Ann Rheum Dis 2001;60:iii81–iii84 iii81 Ann Rheum Dis: first published as 10.1136/ard.60.90003.iii81 on 1 November 2001. Downloaded from Osteoprotegerin

U Feige

Osteoclast diVerentiation and activation resistant alkaline phosphatase (TRAP), cathep- DiVerentiation, activation, and survival of sin K, calcitonin, and vitronectin receptors (for are regulated by the balance be- review see Chambers22). A mature tween RANKL (RANK ligand) and osteopro- forms a ruZed border, an actin ring, and a tegerin (OPG). OPG is also known as osteo- sealed or clear zone and will form a resorption clast inhibitory factor (OCIT) and is a tumour pit on dentin slices.12 20 23 24 However, there are necrosis factor (TNF) receptor family mem- diVerences between in vitro generated osteo- ber, which is found as a “decoy” receptor only clasts and harvested osteoclasts ex vivo. One because of the lack of a membrane spanning such diVerence is the ability of interleukin 1 domain.12 RANKL is a TNF family member (IL1) to induce an actin ring in in vitro gener- 25 and is also known as osteoprotegerin ligand ated but not in ex vivo osteoclasts. (OPG-L), or osteoclast diVerentiation factor (ODF), or TNF related, activation-induced cytokine (TRANCE).3–5 RANKL exhibits its Osteoclast survival activity on cells through RANK (receptor acti- Survival of mature osteoclasts is dependent on vator of NF-êB), another membrane bound the presence of RANKL,16 though other factors member of the TNF receptor family.6–11 In such as IL1 can promote osteoclast survival, marrow, RANKL is produced by osteo- too.25 If osteoclast survival is not suYciently blasts and both osteoblastic and fibroblastic supported osteoclasts will undergo apopto- 17 26 stromal cells. The production of RANKL is sis. In fact, apoptotic osteoclasts in low induced by most (all?) known inducers of bone numbers can be detected after one week in resorption and hypercalcaemia.12–16 Precursor bone marrow cultures driven by M-CSF and 17 cells in bone marrow exposed to M-CSF RANKL. However, if survival factors are (monocyte/macrophage colony stimulating withdrawn, all osteoclasts will undergo apopto- Inflammation Group, sis quickly.17 These eVects can also be demon- Department of factor or CSF-1) and RANKL will diVerentiate 17 26 Pharmacology/Pathology, within one week to mature osteoclasts.3 Both strated in vivo. For example, in mice treated Preclinical with 10 mg/kg OPG intravenously all osteo- M-CSF and RANKL are necessary and suYcient 17 Development, Amgen for this process,17–19 whereas RANKL but not clasts disappear within 48 hours. Interest- Inc, Thousand Oaks, ingly, within 7–10 days osteoclasts return and M-CSF will activate mature osteoclasts to California, USA can be found in normal numbers and typical resorb bone.20 OPG added to such bone U Feige locations in these mice.17 marrow cultures will inhibit the process of Correspondence to: osteoclast generation reversibly.17 18 21 This can

Dr U Feige, Mail Stop http://ard.bmj.com/ 15–2-A, One Amgen Center be shown by either removing the OPG after a Drive, Amgen Inc, Thousand few days or by adding more RANKL to the Lessons from transgenic and knockout Oaks, CA 91320–1799, USA cultures, both of which will result in renewed mice [email protected] osteoclast diVerentiation. Mature osteoclasts Osteoclasts and drive bone remod- Accepted 28 June 2001 are characterised by markers such as tartrate elling, a continuous process of osteoclasts resorbing bone and osteoblasts laying down 1,25(OH)2-D3 new bone. Bone resorption is necessary for skeletal growth as well as tooth eruption.27 on September 28, 2021 by guest. Protected copyright. Excess RANKL activity as in OPG−/− mice PTH results in spontaneous fractures and vertebral 28 PTHrp deformities. Studies using knockout and transgenic mice have shown that skeletal growth is normal in OPG knockout mice Osteoclast (which have high net RANKL levels and show PGE2 RANKL differentiation marked )28 29 and OPG transgenic and activation mice (which have low net RANKL levels and show osteopetrosis).130 In contrast, in IL1 RANKL−/− and RANK−/− mice skeletal 931 TNFα growth is impaired. This illustrates that bio- logically there is quite a diVerence between low OPG RANKL levels or no RANKL at all. Without IL11 inhibits RANKL (or RANK) tooth eruption cannot LIF occur (RANKL−/− or RANK−/− mice), whereas with a little RANKL tooth eruption is IL6 normal (OPG transgenic mice). Although cells of the macrophage lineage also express RANK, it is interesting to note that Figure 1 The balance of RANKL and osteoprotegerin (OPG) controls osteoclast activity. macrophage diVerentiation is normal in OPG Most (all?) known inducers of bone resorption and hypercalcaemia act indirectly through production of RANKL, hence OPG can be used pharmacologically to control osteoclast transgenic and RANKL−/− mice and thus activity independent of the inducing cytokine. seems to be independent of RANKL.131

