Kinetics of Bone Protection by Recombinant OsteoprotegerinTherapy in Lewis Rats With Adjuvant Arthritis
Giuseppe Campagnuolo, Brad Bolon, and Ulrich Feige
Objective. To assess the effect of different dosagesand treatment schedules of osteoprotegerin (OPG) onjoint preservation in an experimental model ofadjuvant-induced arthritis (AIA).
Methods. Male Lewis rats with AIA (6–8 pergroup) were treated with a subcutaneous bolus of re-combinant human OPG according to one of the follow-ing schedules: daily OPG (an efficacious regimen) start-ing at disease onset (days 9–15), early intervention(days 9–11), delayed intervention (days 13–15), andextended therapy (days 9–22). Inflammation (hind pawswelling) was quantified throughout the clinical course;osteoporosis (bone mineral density [BMD], by quanti-tative dual x-ray absorptiometry) and morphologic ap-praisals of inflammation, bone damage, intralesionalosteoclasts (by semiquantitative histopathologic scor-ing), and integrity of the articular cartilage matrix (byretention of toluidine blue stain) were determined inhistology sections of arthritic hind paws.
Results. OPG provided dose- and schedule-dependent preservation of BMD and periarticular bonewhile essentially eliminating intralesional osteoclasts.Dosages >2.5 mg/kg/day preserved or enhanced BMDand prevented essentially all erosions. A dosage of 4mg/kg/day protected joint integrity to a comparabledegree when given for 7 (days 9–15) or 14 (days 9–22)consecutive days. At this dosage, early intervention(days 9–11) was twice as effective as delayed interven-tion (days 13–15) at preventing joint dissolution. Ero-
sions and osteoclast scores were greatly decreased for 26days (measured from the first treatment) after 7 or 14daily doses of OPG (4 mg/kg/day). OPG treatment alsoprevented loss of cartilage matrix proteoglycans, anindirect consequence of protecting the subchondralbone. No OPG dosage or regimen alleviated weight loss,inflammation, or periosteal osteophyte production.
Conclusion. These data indicate that OPG pre-serves articular bone and (indirectly) articular cartilagein arthritic joints in a dose- and schedule-dependentmanner, halts bone erosion when given at any pointduring the course of arthritis, produces sustained anti-erosive activity after a short course, and is most effectivewhen initiated early in the disease.
Bone removal during normal skeletal remodelingas well as in various pathologic conditions, includingarthritis (1,2), is controlled by the balanced interactionsbetween the tumor necrosis factor (TNF) family mole-cules osteoprotegerin (OPG) and receptor activator ofnuclear factor �B ligand (RANKL; also called OPGligand) (3–6). OPG is a soluble decoy receptor thatinhibits osteoclast formation, function, and survival bypreventing the binding of RANKL to RANK, amembrane-bound protein of the TNF receptor familythat is found on chondrocytes, dendritic cells, osteoclastprecursors, and mature osteoclasts (7–9).
Numerous studies of mice with engineered mod-ifications of OPG, RANKL, or RANK have confirmedthe importance of these molecules in bone turnover.Animals with null mutations of the OPG gene exhibitsevere osteoporosis (10), while transgenic mice express-ing supraphysiologic levels of OPG develop profoundosteopetrosis (3). In contrast, mice with ablated RANKLand RANK genes exhibit osteopetrosis (11,12). Exoge-nous OPG has also been shown to prevent bone loss inrodent models of adjuvant-induced arthritis (AIA) (13),collagen-induced arthritis (CIA) (14), bone metastasis(15), and ovariectomy-associated estrogen deficiency
Presented in part at the Annual Congress of Rheumatology ofthe European League Against Rheumatism, Prague, Czech Republic,June 2001.
Mr. Campagnuolo and Dr. Bolon contributed equally to thiswork.
Address correspondence and reprint requests to Ulrich Feige,PhD, Amgen, Inc., One Amgen Center Drive, M/S 15-2-A, ThousandOaks, CA 91320-1789. E-mail: [email protected].
Submitted for publication October 16, 2001; accepted inrevised form March 11, 2002.
1926
(3,16). In humans, a single OPG injection has beenshown to rapidly and profoundly reduce bone turnoverfor a sustained period in postmenopausal women (17).OPG therefore has great potential as a therapy fordiseases characterized by marked bone resorption.
One such condition is rheumatoid arthritis (RA),a major consequence of which is irreversible joint de-struction, which leads to profound disability. Severallines of evidence indicate that an altered balance in theOPG/RANKL/RANK signaling pathway that controlsosteoclast activity contributes significantly to the peri-articular osteoporosis and skeletal erosions that arecharacteristic of RA in humans as well as in animalmodels of inflammatory arthritis. Increased numbers ofosteoclasts and osteoclast-like cells occur both in RApatients (18–22) and in animals with experimental ar-thritis (13,23–27). Primary control of osteoclast activityin such conditions is mediated by RANKL derived fromactivated T cells (13,28) and synovial fibroblasts (29,30).RANKL elaborated by osteoblasts and bone marrowstromal cells also plays a role (5,28,31,32). RANKLdirectly regulates osteoclast populations by controllingthe transformation of circulating monocytes (33), syno-vial macrophages (34,35), and bone marrow hematopoi-etic cells (33,36) into osteoclasts (osteoclastogenesis), aswell as by activating mature (“resting”) osteoclasts (4,37)and by supporting osteoclast survival (38,39). Blockadeof RANKL by the administration of exogenous OPGbeginning at disease onset has been shown to preventjoint erosion in experimental models of immune-mediated AIA (13) and CIA (14) in rats. For thesereasons, OPG therapy to inhibit RANKL-mediated ef-fects on osteoclast activity represents a significant newmeans of ameliorating skeletal destruction in RA.
