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Inhibition of Endogenous TGF-β During Experimental Osteoarthritis Prevents Osteophyte Formation and Impairs Cartilage Repair This information is current as of September 26, 2021. Alwin Scharstuhl, Harrie L. Glansbeek, Henk M. van Beuningen, Elly L. Vitters, Peter M. van der Kraan and Wim B. van den Berg J Immunol 2002; 169:507-514; ;

doi: 10.4049/jimmunol.169.1.507 Downloaded from http://www.jimmunol.org/content/169/1/507

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Inhibition of Endogenous TGF-␤ During Experimental Osteoarthritis Prevents Osteophyte Formation and Impairs Cartilage Repair

Alwin Scharstuhl,1 Harrie L. Glansbeek, Henk M. van Beuningen, Elly L. Vitters, Peter M. van der Kraan, and Wim B. van den Berg

Osteoarthritis has as main characteristics the degradation of articular cartilage and the formation of new bone at the joint edges, so-called osteophytes. In this study enhanced expression of TGF-␤1 and -␤3 was detected in developing osteophytes and articular cartilage during murine experimental osteoarthritis. To determine the role of endogenous TGF-␤ on osteophyte formation and articular cartilage, TGF-␤ activity was blocked via a scavenging soluble TGF-␤-RII. Our results clearly show that inhibition of endogenous TGF-␤ nearly completely prevented osteophyte formation. In contrast, treatment with recombinant soluble TGF-␤- Downloaded from RII markedly enhanced articular cartilage proteoglycan loss and reduced the thickness of articular cartilage. In conclusion, we show for the ﬁrst time that endogenous TGF-␤ is a crucial factor in the process of osteophyte formation and has an important function in protection against cartilage loss. The Journal of Immunology, 2002, 169: 507Ð514.

steoarthritis (OA)2 is a joint disease that is characterized markable ﬁnding was the formation of osteophytes after multiple

by degradation of articular cartilage and the formation of intra-articular (i.a.) injections of TGF-␤ protein or adenoviral http://www.jimmunol.org/ O new bone at the joint margins, so-called osteophytes. overexpression of TGF-␤1 in the knee joint (7, 8, 10). Osteophytes Articular cartilage proteoglycan (PTG) loss and fibrillation of the are newly formed bony structures located at the joint margins, and articular surface are early events in the OA process. At later stages their occurrence is strongly associated with OA. Osteophytes orig- clefts are formed in the cartilage, while end-stage OA shows ero- inate from activated periosteum leading to new cartilaginous out- sion of the articular cartilage. growths that eventually ossify into osteophytes via the process of TGF-␤ is a multifunctional cytokine involved in crucial biolog- endochondral ossification. In developing osteophytes, mRNAs for ical processes such as development, extracellular matrix synthesis, TGF-␤ and TGF-␤ protein expression are strongly up-regulated (11Ð cell proliferation/differentiation, and tissue repair. So far, three 13). These data suggest that TGF-␤ induces osteophyte formation. ␤ ␤ ␤ by guest on September 26, 2021 mammalian isoforms have been described: TGF- 1, - 2, and - 3. Although TGF-␤ seems implicated in pathology, TGF-␤ has ␤ TGF- is produced in a latent form, associated with the latency- also been suggested as a beneficial factor in cartilage repair. We ␤ associated peptide. TGF- can be activated after dissociation of have previously shown that injection of TGF-␤ in naive murine ␤ latency-associated peptide from the mature protein (1). TGF- sig- knee joints results in an increase in PTG synthesis and PTG con- naling starts with binding of TGF-␤ to TGF-␤-RII, a constitutively tent of articular cartilage (14). Moreover, i.a. injection of TGF-␤ active serine/threonine kinase (2). After transphosphorylation of during experimental arthritis resulted in protection from PTG loss TGF-␤-RI by TGF-␤-RII the signal is further propagated involv- (15). In addition, effects of IL-1, such as inhibition of cartilage ing Smad proteins (3, 4). PTG synthesis and release of cartilage PTG content, could be TGF-␤ has been suggested as an important factor in the patho- blocked by local application of TGF-␤ (16, 17). This demonstrates genesis of OA. One indication is the significant levels of active that TGF-␤ is able to counteract the deleterious effects of IL-1, a TGF-␤ in the synovial fluid of OA patients (5, 6). Indeed, OA changes of the cartilage have been described after exposure of cytokine considered to be a key mediator during erosive joint dis- knee joints to TGF-␤, supporting a role for TGF-␤ in the patho- eases. Taken together, these experiments suggest a protective func- ␤ genesis of OA (7, 8). Local administration of TGF-␤ in the knee tion for TGF- in articular cartilage. ␤ joint also induced inflammation and fibrosis (7Ð9). Another re- As can be concluded from the discussion above, TGF- appears to have a dualistic role in OA: protection against cartilage damage but induction of osteophyte formation. Experiments conducted to investigate the role of TGF-␤ in joint diseases are mainly based on Rheumatology Research Laboratory, Department of Rheumatology, University Med- ical Center, Nijmegen, The Netherlands TGF-␤ supplementation. In this study, we aimed to determine the Received for publication February 25, 2002. Accepted for publication April 30, 2002. role of endogenous TGF-␤ during experimental OA. We selec- ␤ The costs of publication of this article were defrayed in part by the payment of page tively blocked endogenous TGF- via systemic treatment with sol- charges. This article must therefore be hereby marked advertisement in accordance uble TGF-␤-RII (solRII), the cloned scavenging extracellular do- with 18 U.S.C. Section 1734 solely to indicate this fact. main of the TGF-␤-RII, an approach that to our knowledge has 1 Address correspondence and reprint requests to Dr. Alwin Scharstuhl, Rheumatol- never been previously used in OA. solRII has a very high affinity ogy Research Laboratory, Department of Rheumatology, University Medical Center, Geert Grooteplein zuid 26-28, 6525 GA, Nijmegen, The Netherlands. E-mail address: for TGF-␤1 and -␤3, the two most abundant isoforms of TGF-␤ in the [email protected] knee joint. Moreover, due to the small size of solRII it can penetrate 2 Abbreviations used in this paper: OA, osteoarthritis; PTG, proteoglycan; solRII, the articular cartilage and affect chondrocytes. We show for the first soluble TGF-␤-RII; i.a., intra-articular; MMP, matrix metalloproteinase; TIMP, tissue ␤ inhibitor of MMP; ADAMTS, a disintegrin and metalloproteinase with throm- time that inhibition of endogenous TGF- during experimental OA bospondin motif; Saf-O, Safranin O; PMB, polymyxin B. dramatically decreases osteophyte size but enhances PTG loss. Our

