� inhibition is unable to halt the process. TNF�itself is a potent down-regulator of bone formation, andits removal might be expected to increase bone forma-tion (44,45). However, TNF� blockade also did notincrease bony spur formation. The clinical consequencesof these observations are obvious, in that TNF� inhibi-tion is not expected to change bony spur formation indiseases such as AS, PsA, and also, potentially, OA.Thus, TNF� inhibition does not actively prevent bonyspur formation, which is potentially beneficial, sincebony spurs allow a certain stabilization of affected joints.Furthermore, TNF� does not promote ankylosis andimmobilization of joints. This finding has potential im-plications for the treatment of AS, but also of RA, whichis sometimes associated with secondary OA and forma-tion of bony spurs and osteosclerosis.
Another important finding is the lack of effect ofosteoclast inhibition on bony spur formation. Becausegrowth of bony spurs requires endochondral bone for-mation, which is characterized by the production of acartilaginous scaffold containing hypertrophic chondro-cytes followed by remodeling into bone, one couldassume that osteoclasts are required for this process.Our data, as well as recent data in a nonerosive model ofarthritis in which treatment with bisphosphonates wasused (46), do not support this concept. Thus, RANKLinhibition by OPG did not influence growth of bonyspurs in either model, which strongly suggests thatosteoclasts are not necessary for the process of bonyspur formation to occur. This notion is consistent withdata from a study in nonhuman primates, which showedthat osteoclast inhibition by estrogen had no significantimpact on periarticular bony spurs (28). Furthermore,an observational study indicated that the antiresorptiveeffects of bisphosphonates were associated with neutraleffects on OA symptoms and bony spur formation inpostmenopausal women (47).
RANKL inhibition is considered a promisingstrategy to treat osteoporosis, bone metastasis, andarthritic bone erosions (48). Our observation thatRANKL inhibition did not affect bony spur formation isof clinical interest, since bony spurs are frequently foundin elderly patients and are a symptom of OA as well.These data suggest that RANKL inhibition would have a
2652 SCHETT ET AL
neutral role in terms of its effects on such lesions;instead, RANKL inhibition would allow the stabilizationof an affected joint, because new bone formation wouldoccur while bony ankylosis would not be provoked.
Another clinical implication of our findings re-lates to the confounding influence of bony spurs on theinterpretation of bone densitometry evaluations by dualx-ray absorptiometry (49). Because the inhibition ofRANKL and TNF� did not influence bony spurs, it isreasonable to suggest that those therapies also might notfurther complicate this scenario.
In summary, these data show that bony spurformation is a response-to-injury mechanism of thejoint, which is turned on rapidly during initial jointdamage. This mechanism occurred independently fromTNF�, a major inflammatory stimulus, and RANKL, thetriggering factor for osteoclast activation and bone loss.In fact, these observations reinforce current molecularand clinical concepts, which suggest that bony spurformation is not influenced by TNF� inhibition andfollows distinct molecular processes that control boneformation.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising itcritically for important intellectual content, and all authors approvedthe final version to be published. Dr. Schett had full access to all of thedata in the study and takes responsibility for the integrity of the dataand the accuracy of the data analysis.Study conception and design. Schett, Stolina, Zack, Kostenuik, Feige.Acquisition of data. Schett, Stolina, Dwyer, Uderhardt, Kronke,Kostenuik, Feige.Analysis and interpretation of data. Schett, Stolina, Dwyer, Zack,Kostenuik.
REFERENCES
1. Firestein GS. Evolving concepts of rheumatoid arthritis. Nature2003;423:356–61.
2. McInnes I, Schett G. Cytokines in the pathogenesis of rheumatoidarthritis. Nat Immunol 2007;7:429–42.
3. Goldring SR, Goldring MB. Eating bone or adding it: the Wntpathway decides. Nat Med 2007;13:133–4.
4. Schett G, Landewe R, van der Heijde D. TNF blockers andstructural remodeling in ankylosing spondylitis—what is realityand what is fiction? Ann Rheum Dis 2007;66:709–11.
5. Lam J, Takeshita S, Barker JE, Kanagawa O, Ross FP, TeitelbaumSL. TNF-� induces osteoclastogenesis by direct stimulation ofmacrophages exposed to permissive levels of RANK ligand. J ClinInvest 2000;106:1481–8.
6. Kong YY, Feige U, Sarosi I, Bolon B, Tafuri A, Morony S, et al.Activated T cells regulate bone loss and joint destruction inadjuvant arthritis through osteoprotegerin ligand. Nature 1999;402:304–9.
7. Redlich K, Hayer S, Ricci R, David JP, Tohidast-Akrad M, KolliasG, et al. Osteoclasts are essential for TNF-�-mediated jointdestruction. J Clin Invest 2002;110:1419–27.
8. Tat SK, Pelletier JP, Lajeunesse D, Fahmi H, Duval N, Martel-
Pelletier J. Differential modulation of RANKL isoforms by humanosteoarthritic subchondral bone osteoblasts: influence of osteotro-pic factors. Bone 2008;43:284–91.