www.annrheumdis.com iii82 Feige Ann Rheum Dis: first published as 10.1136/ard.60.90003.iii81 on 1 November 2001. Downloaded from http://ard.bmj.com/ on September 28, 2021 by guest. Protected copyright.

Figure 2 Administration of recombinant osteoprotegerin (OPG) alleviates bone destruction in arthritis. (A, B, and C) Tibiotarsal (“ankle”) joint of a normal Lewis rat (A) compared with severely inflamed joints of animals with advanced mycobacterial-induced adjuvant arthritis (B, C). Rats given OPG at 4 mg/kg/day subcutaneously for seven days beginning at the onset of clinical arthritis, retained skeletal integrity (C), whereas untreated rats did not (B). The arrowhead denotes the junction of the growth plate with cortical bone; the growth plate is destroyed in untreated arthritic rats (B). (D, E, and F) and cartilage of the proximal intertarsal joint of normal (D) and OPG treated (F) arthritic rats are intact. In contrast, bone is eroded and cartilage matrix is degraded (denoted by pallor) in untreated arthritic rats (E). Asterisks denote inflammation. Stains: haematoxylin and eosin (A-C), toluidine blue (D-F). Photo courtesy of Dr Brad Bolon.

Hypercalcaemia histologically17 but which show functional se- RANKL treatment of mice results in induction, quelae, such as hypercalcaemia.312 Known diVerentiation, and activation of osteoclasts inducers of bone resorption and hypercalcae- which can be demonstrated and quantified mia, such as IL1, TNFá,

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(PTH), PTH related peptide (PTHrp), vitamin allowed for the diVerentiation of osteoclasts 35 39 D3 (1,25(OH)2-D3), etc, have been tested alone only if M-CSF was also present. This eVect and jointly with OPG. In all cases OPG was inhibited by addition of OPG to the bone prevented the induction of hypercalcaemia, marrow cultures. Furthermore, formalin fixed, indicating that all those inducers act indirectly activated T cells could substitute for RANKL, through the induction of RANKL.15 This which indicates that RANKL is active both in conclusion is supported by the fact that injection soluble and in the cell surface bound form. of RANKL, IL1â, TNFá, PTHrp, or Both activated CD4 and CD8 T cells can sup- 40 1,25(OH)2-D3 into RANK−/− mice fails to port osteoclast diVerentiation. As many other induce hypercalcaemia.9 These studies and oth- cytokines (IL1, TNFá, IL6, interferon ã, IL3, ers not reviewed here led to the view, depicted in etc) which induce RANKL in osteoblasts and fig 1, of RANKL as a unique cytokine in osteo- osteoblastic stromal cells are generated in such clast diVerentiation, activation, and survival. T cell cultures, formalin fixed, activated T cells from RANKL−/− mice were added to bone Tumours marrow cells and found not to induce osteo- Many tumour types metastasise into bone. clasts.35 This indicates that it is the surface These eVects can be mediated through PTHrp bound RANKL (and not other factors) on T or IL6, other inducers of bone resorption. Sub- cells which drives osteoclast diVerentiation in jects with a tumour often show hypercalcae- this cell culture system.35 mia. In mouse tumour models, such as the C26 (originally derived from an adenocarcinoma) OPG protects bone in arthritis model, it has been shown that OPG treatment The above findings prompted us to look into T dose dependently prevents tumour-induced cell mediated autoimmune diseases and bone hypercalcaemia.32 Furthermore, OPG can also eVects. Mycobacteria-induced adjuvant arthri- reverse tumour-induced hypercalcaemia in tis in Lewis rats was chosen as a model. Severe these models.32 Interestingly, OPG treatment polyarthritis with extensive joint destruction is can prevent osteolysis and tumour metastasis induced in these rats by an injection of a into bone.33 The data demonstrate that tumour suspension of heat killed mycobacteria in oil at cell derived inducers of bone resorption, such the base of the tail.41 Nine days later clinical as PTHrp, IL6, or TNF , act indirectly á symptoms of disease, paw swelling, and weight through increases in RANKL. loss begin, and disease severity is monitored by their daily measurement. Bone and cartilage Activated T cells produce RANKL destruction in this model is very rapid. By day Other cells besides osteoblasts and osteoblastic 16 significant loss of bone has occurred as stromal cells can also produce RANKL. Inter- shown by erosions and by significant loss of estingly, RANKL is produced during, and is bone mineral density in the remaining bone.41 necessary for, mammary gland development (for We routinely measure bone mineral density in details see Fata et al34). However, in the following the calcaneus on day 16 by dual enhanced x ray the discussion will be restricted to T cells as absorptiometry(DEXAscan).41 Treatment of producers of RANKL.35 During T cell activation