Copyright © 2002 by The American Association of Immunologists, Inc. 0022-1767/02/$02.00 508 INHIBITION OF ENDOGENOUS TGF-␤ DURING OA study implies a crucial role for endogenous TGF-␤ in osteophyte for- beled Ab (Vector Laboratories). Bound complexes were detected by the mation and cartilage maintenance. ECL Plus detection reagents (Amersham Pharmacia Biotech). Activity of solRII Materials and Methods Immunohistochemistry The blocking capacity of the recombinant solRII was determined via a TGF-␤ competition ELISA using solRII as a capture agent. To assure the The expression of TGF-␤1, -␤2, and -␤3 was studied during papain-in- stability of the solRII for the duration of the implantation period, the used duced OA. The papain model is characterized by PTG depletion of articular osmotic pumps were retrieved at the end of the experiment and flushed cartilage, which is followed by attempted replenishment of the articular with 200 ␮l PBS. The diluted samples were also tested via ELISA. Sam- cartilage with PTGs at approximately day 10 (18). The right knee joints of ples of recombinant solRII were mixed with TGF-␤1 in a molar ratio of mice were injected with 1 U papain solution; the left knee joints served as 1000:1Ð8000:1. The TGF-␤1 ELISA was performed as described in Quan- internal controls. Knee joints were dissected at days 7 and 14. Immuno- tification of TGF-␤1 and TGF-␤3 isoforms. histochemistry was performed on cryosections with specific Ab against TGF-␤1, -␤2, and -␤3 (R&D Systems, Abingdon, U.K.). As a negative Animals control, the primary Ab was replaced with chicken IgYs or goat IgGs. Male C57BL/6 mice aged 12 wk were used. Animals were kept in filter-top Biotin-labeled secondary Ab were used (Vector Laboratories, Burlingame, cages with a wood chip bedding. They were fed a standard diet and tap CA) followed by a biotin-streptavidin detection system (Vectra elite kit; water ad libitum. Vector Laboratories). Bound complexes were visualized via reaction with Ј Ј 3 ,3 -diaminobenzidine (Sigma-Aldrich, St. Louis, MO) and H2O2. Sec- Experimental design and histology tions were briefly counterstained with hematoxylin and mounted with permount. Alzet osmotic pumps (models 1007D and 2002; ALZA, Palo Alto, CA) were filled with the solution containing solRII (60 mg/ml)/PMB (2.63 mg/ Quantification of TGF-␤1 and TGF-␤3 isoforms ml) or PMB alone and as a control empty pumps were used. One day after Downloaded from i.p. implantation of the osmotic pumps, 1 U papain solution was injected ␤ ␤ Because TGF- 1 and - 3 were detected by immunohistochemistry, into the right knee joint. We aimed to induce a relatively mild OA to allow ␤ ␤ TGF- 1 and - 3 levels in knee joints were quantified via ELISA. There- for diminished and increased PTG loss of the cartilage due to the treatment ϭ fore, 1 U papain was injected in the right knee joints of mice (n 6); with solRII. Left knee joints served as internal controls for the systemic uninjected left knee joints served as internal controls. After 3 days, patellae treatment with solRII/PMB and PMB. To administer solRII for 3 wk, first were isolated and placed in RPMI medium/0.1% BSA. After 2 or 10 h of an osmotic pump model 2002 was implanted i.p., which after 14 days was culturing, the medium was analyzed for the presence of TGF-␤1 and -␤3 replaced by a model 1007D pump. After 7, 14, or 21 days mice were http://www.jimmunol.org/ via ELISA. In short, ELISA plates were coated with solRII (R&D Sys- sacrificed and whole knee joints were dissected and fixed for 4 days in 4% ␤ ␤ tems). TGF- 1or- 3 standards (R&D Systems) or samples were applied formalin. Subsequently, knee joints were decalcified in 5% formic acid and ␤ ␤ and subsequently incubated with anti-TGF- 1or- 3 Ab. The appropriate processed for paraffin embedding. Tissue sections (7 ␮m) were stained secondary biotinylated Ab were used, followed by incubation with strepta- with Safranin O (Saf-O)/fast red. vidin-polyperoxidase conjugate. Bound complexes were detected by reac- [35S]Sulfate incorporation was studied by i.p. injection of radiolabeled tion with orthophenylenediamine (Sigma-Aldrich) and H2O2. Absorbance sulfate (75 ␮Ci) 4 h before dissection of the knee joints. After histological was read at 492 nm using an ELISA plate reader (Multiscan MCC/340; ␮ processing, 7- m tissue sections were dipped in K5 emulsion (Ilford, Basil- Titertek, Huntsville, AL). don, Essex, U.K.) and exposed for 3Ð5 wk. Then the slides were developed Construction of the yeast expression vector and stained with H&E. All findings are the result of two independent experiments. Construction of the yeast expression vector and screening of solRII-ex- by guest on September 26, 2021 pressing clones are described elsewhere (19). In short, the complete extra- Image analysis of histology ␤ cellular domain of human TGF- type II receptor was cloned in the pPic-9 The surface area of the osteophytes was measured using the Qwin image expression plasmid (Invitrogen, San Diego, CA). During the cloning pro- ␣ analysis system (Leica Imaging Systems, Cambridge, U.K.), using a JVC cedure the cleavage signal of the Saccharomyces cerevisiae -factor se- 3-CCD color video camera. The size of the osteophytes was determined by cretion signal peptide and a sequence coding