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 References This article cites 45 articles, 11 of which you can access for free at: http://www.jimmunol.org/content/169/1/507.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 26, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts 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: 507–514. 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 (11– 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 (7–9). 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 1000:1–8000:1. 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.