9. Crotti T, Smith MD, Weedon H, Ahern MJ, Findlay DM, KraanM, et al. Receptor activator NF-�B ligand (RANKL) expression insynovial tissue from patients with rheumatoid arthritis, spondylo-arthropathy, osteoarthritis, and from normal patients: semiquan-titative and quantitative analysis. Ann Rheum Dis 2002;61:1047–54.
10. Kearns AE, Khosla S, Kostenuik PJ. OPG and RANKL regulationof bone remodeling in health and disease. Endocr Rev 2008;29:155–92.
11. Grimaud E, Soubigou L, Couillaud S, Coipeau P, Moreau A,Passuti N, et al. Receptor activator of nuclear factor �B ligand(RANKL)/osteoprotegerin (OPG) ratio is increased in severeosteolysis. Am J Pathol 2003;163:2021–31.
12. Haynes DR, Barg E, Crotti TN, Holding C, Weedon H, Atkins GJ,et al. Osteoprotegerin expression in synovial tissue from patientswith rheumatoid arthritis, spondyloarthropathies and osteoarthri-tis and normal controls. Rheumatology (Oxford) 2003;42:123–34.
13. Gravallese EM, Harada Y, Wang JT, Gorn AH, Thornhill TS,Goldring SR. Identification of cell types responsible for boneresorption in rheumatoid arthritis and juvenile rheumatoid arthri-tis. Am J Pathol 1998;152:943–51.
14. Sakai T, Kambe F, Mitsuyama H, Ishiguro N, Kurokouchi K,Takigawa M, et al. Tumor necrosis factor � induces expression ofgenes for matrix degradation in human chondrocyte-like HCS-2/8cells through activation of NF-�B: abrogation of the tumornecrosis factor � effect by proteosome inhibitors. J Bone MinerRes 2001;16:1272–80.
15. Steiner G, Tohidast-Akrad M, Witzmann G, Vesely M, Studnicka-Benke A, Gal A, et al. Cytokine production by synovial T cells inrheumatoid arthritis. Rheumatology (Oxford) 1999;38:202–13.
16. Cope AP, Aderka D, Doherty M, Engelmann H, Gibbons D, JonesAC, et al. Increased levels of soluble tumor necrosis factorreceptors in the sera and synovial fluid of patients with rheumaticdiseases. Arthritis Rheum 1992;35:1160–9.
17. Smolen JS, Steiner G. Therapeutic strategies for rheumatoidarthritis. Nat Rev Drug Discov 2003;2:473–88.
18. Ball J. The enthesopathy of ankylosing spondylitis. Br J Rheuma-tol 1983;22(4 Suppl 2):25–8.
19. Sieper J, Appel H, Braun J, Rudwaleit M. Critical appraisal ofassessment of structural damage in ankylosing spondylitis: impli-cations for treatment outcomes [review]. Arthritis Rheum 2008;58:649–56.
20. Lories RJ, Derese I, Luyten FP. Modulation of bone morphoge-netic protein signaling inhibits the onset and progression ofankylosing enthesitis. J Clin Invest 2005;115:1571–9.
21. Scharstuhl A, Vitters EL, van der Kraan PM, van den Berg WB.Reduction of osteophyte formation and synovial thickening byadenoviral overexpression of transforming growth factor �/bonemorphogenetic protein inhibitors during experimental osteoarthri-tis. Arthritis Rheum 2003;48:3442–51.
22. Diarra D, Stolina M, Polzer K, Zwerina J, Ominsky MS, Dwyer D,et al. Dickkopf-1 is a master regulator of joint remodeling. NatMed 2007;13:156–63.
23. Averns HL, Oxtoby J, Taylor HG, Jones PW, Dziedzic K, DawesPT. Radiological outcome in ankylosing spondylitis: use of theStoke Ankylosing Spondylitis Spine Score (SASSS). Br J Rheuma-tol 1996;35:373–6.
24. Creemers MC, Franssen MJ, van ’t Hof MA, Gribnau FW, van dePutte LB, van Riel PL. Assessment of outcome in ankylosingspondylitis: an extended radiographic scoring system. Ann RheumDis 2005;64:127–9.
25. Van der Heijde D, Landewe R, Einstein S, Ory P, Vosse D, Ni L,et al. Radiographic progression of ankylosing spondylitis after up
TNF� AND RANKL BLOCKADE IN EXPERIMENTAL INFLAMMATORY ARTHRITIS 2653
to two years of treatment with etanercept. Arthritis Rheum2008;58:1324–31.
26. Lories RJ, Derese I, De Bari C, Luyten FP. Evidence for uncou-pling of inflammation and joint remodeling in a mouse model ofspondylarthritis. Arthritis Rheum 2007;56:489–97.
27. Feige U, Hu YL, Gasser J, Campagnuolo G, Munyakazi L, BolonB. Anti-interleukin-1 and anti-tumor necrosis factor-� synergisti-cally inhibit adjuvant arthritis in Lewis rats. Cell Mol Life Sci2000;57:1457–70.
28. Bolon B, Morony S, Cheng Y, Hu YL, Feige U. Osteoclastnumbers in Lewis rats with adjuvant-induced arthritis: identifica-tion of preferred sites and parameters for rapid quantitativeanalysis. Vet Pathol 2004;41:30–6.