While it has been shown that daily treatment withOPG significantly reduces joint destruction in immune-mediated arthritis, the finding that a single OPG treat-ment delivers long-term, dose-dependent reductions inbone turnover (17) indicates that the optimum dosageand treatment schedule have yet to be defined. There-fore, the principal objective of our experiments was toexplore the effect of various OPG dosages and regimenson osteoclast kinetics in a rat model of immune-mediated arthritis. Our data indicate that OPG treat-ment yields dose- and schedule-dependent preservationof bone, and indirectly of cartilage, in arthritic jointsand show that protection can be maintained for anextended period even in the presence of continuedinflammation.
MATERIALS AND METHODS
Animals. Male Lewis rats (Charles River, Wilmington,MA) weighing 180–200 gm were acclimated for 1 week andthen randomly assigned to treatment groups (6–8 rats pergroup). This group size was used because interindividualvariability between arthritic rats is minimal in this model (40).Animals were given tap water and fed pelleted rodent chow(no. 8640; Harlan Teklad, Madison, WI) ad libitum; calciumand phosphorus contents were 1.2% and 1.0%, respectively. Atnecropsy, all animals were killed by carbon dioxide. Thesestudies were conducted in accordance with federal animal careguidelines and were preapproved by the Institutional AnimalCare and Use Committee of Amgen.
Induction of AIA. AIA was induced on day 0 by a singleintradermal injection of heat-killed Mycobacterium tuberculosisH37Ra (0.5 mg; Difco, Detroit, MI) suspended in 0.05 ml ofparaffin oil (Crescent Chemical, Hauppauge, NY) into thebase of the tail, as described elsewhere (40). The clinical onsetof arthritis occurred on day 9, as indicated by swelling of thehind paws and difficulty walking.
Experimental design. Three serial studies were per-formed using a recombinant human fusion protein (Amgen,Thousand Oaks, CA) that combined the OPG ligand-bindingdomain with the constant domain of human IgG1 (3). OPG inphosphate buffered saline (PBS) was injected by subcutaneousbolus. Concurrent control groups were injected with PBS orwere not treated, since our experience with AIA has shownthat clinical and histopathologic responses for these “treat-ments” are similar (Feige U, et al: unpublished observations).
Dose-dependent efficacy of OPG (experiment 1). OPGwas administered at 6 dosages (0.01, 0.04, 0.16, 0.625, 2.5, or 10mg/kg/day), which were selected to bracket a dosage that isknown to protect joint integrity in AIA (1 mg/kg/day) (13). Allrats were given OPG daily for 7 days (days 9–15) and thennecropsied on day 16.
Schedule-dependent efficacy of OPG (experiment 2).Rats were given OPG (4 mg/kg/day) according to 1 of thefollowing 3 schedules (Table 1): daily OPG beginning at theonset of arthritis (days 9–15), a regimen that has been shownto be effective against AIA (13), an early intervention protocol(days 9–11), or a delayed intervention protocol (days 13–15).All animals were necropsied 1 day after the end of treatment.
Duration of OPG efficacy (experiment 3). Rats weregiven OPG at a dosage of 4 mg/kg/day for 7 days (days 9–15).This regimen has been shown to prevent structural dissolutionin AIA on day 16 (13). Additional cohorts were given the samedosage for 14 days (days 9–22) to explore whether extendedtherapy would yield sustained efficacy. Rats were necropsiedon days 16 (arthritis onset � 7 days), 23 (onset � 14 days), 25(onset � 16 days), 28 (onset � 19 days), or 35 (onset � 26 days).
Assessment of arthritis. Clinical evaluation. Total bodyweights were recorded daily beginning on day 8 (arthritisonset � 1 day) until the day of necropsy. On the same days,mobility was assessed qualitatively using a binary scale (normalor impaired).
Hind paw volume appraisal (plethysmography). Pawswelling (40) was used as the indicator of the onset andprogression of arthritis. Measurements were taken starting onday 8 and continued until the time of necropsy. Each paw wasimmersed in a preweighed water-filled beaker (to just above
BONE PROTECTION AND OPG THERAPY IN RATS WITH AIA 1927
the tibiotarsal joint [hock]) and weighed. The paw volume wasthen calculated as the difference between the “water � paw”and “water only” readings. The mean of 2 consecutive readingsfor each paw was computed and used for further analysis.Inhibition of inflammation was calculated based on the areaunder the curve, using the trapezoidal rule:
Bone densitometry. Bone mineral density (BMD) in thehock was measured by dual x-ray absorptiometry (DXA) usinga model QDR-4500A densitometer (Hologic, Waltham, MA).At necropsy, paws were removed at the fur line (just proximalto the hock) and stored in 70% ethanol. Paws were orientedhorizontally relative to the detector. Following scanning, wecalculated the BMD in a 29 � 25–mm rectangle of tissuecentered at the calcaneus, using proprietary algorithms.
Lewis rats with AIA gradually lose BMD during thefirst week following disease onset, with significant deficitsdeveloping by day 16 (onset � 7 days) after adjuvant inocula-tion (40). In contrast, the BMD in normal, young adult, maleLewis rats increased throughout the period during which theseexperiments were conducted (Figure 1).
Histopathologic assessment. After DXA analysis,ethanol-fixed paws were decalcified, divided longitudinallyalong the median axis, processed, and embedded into paraffin.A 4 �m–thick section was stained for osteoclasts using anindirect immunoperoxidase method and proprietary rabbitanti-human monoclonal antibody (Amgen) directed againstcathepsin K; sections were counterstained with hematoxylinand eosin.