for six consecutive histidine manual selection of the surface area of the osteophyte in five semiserial residues (6xHis tag) were introduced via PCR. sections per knee, spaced ϳ50 ␮m apart. Measurements of eight knees per Production and purification of solRII group were averaged. The PTG content of articular cartilage was also measured using an im- For the production of solRII the Pichia pastoris expression system (In- age analysis system as described elsewhere (21). The noncalcified layer of vitrogen) was used. P. pastoris was cultured in the Bioflow 3000 tabletop the patellar and femoral cartilage and cartilage of both the lateral and me- fermenter (New Brunswick Scientific, Edison, NJ). In essence, the fermen- dial sides of the tibia-femur plateau was selected by hand. Loss of red tation process was conducted as described elsewhere (20). Yeast culture staining (loss of PTGs) was calculated by measuring the amount of blue supernatant was filtered through a 0.2-␮m filter (Schleicher & Schull, Das- light passing through the tissue section. Of each knee joint, three sections sel, Germany). solRII was purified from the supernatant by means of a per cartilage compartment were measured and the average was taken. Of 6xHis tag and a nickel-nitrilotriacetic acid column (Qiagen, Leusden, The each compartment the most severe depletion was considered as maximal Netherlands). Bound protein was eluted from the column with 300 mM PTG loss. The noncalcified cartilage of the control left knees were also imidazole in wash buffer. Imidazole was removed via a HiPrep 26/10 de- measured and averaged and the PTG content was stated as being 100%. All salting column (Amersham Pharmacia Biotech, Little Chalfont, U.K.). The measurements were expressed on a scale of 0Ð100% PTG content as mea- protein was subsequently further purified and concentrated using m.w. cut- sured by Saf-O staining. off 100,000 and 10,000 filters (Millipore, Etten-Leur, The Netherlands) to We also noted a thinning of the articular cartilage, which resulted in a a final concentration of ϳ60 mg/ml. Although yeast cells do not contain decrease in the thickness of the cartilage. This loss of noncalcified articular LPS, the endotoxin level of purified protein was analyzed via the endosafe cartilage of the patella and femur was measured in three sections, spaced kinetic turbidimetric assay (Charles River Endosafe, Kent, U.K.). How- ϳ50 ␮m apart by manual selection of the noncalcified cartilage. Then the ever, yeast supernatant contained an unknown interfering substance that surface area was determined and values were averaged. confounded test results (data not shown). Therefore, an endotoxin inhibitor, polymyxin B (PMB; Sigma-Aldrich), was added before implantation as a MMP plus ADAMTS plus TIMP mRNA expression precautionary measure. Treatment with solRII during experimental OA increases PTG loss com- SDS-PAGE and Western blotting procedures pared with untreated animals. We investigated whether solRII had an effect on the mRNA expression of matrix metalloproteinase (MMP)-3, -13, and SDS-PAGE analysis of recombinant solRII was performed on a 12% gel -14, a disintegrin and metalloproteinase with thrombospondin motif (AD- under denaturating conditions. Proteins were visualized via a standard sil- AMTS)-4, and -5, and tissue inhibitors of MMPs (TIMP)-1 and -3. There- verstaining procedure. fore, papain (1 U) was injected in the right knee joints of mice (n ϭ 6 per For Western blotting analysis, solRII protein was blotted on a polyvi- group). Uninjected left knee joints served as internal controls. After 3 days, nylidene difluoride membrane (Amersham Pharmacia Biotech). solRII pro- patellae were isolated and placed in 200 ␮l RPMI 1640 (Dutch modifica- tein was detected by an Ab against the extracellular part of the TGF-␤-RII tion) medium (Life Technologies, Breda, The Netherlands) for 24 h as (R&D Systems) in combination with the appropriate secondary biotin-la- described previously (22, 23). Patellae were treated with solRII (10 ␮g/ml) The Journal of Immunology 509 or IL-1 (10 ng/ml) as a positive control or were not treated. Next, RNA was isolated and treated with reverse transcriptase (Life Technologies). The RT-PCR protocol started with a 1-min denaturation at 92¡C followed by annealing at 60¡C for 1 min (58¡C for TIMP-3) and a 1-min extension at 72¡C. PCR products were subjected to electrophoresis in a 1% low-melting agarose gel (Seaplaque; SanverTech, Boechout, Belgium) containing ethidium bromide. For each primer set, the cycle number at which the PCR product was first detected on the agarose gel was identified. It was determined that in this phase the PCR still performs linearly. The PCR products were excised from the gel. PCR products were quantified using PicoGreen (Molecular Probes, Eugene, OR), a fluorescent dye that selectively stains dsDNA, according to the manufacturer’s recommendations. GAPDH levels were used to correct for the amount of template added to the PCR mix. For control and papain-injected knee joints the mRNA levels in the nontreated group were stated as 100%. Each PCR was performed in triplicate and results were averaged. Statistical analysis Results were analyzed via a Student t test. Results were considered significant at p Ͻ 0.05.