29. Frischholz S, Beier F, Girkontaite I, Wagner K, Poschl E, TurnayJ, et al. Characterization of human type X procollagen and itsNC-1 domain expressed as recombinant proteins in HEK293 cells.J Biol Chem 1998;273:4547–55.
30. Campagnuolo G, Bolon B, Feige U. Kinetics of bone protection byrecombinant osteoprotegerin therapy in Lewis rats with adjuvantarthritis. Arthritis Rheum 2002;46:1926–36.
31. Schett G, Stolina M, Bolon B, Middleton S, Adlam M, Brown H,et al. Analysis of the kinetics of osteoclastogenesis in arthritic rats.Arthritis Rheum 2005;52:3192–201.
32. Schett G, Middleton S, Bolon B, Stolina M, Brown H, Zhu L, et al.Additive bone-protective effects of anabolic treatment when usedin conjuction with RANKL and tumor necrosis factor inhibition intwo rat arthritis models. Arthritis Rheum 2005;52:1604–11.
33. Benjamin M, McGonagle D. The anatomical basis for diseaselocalization in seronegative spondylarthropathy at entheses andrelated sites. J Anat 2001;199:503–26.
34. Iwata K, Li J, Follet H, Phipps RJ, Burr DB. Bisphosphonatessuppress periosteal osteoblast activity independently of resorptionin rat femur and tibia. Bone 2006;39:1053–8.
35. Barnes GL, Kostenuik PJ, Gerstenfeld LC, Einhorn TA. Growthfactor regulation of fracture repair. J Bone Miner Res 1999;14:1805–15.
36. Kitaori T, Ito H, Schwarz EM, Tsutsumi R, Yoshitomi H, Oishi S,et al. Stromal cell–derived factor 1/CXCR4 signaling is critical forthe recruitment of mesenchymal stem cells to the fracture siteduring skeletal repair in a mouse model. Arthritis Rheum 2009;60:813–23.
37. Olson EJ, Lindgren BR, Carlson CS. Effects of long-term estrogenreplacement therapy on bone turnover in periarticular tibialosteophytes in surgically postmenopausal cynomolgus monkeys.Bone 2008;42:907–13.
38. Flick LM, Weaver JM, Ulrich-Vinther M, Abuzzahab F, Zhang X,Dougall WC, et al. Effects of receptor activator of NF�B (RANK)signaling blockade on fracture healing. J Orthop Res 2003;21:676–84.
39. Amanat N, McDonald M, Godfrey C, Bilston L, Little D. Optimaltiming of a single dose of zoledronic acid to increase strength in ratfracture repair. J Bone Miner Res 2007;22:867–76.
40. Ulrich-Vinther M, Andreassen TT. Osteoprotegerin treatmentimpairs remodeling and apparent material properties of callustissue without influencing structural fracture strength. Calcif Tis-sue Int 2005;76:280–6.
41. Keffer J, Probert L, Cazlaris H, Georgopoulos S, Kaslaris E,Kioussis D, et al. Transgenic mice expressing human tumournecrosis factor: a predictive genetic model of arthritis. EMBO J1991;10:4025–31.
42. Lories RJ, Matthys P, de Vlam K, Derese I, Luyten FP. Ankylosingenthesitis, dactylitis, and onychoperiostitis in male DBA/1 mice: amodel of psoriatic arthritis. Ann Rheum Dis 2004;63:595–8.
43. De Vries BJ, van den Berg WB. Impact of NSAIDS on murineantigen induced arthritis. II. A light microscopic investigation ofantiinflammatory and bone protective effects. J Rheumatol 1990;17:295–303.
44. Bertolini DR, Nedwin GE, Bringman TS, Smith DD, Mundy GR.Stimulation of bone resorption and inhibition of bone formation invitro by human tumor necrosis factor. Nature 1986;319:516–8.
45. Canalis E. Effects of tumor necrosis factor on bone formation invitro. Endocrinology 1987;121:1596–604.
46. Lories RJ, Derese I, Luyten FP. Inhibition of osteoclasts does notprevent joint ankylosis in a mouse model of spondyloarthritis.Rheumatology (Oxford) 2008;47:605–8.
47. Carbone LD, Nevitt MC, Wildy K, Barrow KD, Harris F, FelsonD, et al, for the Health, Aging and Body Composition Study. Therelationship of antiresorptive drug use to structural findings andsymptoms of knee osteoarthritis. Arthritis Rheum 2004;50:3516–25.
48. McClung MR, Lewiecki EM, Cohen SB, Bolognese MA, WoodsonGC, Moffett AH, et al, for the AMG 162 Bone Loss Study Group.Denosumab in postmenopausal women with low bone mineraldensity. N Engl J Med 2006;354:821–31.
49. Muraki S, Yamamoto S, Ishibashi H, Horiuchi T, Hosoi T, OrimoH, et al. Impact of degenerative spinal diseases on bone mineraldensity of the lumbar spine in elderly women. Osteoporos Int2004;15:724–8.