Four components of the arthritic process in the distaltibia and tarsal bones as well as the surrounding fascia wereevaluated for each paw by one of us (BB), a board-certifiedveterinary pathologist, using tiered, semiquantitative gradingcriteria (Table 2) and a “blinded” analytical paradigm. Scoresfor inflammation (in periarticular soft tissues and bone mar-row), skeletal remodeling (chiefly for bony repair), and boneerosion (for bone destruction) were acquired using previouslydescribed criteria (40). In addition, an osteoclast score wasobtained by assessing cathepsin K–labeled multinucleated cellsin bones of the tibiotarsal and intertarsal joints; this score is
taken more rapidly but yields results comparable to absolutecounts of intralesional osteoclasts (41). Cartilage matrix integ-rity was assessed qualitatively in a serial section stained withtoluidine blue (13).
Statistical analysis. Results were expressed as thegroup mean � SEM. Clinical data (continuous variables) wereassessed using Kruskal-Wallis analysis of variance and theMann-Whitney U test, while the histopathologic lesion scores(ordinal variables) were analyzed using the chi-square test. A Pvalue of 0.05 was used to delineate significant differencesbetween groups.
RESULTS
Findings of dose-dependent efficacy studies (ex-periment 1). A 7-day course of OPG (2.5 or 10 mg/kg/day beginning at disease onset [day 9]) significantly
Figure 1. Bone mineral density (BMD) of the skeleton in the tibio-tarsal (hock) region of normal, young adult, male Lewis rats. TheBMD in this region increased as the animals grew. Values are themean and SEM.
Table 1. Design of experiment for the schedule-dependent efficacy of OPG in rats with adjuvant-induced arthritis (experiment 2)
Experimental group TreatmentDosage
(mg/kg/day)Dosing
schedule Necropsy dayDiseasestatus
Control cohortsGroup 1 None – None Day 9 (onset) ArthritisGroup 2 Vehicle – Days 9–15 Day 16 (onset � 7 days) Arthritis
Continued treatmentGroup 3* OPG 4 Days 9–15 Day 16 (onset � 7 days) Arthritis
Early interventionGroup 4 OPG 4 Days 9–11 Day 12 (onset � 3 days) ArthritisGroup 5 Vehicle – Days 9–11 Day 12 (onset � 3 days) Arthritis
Delayed interventionGroup 6 None – None Day 13 (onset � 4 days) ArthritisGroup 7 OPG 4 Days 13–15 Day 16 (onset � 7 days) ArthritisGroup 8 Vehicle – Days 13–15 Day 16 (onset � 7 days) Arthritis
* The osteoprotegerin (OPG) dosage and schedule used for group 3 rats have been shown to protect jointintegrity in rats with adjuvant arthritis (13).
1928 CAMPAGNUOLO ET AL
preserved hind paw BMD in the arthritic joints of ratswith AIA (Figure 2A). Similarly, these 2 OPG dosagesyielded significant, dose-dependent reductions in scoresfor skeletal remodeling (Figure 2B) and erosions (Figure2C). The extent of the decrease in the erosion scoreexceeded that of the remodeling score at the 2 highestdosages (compare Figures 2D and E).
The next lowest OPG dosage, 0.625 mg/kg/day,generated modest, but not significant, decreases in lossof BMD (Figure 2A), osteophyte formation (Figure 2B),and erosions (Figure 2C) when given for the sameperiod. The lowest OPG dosages (0.01, 0.04, and 0.16mg/kg/day) provided no substantial efficacy.
Findings of schedule-dependent efficacy studies(experiment 2). As expected, relative to nonarthriticanimals, rats with AIA treated with OPG at a dosage of4 mg/kg/day for 7 days beginning at disease onset (day 9)had substantially higher hind paw BMD on day 16, while
those receiving vehicle during the same period hadmarkedly lower BMD (Figure 3A). Similarly, his-topathologic scores for remodeling (data not shown),erosions (Figure 3B), and intralesional osteoclasts (Fig-ure 3C) were minimal in rats given OPG for 7 days butsevere in vehicle-treated animals.
Abbreviated (3-day) courses of OPG at a dosageof 4 mg/kg/day were effective at protecting hind pawBMD regardless of whether intervention was initiated atdisease onset (day 9) or at onset plus 4 days (day 13)(Figure 3A). In contrast, mean erosion scores weresignificantly lower when treatment was begun at diseaseonset than when begun at onset plus 4 days (Figure 3B).The erosion scores for animals in which OPG treatmentwas delayed until day 13 (onset � 4 days) were similar tothose found in untreated AIA rats necropsied on day 13(Figure 3B). Osteoclast scores assessed the day after thefinal OPG injection were comparable for both the early
Table 2. Histopathology criteria for scoring lesions in adjuvant-induced arthritis
Parameter,score* Feature
Inflammation0 Normal1 Few inflammatory cells2 Mild inflammation3 Moderate inflammation (often, but not always, diffuse)4 Marked inflammation (diffuse and dense, with synovial abscesses)
Erosions0 Normal1 Minimal loss of cortical or trabecular bone at rare sites2 Mild loss of cortical or trabecular bone at a few sites (generally the
tarsals)3 Moderate loss of bone at many sites (usually the trabeculae of the tarsals,
but sometimes, the cortex of the distal tibia)4 Marked loss of bone at many sites (usually as extensive destruction of
trabeculae in the tarsals, but sometimes, with partial loss of corticalbone in the distal tibia)
5 Marked loss of bone at many sites (with fragmenting of tarsal trabeculaeand full-thickness penetration of cortical bone in the distal tibia)
Osteoclasts0 Normal (essentially no osteoclasts)1 Few osteoclasts (lining �5% of most affected bone surfaces)2 Some osteoclasts (lining �5% to 25% of most affected bone surfaces)3 Many osteoclasts (lining �25% to 50% of most affected bone surfaces)4 Myriad osteoclasts (lining �50% of most affected bone surfaces)
of cortical bone of either the tarsals or (less frequently) the tibia5 Marked periosteal and/or endosteal proliferation with full-thickness
erosion of cortical bone of the tarsals and the tibia
* The erosion score assesses destruction. The skeletal remodeling score chiefly assesses bone repair.