Results Downloaded from Immunohistochemistry The expression of TGF-␤ isoforms was studied in papain-injected joints and compared with normal knee joints, using immunohisto- chemical methods on cryosections of whole knee joints. In the periosteum of normal knee joints, hardly any expression of TGF-␤1 and TGF-␤3 was observed. After injection of papain http://www.jimmunol.org/ the expression of both TGF-␤ isoforms was enhanced in the periosteum, where the expression of TGF-␤3 seemed more intense than TGF-␤1 expression (Fig. 1, AÐD). The expression was present at locations known to develop osteophytes. TGF-␤2 expression was not detected in normal periosteum or in periosteum after papain injection. Thus, TGF-␤1 and -␤3 seem to be up-regulated after papain injection at locations in the periosteum linked to osteophyte formation. Besides expression in the periosteum, chondrocytes in the ar- by guest on September 26, 2021 ticular cartilage expressed TGF-␤1 and -␤3 isoforms. The expression was mostly confined to the noncalcified layer of the cartilage. As in the periosteum, this expression was up-regulated after papain injection compared with noninjected left knee joints (Fig. 1, EÐH). ␤ In addition, in the cartilage the staining for TGF- 3 seemed more FIGURE 1. Immunohistochemistry of TGF-␤ isoforms of normal and pa- ␤ ␤ intense than TGF- 1 staining. Again, no staining for TGF- 2 was pain-injected knee joints (day 7). Right knee joints were injected with papain observed. Negative controls, such as substitution of the primary solution. The noninjected left knee joints severed as internal controls. TGF-␤ Ab with chicken IgYs or goat IgGs, did not result in a color re- expression in periosteum (AÐD) and cartilage (EÐH). TGF-␤1 expression in action (Fig. 1, I and J). normal knee joint (A) and after papain injection (B). TGF-␤3 expression in The results show that TGF-␤1 and TGF-␤3 isoforms are ex- normal knee joint (C) and after papain injection (D). TGF-␤1 expression in left pressed in periosteum and articular cartilage and are up-regulated knee joint (E) and after papain injection (F). TGF-␤3 expression in left knee after papain injection. Next, we tried to quantify TGF-␤1 and -␤3 joint (G) and after papain injection (H). Negative control of noninjected left levels in knee joints and we studied the effect of papain injection. knee joint (I) and of papain-injected joint (J). Arrowhead, Synovial layer; arrow, periosteum; broad arrows, TGF-␤1-positive cells; js, joint space. Quantification of TGF-␤1 and TGF-␤3 isoforms The effect of papain injection on TGF-␤1 and TGF-␤3 protein levels in knee joints was compared with noninjected knee joints We proceeded to study the effect of TGF-␤ inhibition during ex- via ELISA. TGF-␤1 was detected in 2-h patellae wash-outs from perimental OA on these joint structures. We made use of solRII, control left knee joints (Table I). Part of this released TGF-␤ is the extracellular part of the TGF-␤-RII, as a specific TGF-␤ in- probably the result of the patella isolation procedure itself. Nev- hibitor. solRII has a high affinity for TGF-␤1 and -␤3 and only low ertheless, injection of papain increased TGF-␤1 protein expression affinity for TGF-␤2. significantly from 10 pg/ml in control patellae to 30 pg/ml ( p Ͻ 0.05). TGF-␤3 protein was also detected in wash-outs from control SDS-PAGE and Western blotting of solRII produced by P. patellae (200 pg/ml, Table I). Injection of papain increased the pastoris expression of TGF-␤3 2-fold in 2-h wash-outs ( p Ͻ 0.001). Ap- Before using solRII to inhibit TGF-␤ in vivo we characterized the proximately 20 times more TGF-␤3 than TGF-␤1 was detected, recombinant solRII as produced by P. pastoris. Therefore, purified which supports the observation from the immunohistochemistry solRII was analyzed via SDS-PAGE and Western blot analysis. experiment that murine knee joints seem to contain more TGF-␤3 Recombinant solRII showed a pattern of protein bands ranging than TGF-␤1. The up-regulation of TGF-␤1 and -␤3 protein was from ϳ25 to 40 kDa (data not shown). The different bands are the also found in 10-h wash-outs with similar results (data not shown). result of heterogeneous glycosylation (19). The Ab used did not 510 INHIBITION OF ENDOGENOUS TGF-␤ DURING OA