BONE PROTECTION AND OPG THERAPY IN RATS WITH AIA 1929
Figure 2. Dose-dependent efficacy of osteoprotegerin (OPG) in rats with adjuvant-induced arthritis (experiment 1). A 7-day course of OPG startingat disease onset (day 9) preserved hind paw bone mineral density (BMD) (A) and inhibited skeletal remodeling (B) and erosion (C) in a dose-dependentmanner. Open bars in A–C represent normal rats; solid bars represent arthritic rats. Values are the mean and SEM. Asterisks denote a significant differencecompared with animals given vehicle for 7 days. As shown in hematoxylin and eosin–stained sections, the effect of OPG on remodeling (an evaluation ofrepair) was less profound than the effect on erosions because there was little impact on the formation of osteophytes (arrowheads) in OPG-treated rats(D) compared with arthritic rats (E). A section from a normal rat (F) is shown for comparison. N � navicular tarsal; T � talus.
Figure 3. Schedule-dependent efficacy of osteoprotegerin (OPG) in rats with adjuvant-induced arthritis (AIA) (experiment 2). The effect of OPGinjection at 4 mg/kg/day on bone mineral density (BMD) (A), erosions (B), and intralesional osteoclasts (C) in the hind paw varied with the treatmentschedule. Untreated rats with AIA lost BMD and developed marked erosions and large osteoclast populations, while AIA rats given OPG for 3 or7 days starting at disease onset (day 9) had substantially more BMD, with fewer erosions and osteoclasts. However, a 3-day course of OPG initiatedon day 13 (onset � 4 days) did not reduce erosions as effectively as a 3-day course started on day 9 (onset), indicating that aggressive bone-protectivetherapy is essential early in the course of arthritis to prevent joint destruction. Values are the mean and SEM. Asterisks denote a significantdifference compared with animals given vehicle for the same number of days.
1930 CAMPAGNUOLO ET AL
and late OPG treatment groups (Figure 3C). In the caseof the delayed OPG cohort, the osteoclast scores after 3days were significantly reduced relative to those ofuntreated AIA rats examined on day 13 (Figure 3C).
Duration of joint protection (experiment 3). Asdemonstrated previously (40), hind paw BMD in un-treated (Figure 4A) or vehicle-treated (data not shown)rats with AIA was lower by 12% on day 16 (onset � 7days) relative to that in nonarthritic control rats. TheBMD decreased by an additional 11% by day 23 (on-set � 14 days), remained stable through day 28 (onset �19 days), and then decreased by a further 9% by day 35(onset � 26 days) (Figure 4A). In contrast, hind pawBMD in AIA rats given OPG at a dosage of 4 mg/kg/dayon days 9–15 was elevated on day 16 and continued toincrease until day 35 (Figure 4A) in a pattern consistentwith that in nonarthritic control rats (Figure 1). On day35, the BMD in OPG-treated rats with AIA was twice ashigh as that in untreated rats with AIA. Joint integritywas preserved to an equivalent degree in arthritic ratsgiven OPG for 14 days (data not shown).
Scores for skeletal remodeling (Figure 4B), ero-
sion (Figure 4C), and osteoclasts (Figure 4D) weremarked in untreated or vehicle-treated (data not shown)rats with AIA starting on day 16 (onset � 7 days) andcontinuing through day 35 (onset � 26 days). The soleexception was the mean osteoclast score at day 35, whichwas significantly higher than the scores in animalsexamined at earlier time points (Figure 4D). Treatmentwith OPG at a dosage of 4 mg/kg/day for 7 daysbeginning at disease onset (day 9) significantly reducedthe mean skeletal remodeling score on day 16 (onset �7 days), while scores in OPG-injected rats at all latertime points were comparable to those in rats withuntreated AIA (Figure 4B). In contrast, erosion (Figure4C) and osteoclast (Figure 4D) scores in rats given OPGwere uniformly minimal from day 16 through day 28(onset � 19 days), a finding consistent with the completeabsence of intralesional osteoclasts at these time points.Interestingly, on day 35, OPG-treated rats still had minimalerosions even though the osteoclast score, while low, washigher than the scores in the other OPG-treated cohortsevaluated at earlier time points (Figure 4D).
Articular cartilage in markedly inflamed joints
Figure 4. Duration of joint protection in rats with adjuvant-induced arthritis (AIA) treated with osteoprotegerin (OPG)at a dosage of 4 mg/kg/day (experiment 3). OPG given for 7 days starting at disease onset (day 9) enhanced hind pawbone mineral density (BMD) (A) and inhibited skeletal remodeling (B), erosions (C), and intralesional osteoclasts (D)in rats with AIA, as examined on day 16. The effects of OPG on BMD and erosions were maintained until day 35(onset � 26 days), while osteoclasts remained low through day 28 (onset � 19 days) before starting to recover. Asterisksdenote a significant difference compared with untreated AIA rats on the same necropsy day.
BONE PROTECTION AND OPG THERAPY IN RATS WITH AIA 1931
typically was preserved by systemic treatment with OPG,as indicated by retention of toluidine blue staining of thematrix (Figure 5H), while full-thickness loss of staining
occurred in arthritic joints in the absence of OPG(Figure 5G). However, OPG did not protect cartilage intwo circumstances. First, loss of staining was pro-
Figure 5. Preservation of articular cartilage, an indirect effect of osteoprotegerin (OPG) treatment. In normal joints (A and F), toluidine bluestained matrix proteoglycans throughout the articular cartilage. Inflamed joints exhibited full-thickness loss of toluidine blue staining in the absenceof OPG treatment (B and G), while systemic OPG therapy usually prevented its loss (C and H). Two patterns of staining loss occurred in the cartilageof arthritic joints in OPG-treated rats: loss of staining in the superficial layers (arrowheads) associated with pannus in the synovial cavity (D and I),and loss of staining in deep layers adjacent to regions of eroded subchondral bone (E and J). Sections were stained by cathepsin K immunochemistrywith hematoxylin and eosin counterstain (A–E) or were stained with toluidine blue (F–J). s � subchondral bone; c � articular cartilage; p � pannus;� � leukocytic infiltrate (eroding subchondral bone).
1932 CAMPAGNUOLO ET AL
nounced in the superficial layers if pannus extended overthe articular surface (Figure 5I). Second, loss of stainingprincipally affected the deep cartilage layers locatedadjacent to eroded subchondral bone (Figure 5J).
Effect of OPG on weight loss and mobility.Injection of OPG had no effect on weight loss ormobility at any dosage in any experiment (data notshown).
Effect of OPG on inflammation. Administrationof OPG affected neither clinical (paw volume) norhistopathologic (leukocyte infiltration in periarticulartissues) measures of inflammation in any experiment,regardless of the dosage or schedule (data not shown).In histologic sections, the cell infiltrate was decreased atlater time points (always at day 35 [onset � 26 days], andsometimes earlier) regardless of OPG therapy (Figure6).
DISCUSSION
Irreversible dissolution of bone and cartilage inaffected joints is a hallmark of RA. Because jointdestruction results in disability, prevention of bone andcartilage damage is the most critical issue in treating RA.This objective has not been fully attained with the use ofstandard therapies for RA, which indirectly preserveskeletal integrity by targeting signs and symptoms (i.e.,reducing inflammation). In contrast, OPG essentiallyhalts joint erosions in experimental arthritis (13,14).OPG performs this feat by inhibiting RANKL, one ofthe two molecules (along with colony-stimulating factor1) that are necessary and sufficient for osteoclast induc-tion (42). The chief sources of RANKL in arthritis are
the activated T cells (13,28) and synovial fibroblasts(29,30) that initiate and sustain inflammation. In turn,RANKL acts reciprocally to maintain inflammation (43)while enhancing osteoclast populations in the arthriticjoint (13,27,35) as well as at distant sites (44). The jointerosions in RA likely reflect an imbalance in theRANKL signaling pathway, such that endogenous OPGlevels are insufficient to counteract the excess RANKLelaborated in this condition (2,30,35).
Data from the present experiments extend ourprevious findings (13) regarding the bone-protectiveefficacy of OPG in the Lewis rat model of mycobacteria-induced AIA. In our previous studies, BMD loss, boneerosions, and expansion of intralesional osteoclast num-bers were significantly alleviated by injection of OPG(subcutaneously for 7 days, beginning at disease onset)at a dosage of 1 mg/kg/day, but not at two lower dosages(0.016 and 0.0625 mg/kg/day). In the current study(experiment 1), we used an identical treatment scheduleand dosages bracketing this known effective dosage todefine the OPG dose-response curve for bone protec-tion. As anticipated, we found that 2.5 or 10 mg/kg/dayof OPG yielded significant reductions in clinical andhistopathologic measurements of bone damage (Figure2). For both dosages, the decrease in the skeletalremodeling score was substantially less than that ob-served for the erosion score. The explanation for thisdivergence is that OPG strongly inhibited bone destruc-tion (erosions) but did not influence repair (periostealosteophyte production). An OPG dosage of 0.625 mg/kg/day generated modest decreases in bone destructionand remodeling, while lower dosages were ineffective.Together, these findings indicate that the minimal OPGdosage that provides essentially complete protection toinflamed joints in AIA is 1 mg/kg/day. By comparison,administration of OPG (at dosages of 0.001, 0.004, 0.016,0.06, 0.25, or 1.0 mg/kg/day subcutaneously for 7 daysstarting at disease onset) to Lewis rats with CIA sparesbone at dosages �0.06 mg/kg/day (Campagnuolo G, etal: unpublished observations). Moreover, a single OPGdose �0.3 mg/kg has been shown to inhibit bone turn-over for several weeks in postmenopausal women (17).Thus, we anticipate that the effective OPG dose in RAwill fall between 0.1 mg/kg and 1.0 mg/kg, with the actualdosage depending on treatment frequency.
More importantly, our present data provide sig-nificant new information about the use of OPG toprotect bone and cartilage in arthritis. One key conclu-sion is that bone protection can vary with the treatmentschedule (experiment 2). As expected, arthritic ratsgiven a 7-day course of OPG at a dosage of 4 mg/kg/day
Figure 6. Resolution of inflammation occurred independently of os-teoprotegerin (OPG) treatment in rats with adjuvant-inducted arthritis(AIA). Leukocyte infiltration in the hind paws of rats with AIAregressed beginning by day 25 (onset � 16 days) and progressed overtime. OPG therapy at a dosage of 4 mg/kg/day for 7 days beginning atdisease onset (day 9) had no effect on inflammation.