Table I. Quantiﬁcation of TGF-␤1 and TGF-␤3 isoformsa

Injection TGF-␤1 TGF-␤3

Control 8.95 Ϯ 7.35 184.66 Ϯ 28.17 Papain 33.36 Ϯ 18.07* 387.83 Ϯ 65.25**

a Right knee joints were injected with 1 U papain solution. The noninjected left knee joints severed as internal controls. Three days after papain injection, patellae with surrounding tissue were isolated and transferred to RPMI medium supplemented with 0.1% BSA. Wash-outs of 2 h were analyzed for the presence of TGF-␤1 and -␤3 via ELISA. The concentration of TGF-␤ is shown. Results are the mean Ϯ SD of six patellae wash-outs per group. .p Ͻ 0.05 ,ء .p Ͻ 0.001 ,ءء FIGURE 3. Measurement of osteophyte size. The surface area of osteophytes was determined. From each mouse (n ϭ 8 per treatment), ﬁve cross-react with BSA. Our recombinant solRII was estimated to be semiserial tissue sections from the knee joint were measured and the av- Ͼ95% pure. erage was taken. The eight values obtained per treatment were averaged .(p ϭ 0.001 ,ءء ;p Ͻ 0.05 ,ء) and analyzed via a Student t test Neutralizing activity of solRII

We proceeded to determine the blocking activity of freshly pre- Downloaded from pared solRII with an ELISA. To fully neutralize TGF-␤, a 4000- Osteophytes fold excess of solRII was required (data not shown). solRII in To determine the role of endogenous TGF-␤ in osteophyte forma- excess of 2000- and 1000-fold prevented detection of TGF-␤ in the tion we treated mice with solRII during experimental OA. Papain order of 84 and 68%, respectively. injection in the knee joint was used to cause the formation of To apply the TGF-␤ inhibitor in vivo, we first checked the sta- osteophytes at specific locations. Osteophytes developed on the bility of the solRII in osmotic pumps, implanted i.p. in mice. femur close to the medial collateral ligament and on the tibia be- http://www.jimmunol.org/ Therefore, after 7 and 14 days the remainder of the protein was tween the articular cartilage and where the growth plate meets the flushed from the retrieved osmotic pumps. Comparable concentra- joint space. Early osteophytes consisted of large round chondro- tions of freshly and retrieved solRII were equally effective in cytes, which stained with Saf-O and had not yet undergone the blocking TGF-␤, indicating no loss of blocking activity of the process of endochondral ossification (Fig. 2, A and E). Autoradio- retrieved solRII protein (data not shown). This implies that, during graphic analysis showed that these chondrocytes had incorporated the complete period, active solRII was administered. radioactive sulfate in high amounts, suggesting a very active cell by guest on September 26, 2021