BONE PROTECTION AND OPG THERAPY IN RATS WITH AIA 1933
beginning at disease onset exhibited markedly betterpreservation of BMD with fewer erosions and intrale-sional osteoclasts than did vehicle-treated animals (Fig-ure 3). A similar degree of protection was provided by a3-day OPG course initiated at disease onset but not ifinitiated after a modest delay (onset � 4 days) (Figure3). Since OPG effectively reduced osteoclast popula-tions regardless of the treatment schedule, the explana-tion for this disparity is that bone destruction is minimalat disease onset but is already pronounced by onset plus4 days. With regard to cartilage preservation, our datasuggest that OPG prevents the destruction of matrixproteoglycans indirectly, rather than through a directeffect on chondrocytes (experiment 3). This conclusionwas based on our finding that OPG protected cartilageintegrity in AIA only if subchondral bone was intact andinflammatory cells were not present in the joint cavity(Figure 5). This situation likely reflects conditions thatexist in RA joints, where superficial (unmineralized)cartilage is destroyed from above by pannus, whilemineralized cartilage is razed from below as subchondralbone is eroded (18). Furthermore, OPG could be ex-pected to protect articular cartilage in RA joints, asindicated by the ability of bisphosphonates to reducecartilage destruction by defending the integrity of sub-chondral bone (21). Thus, our data indicate that aggres-sive bone-sparing therapy should be initiated as early aspossible in RA to reduce the degree of bone destructionand, by extension, cartilage destruction.
Another fundamental outcome of the presentstudy was that the interval between bone-sparing treat-ments will vary with the schedule (experiment 3). A7-day course of OPG at a dosage of 4 mg/kg/day initiatedat disease onset significantly reduced the erosion scoresin arthritic rats for 19 days after the final OPG dose hadbeen given (Figure 4). The same regimen completelyabolished intralesional osteoclasts for 12 days. It isinteresting that scattered osteoclasts were observed ininflamed bones by 19 days after the last dose eventhough erosions were essentially absent (Figure 4). Twoimportant deductions may be inferred from these data.First, intralesional osteoclast populations will rebuildover time in the presence of continued inflammation.This fact indicates that periodic re-administration ofOPG throughout the course of RA will be necessary toensure that joint integrity is preserved.
In addition, because OPG does not ameliorateinflammation (Figure 6), a corollary conclusion is thatOPG will have to be used in combination with one ormore antiinflammatory agents. Two logical candidatesfor combination therapy with OPG are inhibitors of
interleukin-1 (IL-1) and TNF�. These two cytokines arethe dominant drivers of RA (45). Both anti–IL-1 andanti-TNF treatments have been demonstrated to pre-serve joint architecture in RA in their own right(40,46,47), thereby raising the possibility of additive orsynergistic activity with OPG in protecting bone. Severallines of evidence support this speculation. For example,both IL-1 and TNF� are produced at the cartilage–pannus junction in RA (48). Pannus is rich in newlyformed blood vessels. Human vascular endothelial cellsactivated by in vitro exposure to either IL-1 or TNF�exhibit an early, transient increase in OPG expressionfollowed by a later, but sustained, upswing in RANKL(49). IL-1 has been shown to enhance osteoclastogenesisin vitro in the presence of RANKL-producing activatedT cells (50); a possible basis for this effect is the presenceof IL-1 type I and type II receptors on osteoclasts (51).Similarly, TNF� has been reported to stimulate oste-oclast differentiation in vitro by a pathway driven bymacrophage colony-stimulating factor but independentof OPG/RANKL (52). Other studies suggest that theTNF� and RANKL pathways that control osteoclasto-genesis are coupled (53), although the effect of differentTNF receptors on bone resorption may be divergent(54).
The major inference that can be derived fromthese studies is that the combination of OPG and eitheran anti–IL-1 or an anti-TNF molecule might achievebetter bone protection than could any of the 3 agentsalone. This possibility will need to be tested. However,we anticipate that OPG will provide the main contribu-tion toward joint-sparing efficacy and that the otheragents will add only incremental improvements in thisregard.
ACKNOWLEDGMENTS
The authors thank Yan Cheng, Chris De La Torre,Diane Duryea, Darlene Kratavil, Ruiyuan A. Luo, EfrainPacheco, and Li Zhu for technical assistance.
REFERENCES
1. Gravallese EM, Galson DL, Goldring SR, Auron PE. The role ofTNF-receptor family members and other TRAF-dependent recep-tors in bone resorption. Arthritis Res 2001;3:6–12.
2. Haynes DR, Crotti TN, Loric M, Bain GI, Atkins GJ, Findlay DM.Osteoprotegerin and receptor activator of nuclear factor � Bligand (RANKL) regulate osteoclast formation by cells in thehuman rheumatoid arthritic joint. Rheumatology 2001;40:623–30.
3. Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS,Luethy R, et al. Osteoprotegerin: a novel secreted protein involvedin the regulation of bone density. Cell 1997;89:309–19.
4. Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T,
et al. Osteoprotegerin ligand is a cytokine that regulates osteoclastdifferentiation and activation. Cell 1998;93:165–76.
5. Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M,Mochizuki S, et al. Osteoclast differentiation factor is a ligand forosteoprotegerin/osteoclastogenesis-inhibitory factor and is identi-cal to TRANCE/RANKL. Proc Natl Acad Sci U S A 1998;95:3597–602.
6. Hofbauer LC, Khosla S, Dunstan CR, Lacey DL, Boyle WJ, RiggsBL. The roles of osteoprotegerin and osteoprotegerin ligand in theparacrine regulation of bone resorption. J Bone Miner Res2000;15:2–12.
7. Burgess TL, Qian Y, Kaufman S, Ring BD, Van G, Capparelli C,et al. The ligand for osteoprotegerin (OPGL) directly activatesmature osteoclasts. J Cell Biol 1999;145:527–38.
8. Hsu H, Lacey DL, Dunstan CR, Solovyev I, Colombero A, TimmsE, et al. Tumor necrosis factor receptor family member RANKmediates osteoclast differentiation and activation induced byosteoprotegerin ligand. Proc Natl Acad Sci U S A 1999;96:3540–5.
9. Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, MartinTJ. Modulation of osteoclast differentiation and function by thenew members of the tumor necrosis factor receptor and ligandfamilies. Endocr Rev 1999;20:345–57.
10. Bucay N, Sarosi I, Dunstan CR, Morony S, Tarpley J, CapparelliC, et al. Osteoprotegerin-deficient mice develop early onset osteo-porosis and arterial calcification. Genes Dev 1998;12:1260–8.
11. Kong YY, Yoshida H, Sarosi I, Tan HL, Timms E, Capparelli C,et al. OPGL is a key regulator of osteoclastogenesis, lymphocytedevelopment and lymph node organogenesis. Nature 1999;397:315–23.
12. Li J, Sarosi I, Yan X-Q, Morony S, Capparelli C, Tan HL, et al.RANK is the intrinsic hematopoietic cell surface receptor thatcontrols osteoclastogenesis and regulation of bone mass andcalcium metabolism. Proc Natl Acad Sci U S A 2000;97:1566–71.
13. 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.
14. Romas E, Sims NA, Hards D, Lindsay M, Ryan PFJ, Quinn JA, etal. Fc-osteoprotegerin fusion protein (Fc-OPG) reduces osteoclastnumbers and prevents bone destruction in rats with collagen-induced arthritis [abstract]. Arthritis Rheum 2001;44 Suppl 9:S267.
15. Morony S, Capparelli C, Sarosi I, Lacey DL, Dunstan CR,Kostenuik PJ. Osteoprotegerin inhibits osteolysis and decreasesskeletal tumor burden in syngeneic and nude mouse models ofexperimental bone metastasis. Cancer Res 2001;61:4432–6.
16. Bolon B, Carter C, Daris M, Morony S, Capparelli C, Hsieh A, etal. Adenoviral delivery of osteoprotegerin ameliorates bone re-sorption in a mouse ovariectomy model of osteoporosis. Mol Ther2001;3:197–205.
17. Bekker PJ, Holloway D, Nakanishi A, Arrighi HM, Leese PT,Dunstan CR. The effect of a single dose of osteoprotegerin inpostmenopausal women. J Bone Miner Res 2001;16:348–60.
18. Bromley M, Woolley DE. Chondroclasts and osteoclasts at sub-chondral sites of erosion in the rheumatoid joint. Arthritis Rheum1984;27:968–75.
19. Shimizu S, Shiozawa S, Shiozawa K, Imura S, Fujita T. Quantita-tive histologic studies on the pathogenesis of periarticular osteo-porosis in rheumatoid arthritis. Arthritis Rheum 1985;28:25–31.
20. Takayanagi H, Oda H, Yamamoto S, Kawaguchi H, Tanaka S,Nishikawa T, et al. A new mechanism of bone destruction inrheumatoid arthritis: synovial fibroblasts induce osteoclastogen-esis. Biochem Biophys Res Commun 1997;240:279–86.
21. Bogoch ER, Moran E. Abnormal bone remodelling in inflamma-tory arthritis. Can J Surg 1998;41:264–71.
22. Gravallese EM, Harada Y, Wang JT, Gorn AH, Thornhill TS,Goldring SR. Identification of cell types responsible for bone
resorption in rheumatoid arthritis and juvenile rheumatoid arthri-tis. Am J Pathol 1998;152:943–51.
23. Kuratani T, Nagata K, Kukita T, Hotokebuchi T, Nakasima A,Iijima T. Induction of abundant osteoclast-like multinucleatedgiant cells in adjuvant arthritic rats with accompanying disorderedhigh bone turnover. Histol Histopathol 1998;13:751–9.
24. Suzuki Y, Nishikaku F, Nakatuka M, Koga Y. Osteoclast-like cellsin murine collagen induced arthritis. J Rheumatol 1998;25:1154–60.
25. Suzuki M, Suzuki M, Uetsuka K, Shinozuka J, Nakayama H, DoiK. Changes in location and number of tartrate-resistant acidphosphatase (TRAP)-positive cells during the development oftype II collagen-induced arthritis in DBA/1J mice. Exp Anim1998;47:211–4.
26. Bolon B, Morony S, Capparelli C, Hu Y-L, Campagnuolo G,Duryea D, et al. IL-1ra and PEG sTNF-RI reduce osteoclastnumbers in rats with adjuvant arthritis [abstract]. Arthritis Rheum2000;43 Suppl 9:S233.
27. Romas E, Bakharevski O, Hards DK, Kartsogiannis V, QuinnJMW, Ryan PFJ, et al. Expression of osteoclast differentiationfactor at sites of bone erosion in collagen-induced arthritis.Arthritis Rheum 2000;43:821–6.
28. Wong BR, Josien R, Choi Y. TRANCE is a TNF family memberthat regulates dendritic cell and osteoclast function. J LeukocyteBiol 1999;65:715–24.
29. Shigeyama Y, Pap T, Kunzler P, Simmen BR, Gay RE, Gay S.Expression of osteoclast differentiation factor in rheumatoid ar-thritis. Arthritis Rheum 2000;43:2523–30.
30. Takayanagi H, Iizuka H, Juji T, Nakagawa T, Yamamoto A,Miyazaki T, et al. Involvement of receptor activator of nuclearfactor �B ligand/osteoclast differentiation factor in osteoclasto-genesis from synoviocytes in rheumatoid arthritis. Arthritis Rheum2000;43:259–69.
31. Udagawa N, Takahashi N, Jimi E, Matsuzaki K, Tsurukai T, ItohK, et al. Osteoblasts/stromal cells stimulate osteoclast activationthrough expression of osteoclast differentiation factor/RANKLbut not macrophage colony-stimulating factor: receptor activatorof NF-�B ligand. Bone 1999;25:517–23.