FIGURE 2. Reduced osteophyte formation after solRII treatment during papain-induced OA. Frontal sections of whole knee joints were made and stained with Saf-O (A, C, E, and G). PTG synthesis was visualized via the incorporation of [35S]sulfate and H&E staining (B, D, F, and H). A, Osteophyte on tibia in solvent-treated animal. B, Autoradiography of A. Note intense 35S incorporation at the location of osteophyte. C, For comparison, the same region as A in solRII-treated animal. D, Autoradiography of C. E, Osteophyte on femur in solvent-treated animal. F, Autoradiography of E. Note intense 35S incorporation at the location of osteophyte. G, For comparison, the same region as E in solRII-treated animal. H, Autoradiography of G. Osteophytes are indicated by arrowheads. Original magnification, ϫ100. The Journal of Immunology 511 metabolism (Fig. 2, B and F). Treatment with recombinant solRII Cartilage reduced the average size of the osteophytes located on the femur ␤ significantly ( p Ͻ 0.02) from 9616 ␮m2 in the solvent-treated The role of endogenous TGF- on articular cartilage PTG content animals to 2746 ␮m2 in the solRII-treated mice (Fig. 3). On the after papain injection was studied using recombinant solRII. Inhi- tibia, the average size of the osteophytes was reduced from 9849 bition of TGF-␤ activity for 7 days with solRII decreased PTG ␮m2 in solvent-treated mice to 2653 ␮m2 in solRII-treated animals staining in five of six cartilage compartments compared with treat- ( p ϭ 0.007). Autoradiographic analysis showed a less-active cell ment with solvent (Fig. 4, CÐF, and Fig. 5). The effect of TGF-␤ metabolism in the solRII group than in the solvent-treated group. inhibition was most striking in patella and femoral cartilage. In These results strongly implicate endogenous TGF-␤ as an impor- these cartilage compartments a significant reduction ( p Ͻ 0.001) tant factor in osteophyte development. in relative PTG content of 43 and 38%, respectively, was observed Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 4. Reduced cartilage PTG content after solRII treatment during papain-induced OA. Systemic administration of solRII for 7 or 14 days induces changes in patella-femoral cartilage (AÐD) and lateral femoral-tibia cartilage (EÐH) after papain injection. Saf-O stained frontal sections of murine knee joints. Shown are the control left knee joint of solvent-treated animal (A) and of solRII-treated animal (B). Also shown is cartilage of solvent-treated animal (C) and of solRII-treated animal (D). Note the decreased staining intensity of the cartilage and the decrease in cartilage thickness. E and F, Cartilage of solvent-treated animal (E) and of solRII-treated animal (F). Again decreased staining intensity is observed. G and H, Day 14 of solvent-treated animal (G) and solRII-treated animal (H). P, Patella; F, femur, T, tibia; js, joint space. Original magniﬁcation, ϫ100. Inset shows PTG content of the medial tibia at .(p Ͻ 0.05 ,ء) day 14 as a percentage of normal cartilage. Open bar, Solvent treated; ﬁlled bar, solRII treated 512 INHIBITION OF ENDOGENOUS TGF-␤ DURING OA

ness, indicating a pivotal protective role for endogenous TGF-␤ in cartilage.

MMP plus ADAMTS plus TIMP mRNA expression Inhibition of endogenous TGF-␤ during experimental OA reduces articular cartilage PTG content. To gain a better understanding of how TGF-␤ inhibition leads to enhanced PTG loss, we investigated the effect of solRII on the expression of cartilage-degrading proteinases MMP-3, -13, and -14, and ADAMTS-4 and -5, or inhibitors of degradation TIMP-1 and -3. Treatment with solRII had no significant effect on the MMP-3 FIGURE 5. Measurement of cartilage PTG content. For each knee joint expression in patellae of noninjected knee joints. However, after (n ϭ 8 per treatment), the PTG content of six noncalcified articular carti- papain injection, solRII treatment resulted in an ϳ4-fold up-reg- lage surfaces was determined. Loss of Saf-O staining of the articular cartilage was measured in three semiserial tissue sections and values were ulation of MMP-3 mRNA (Table III). Although no effect of solRII averaged. The eight values obtained per compartment were averaged and was seen on MMP-13 expression in control knee joints, treatment differences were analyzed via a Student t test. pat., Patella; fem., femur; with solRII after papain injection resulted in an ϳ4-fold up-regu- tib., tibia; lat., lateral; med., medial. Open bar, Solvent treated; filled bar, lation of MMP-13 mRNA (Table III). We could not identify sig- .p ϭϽ0.001 ,ءء ;p Ͻ 0.05 ,ء .solRII treated

nificant effects of solRII treatment on MMP-14, ADAMTS-4 and Downloaded from -5, or TIMP-1 and -3 mRNA expression in patellae from normal and papain-injected joints (data not shown). (Fig. 4, C and D, and Fig. 5). Also, the lateral femur and lateral We used stimulation with IL-1 as a validation of the method tibia, as well as the medial femur, showed a significant decrease in used to determine the up-regulation of MMP mRNA levels. As PTG content ( p Ͻ 0.05). No significant effects after 7 days of expected, treatment with IL-1 up-regulated MMP-3 and -13 solRII treatment were observed in medial tibia cartilage. However, mRNA expression significantly in both noninjected and papain- after 14 days of treatment with solRII, the cartilage PTG content of http://www.jimmunol.org/ injected knee joints (data not shown). These results indicate the the medial tibia was significantly less than in the solvent-treated ability of isolated patella tissue to respond to stimuli as IL-1. animals ( p Ͻ 0.05) (Fig. 4, G and H). The other cartilage com- From these data we conclude that the observed enhanced PTG partments did not significantly differ between treatments at day 14 loss in articular cartilage after solRII treatment is probably the (data not shown). After 21 days of treatment with solRII, the over- result of up-regulated MMP-3 and -13 expression. all PTG content of the articular cartilage of the tibia/femur plateau was significantly lower than in the control-treated mice ( p Ͻ 0.001) (data not shown). Discussion Treatment with solvent, compared with animals receiving an Our experiments identified for the first time the role of endogenous empty pump, had no significant effects on PTG content in any of TGF-␤ during experimental OA. Via administration of the solRII by guest on September 26, 2021 the cartilage surfaces on days 7, 14, or 21 (data not shown). The we inhibited endogenous TGF-␤. This enabled us to study the control left knee joints of mice receiving solRII for 7, 14, or 21 impact of neutralization TGF-␤ on osteophyte formation and ar- days were indistinguishable from left knee joints of solvent-treated ticular cartilage degradation during experimental OA. Blocking of animals (Fig. 4, A and B), indicating that systemic treatment with endogenous TGF-␤ in the knee joint resulted in an almost com- solRII itself did not induce pathological effects in the knee joint. plete inhibition of osteophyte formation. Furthermore, systemic Besides a decrease in PTG content, solRII treatment also had an delivery of solRII not only decreased articular cartilage PTG con- effect on the thickness of uncalcified cartilage (Fig. 4, C and D). tent but also resulted in cartilage loss, probably via the up-regu- Measurements of patellar cartilage revealed a significant decrease lation of MMP expression. in surface area between solvent- and solRII-treated animals, We chose the extracellular part of TGF-␤-RII as a TGF-␤ an- 22,610 and 16,351 ␮m2, respectively, and p Ͻ 0.001 (Table II). tagonist because it has a very high affinity for TGF-␤1 and -␤3 (24, The femoral uncalcified cartilage also showed a significant de- 25), which are both abundantly present in the joint (12, 26, 27). crease ( p Ͻ 0.05) after solRII treatment (22,463 vs 26,077 ␮m2 in Moreover, because of its small size of 25 kDa, solRII can penetrate solvent-treated animals). These findings further imply a role for cartilage and directly affect chondrocytes (28). Chondrocytes are endogenous TGF-␤ in cartilage maintenance. difficult cells to target due to the dense network of collagen fibrils Taken together, inhibition of endogenous TGF-␤ activity re- and PTGs that make up articular cartilage and render it virtually sulted in a decreased PTG content and decreased cartilage thick- impenetrable for large molecules such as Abs.