32. Tsurukai T, Udagawa N, Matsuzaki K, Takahashi N, Suda T.Roles of macrophage-colony stimulating factor and osteoclastdifferentiation factor in osteoclastogenesis. J Bone Miner Res2000;18:177–84.
33. Quinn JM, Neale S, Fujikawa Y, McGee JO, Athanasou NA.Human osteoclast formation from blood monocytes, peritonealmacrophages, and bone marrow cells. Calcif Tissue Int 1998;62:527–31.
34. Fujikawa Y, Sabokbar A, Neale S, Athanasou NA. Human oste-oclast formation and bone resorption by monocytes and synovialmacrophages in rheumatoid arthritis. Ann Rheum Dis 1996;55:816–22.
35. Itonaga I, Fujikawa Y, Sabokbar A, Murray DW, Athanasou NA.Rheumatoid arthritis synovial macrophage-osteoclast differentia-tion is osteoprotegerin ligand-dependent. J Pathol 2000;192:97–104.
36. Lean JM, Matsuo K, Fox SW, Fuller K, Gibson FM, Draycott G,et al. Osteoclast lineage commitment of bone marrow precursorsthrough expression of membrane-bound TRANCE. Bone 2000;27:29–40.
37. Yasuda H, Shima N, Nakagawa N, Mochizuki SI, Yano K, FujiseN, et al. Identity of osteoclastogenesis inhibitory factor (OCIF)and osteoprotegerin (OPG): a mechanism by which OPG/OCIFinhibits osteoclastogenesis in vitro. Endocrinology 1998;139:1329–37.
38. Akatsu T, Murakami T, Nishikawa M, Ono K, Shinomiya N, TsudaE, et al. Osteoclastogenesis inhibitory factor suppresses osteoclastsurvival by interfering in the interaction of stromal cells withosteoclasts. Biochem Biophys Res Commun 1998;250:229–34.
BONE PROTECTION AND OPG THERAPY IN RATS WITH AIA 1935
39. Lacey DL, Tan HL, Lu J, Kaufman S, Van G, Qiu WR, et al.Osteoprotegerin ligand modulates murine osteoclast survival invitro and in vivo. Am J Pathol 2000;157:435–48.
40. Feige U, Hu Y-L, 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.
41. Bolon B, Morony S, Hu Y-L, Duryea D, Feige U. Preferred sitesand measurements for rapid osteoclast quantification in rat adju-vant arthritis [abstract]. Vet Pathol 2001;38:599.
42. Feige U. Osteoprotegerin. Ann Rheum Dis 2001;60 Suppl iii:81–4.43. Josien R, Li HL, Ingulli E, Sarma S, Wong BR, Vologodskaia M,
et al. TRANCE, a tumor necrosis factor family member, enhancesthe longevity and adjuvant properties of dendritic cells in vivo. JExp Med 2000;191:495–502.
44. Toritsuka Y, Nakamura N, Lee SB, Hashimoto J, Yasui N, ShinoK, et al. Osteoclastogenesis in iliac bone marrow of patients withrheumatoid arthritis. J Rheumatol 1997;24:1690–6.
45. Brennan FM, Maini RN, Feldmann M. Role of pro-inflammatorycytokines in rheumatoid arthritis. Springer Semin Immunopathol1998;20:133–47.
46. Bresnihan B, Alvaro-Gracia JM, Cobby M, Doherty M, DomljanZ, Emery P, et al. Treatment of rheumatoid arthritis with recom-binant human interleukin-1 receptor antagonist. Arthritis Rheum1998;41:2196–204.
47. Peterfy C, Palmer W, Schweitzer M, White D, Finck B. Compar-ison of etanercept vs. methotrexate in early rheumatoid arthritisusing gadolinium-enhanced magnetic resonance imaging [ab-stract]. Arthritis Rheum 1999;42 Suppl 9:S241.
48. Chu CQ, Field M, Allard S, Abney E, Feldmann M, Maini RN.Detection of cytokines at the cartilage/pannus junction in patientswith rheumatoid arthritis: implications for the role of cytokines incartilage destruction and repair. Br J Rheumatol 1992;31:653–61.
49. Collin-Osdoby P, Rothe L, Anderson F, Nelson M, Maloney W,Osdoby P. Receptor activator of NF-�B and osteoprotegerinexpression by human microvascular endothelial cells, regulation byinflammatory cytokines, and role in human osteoclastogenesis.J Biol Chem 2001;276:20659–72.
50. Horwood NJ, Kartsogiannis V, Quinn JM, Romas E, Martin TJ,Gillespie MT. Activated T lymphocytes support osteoclast forma-tion in vitro. Biochem Biophys Res Commun 1999;265:144–50.
51. Xu LX, Kukita T, Nakano Y, Yu H, Hotokebuchi T, Kuratani T,et al. Osteoclasts in normal and adjuvant arthritis bone tissuesexpress the mRNA for both type I and II interleukin-1 receptors.Lab Invest 1996;75:677–87.
52. Kobayashi K, Takahashi N, Jimi E, Udagawa N, Takami M,Kotake S, et al. Tumor necrosis factor � stimulates osteoclastdifferentiation by a mechanism independent of the ODF/RANKL-RANK interaction. J Exp Med 2000;191:275–86.
53. Zhang YH, Heulsmann A, Tondravi MM, Mukherjee A, Abu-Amer Y. Tumor necrosis factor-� (TNF) stimulates RANKL-induced osteoclastogenesis via coupling of TNF type 1 receptorand RANK signaling pathways. J Biol Chem 2001;276:563–8.
54. Abu-Amer Y, Erdmann J, Alexopoulou L, Kollias G, Ross FP,Teitelbaum SL. Tumor necrosis factor receptors types 1 and 2differentially regulate osteoclastogenesis. J Biol Chem 2000;275:27307–10.