Table II. Reduced cartilage thickness after solRII treatment at day 7a Table III. Expression levels of MMP-3 and MMP-13 mRNA after solRII treatmenta Treatment Patella Femur Knee Joint MMP-3 MMP-13 Solvent 22,610 Ϯ 750 26,077 Ϯ 690 solRII 16,351 Ϯ 875* 22,463 Ϯ 748** Left (Ϫ papain) 33 Ϯ 20 87 Ϯ 35 Right (ϩ papain) 404 Ϯ 162 382 Ϯ 103 a Osmotic pumps filled with solRII or solvent were implanted i.p. in mice (n ϭ 8 per treatment). The next day, right knee joints were injected with 1 U papain solution. a Right knee joints were injected with 1 U papain solution and left knee joints At day 7, knee joints were dissected and frontal sections of whole knee joints were served as internal controls. After 3 days, patellae with surrounding tissue were iso- made and stained with Saf-O. For each knee joint, the surface area of the noncalcified lated and transferred to RPMI medium supplemented with solRII (10 ␮g/ml). MMP-3 layer of articular cartilage from the patella-femur junction was determined in three and -13 RT-PCR products were quantified using PicoGreen. GAPDH levels were used semiserial tissue sections. The eight values obtained per compartment were averaged to correct for the amount of template used. For left and papain-injected knee joints the and analyzed via a Student t test. Shown is a representative experiment of three Ϯ SD. mRNA levels of the nontreated patellae were stated as 100%. Each PCR was per- p Ͻ 0.05. formed in triplicate and results were averaged. Results were analyzed via a Student t ,ء .p Ͻ 0.001. test. Shown is the mean Ϯ SD ,ءء The Journal of Immunology 513

The solRII we produced via the P. pastoris expression system is duced PTG content. In conclusion, endogenous TGF-␤ is a nec- a biologically active protein. A 4000-fold excess of recombinant essary agent to protect articular cartilage from PTG loss. solRII to TGF-␤ was able to completely neutralize TGF-␤ activity. Besides effects on PTG content, TGF-␤ deprivation also ap- This is well in range with other studies in which a 1,600- to peared to increase cartilage loss. We found that the thickness of 20,000-fold excess of solRII was needed to fully inhibit TGF-␤ cartilage was decreased after blocking TGF-␤. Increased cartilage activity (25, 29). The solRII obtained from the osmotic pumps loss might be related to a decreased resistance to mechanical load- retrieved from the mice after implantation showed no loss of ing. Because inhibition of endogenous TGF-␤ decreased PTG con- blocking activity compared with freshly produced solRII. This in- tent, and because PTGs are important molecules responsible for dicates that during the complete duration of the experiment we the resistance of articular cartilage (43Ð45), loss of PTGs may lead were able to inhibit TGF-␤ activity. to loss of cartilage. Therefore, endogenous TGF-␤ appears impor- One of the main characteristics of OA is the formation of new tant in the prevention of cartilage loss. cartilage and bone on the joint edges, so-called osteophytes. Os- We found that inhibition of endogenous TGF-␤ leads to en- teophytes are the principal cause of pain in OA patients and thus hanced cartilage PTG loss. This can be the result of either up- an unwanted feature of the disease. Osteophytes are believed to regulation of cartilage-degrading proteases or down-regulation of originate from the periosteum. Periosteal cells have the potential to the natural inhibitors of these enzymes. To clarify the mechanistics undergo chondrogenesis and osteogenesis in vivo and in vitro (30Ð of TGF-␤ inhibition on articular cartilage, the mRNA levels of 32). In developing osteophytes, not only is TGF-␤ mRNA strongly several MMPs, ADAMTS, and TIMPs in the knee joint were de- expressed but also TGF-␤-R expression can be found (12, 13). termined after exposure to solRII. Our results suggest that inhibi- Chondrogenesis of periosteal cells can be enhanced by TGF-␤ in tion of endogenous TGF-␤ via solRII enhances cartilage PTG loss Downloaded from a dose-dependent manner (33Ð35). We previously showed that al- via up-regulation of MMP-3 and MMP-13. We observed only a ready 1 day after i.a. injection of TGF-␤ increased PTG synthesis small stimulation on ADAMTS-4 and -5 mRNA expression after was observed in periosteal cells at sites that later show massive solRII treatment. The expression levels of TIMP-1 and -3 were osteophyte development (14). However, these studies did not unchanged after solRII and IL-1 treatment. Thus, endogenous prove a role of endogenous TGF-␤ during osteophyte formation. In TGF-␤ suppresses the expression of several MMPs, such as this study we show expression of TGF-␤1 and -␤3 in periosteum MMP-3 and -13. http://www.jimmunol.org/ after papain injection. Blocking of TGF-␤1 and -␤3 activity by use In summary, inhibition of endogenous TGF-␤ leads to an in- of the solRII resulted in a dramatic reduction in osteophyte for- creased loss of PTGs from the cartilage and enhances cartilage loss mation. So, here we show for the first time that endogenous TGF-␤ during experimental OA, most likely through the up-regulation of is essential in osteophyte formation during experimental OA. MMPs. This indicates a protective role for endogenous TGF-␤ on One of the first signs of OA is the loss of PTGs from the artic- cartilage. 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