arthritic rats with OPG daily subcutaneously http://ard.bmj.com/ a first signal occurs through the interaction of from day 9 to 16 resulted in a dose dependent peptide in the groove of the major histocompat- inhibition of the loss of bone mineral density ibility complex class II on the surface of antigen and bone erosive processes.35 In fact, osteoclast presenting cells and the T cell receptor, and a numbers, which were high in the arthritis second signal or costimulatory signal occurs group (80 ± 20) osteoclasts/mm2), were dose through the interaction of B7 on antigen dependently reduced to levels found in normal presenting cells and CD28 on T cells. Activation rats (9 ± 2) osteoclasts/mm2). Interestingly, of T cells leads to induction of CTLA-4, a inflammatory parameters, paw swelling and on September 28, 2021 by guest. Protected copyright. member of the CD28 family. In contrast with body weight loss, were not aVected by OPG the activatory properties of CD28, CTLA-4 is a treatment at all. OPG inhibits these bone down regulatory element. CTLA-4 binds 50- erosive processes leaving less room for infiltrat- fold better to B7 on antigen presenting cells, ing inflammatory cells (fig 2). The results indi- resulting in down regulation of T cell activity. cate that at least in this animal model of arthri- Samson recently reviewed and discussed the tis all eVects which lead to bone erosive relative roles of CD28 and CTLA-4.36 Not processes go through (the induction of) unexpectedly, in CTLA-4−/− mice a hyperacti- RANKL and, furthermore, the eVects of IL1, vated T cell compartment is seen.37 38 The TNFá, and other mediators of bone resorption activated T cells in CTLA-4−/− mice produce seem to be indirect through the induction of RANKL and, consequently, CTLA-4−/− ex- RANKL.35 hibit severe osteoporosis.35 OPG treatment of CTLA-4−/− mice results in increased bone mineral density and a significantly reduced Conclusions number of osteoclasts.35 + (All) known stimulators of bone resorption RANKL is induced in T cells upon activa- seem to work indirectly through the induc- tion through the T cell receptor. Several stud- tion of RANKL. ies have considered the function of RANKL/ + Activated T cells produce RANKL, which is RANK in the interaction of T cells and active both when cell surface bound and dendritic cells.56 Supernatants of activated T when soluble. cell cultures added in place of RANKL in the + The balance of RANKL and OPG deter- above described bone marrow culture system mines osteoclast activity (bone resorption).

www.annrheumdis.com iii84 Feige Ann Rheum Dis: first published as 10.1136/ard.60.90003.iii81 on 1 November 2001. Downloaded from

+ OPG can inhibit bone metastasis of tumours 20 Burgess TL, Qian Y, Kaufman S, Ring BD, Van G, Capparelli C, et al. The ligand for osteoprotegerin (OPGL) by inhibiting bone resorption. directly activates mature osteoclasts. J Cell Biol 1999;145: + OPG prevents bone loss in adjuvant arthritis 527–38. 21 Yasuda H, Shima N, Nakagawa N, Mochizuki SI, Yano K, without eVect on inflammation. Fujise N, et al. Identity of osteoclastogenesis inhibitory fac- + OPG may provide a pharmacological tool tor (OCIF) and osteoprotegerin (OPG): a mechanism by for osteoporotic and erosive bone disor- which OPG/OCIF inhibits osteoclastogenesis in vitro. 42 Endocrinology 1998;139:1329–37. ders. 22 Chambers TJ. Regulation of the diVerentiation and function of osteoclasts. J Pathol 2000;192:4–13. 23 Chambers TJ, Revell PA, Fuller K, Athanasou NA. Resorp- I thank Brad Bolon and Giuseppe Campagnuolo for their con- tion of bone by isolated rabbit osteoclasts. J Cell Sci 1984; tributions and stimulating discussions. 66:383–99. 24 Hakeda Y, Kobayashi Y, Yamaguchi K, Yasuda H, Tsuda E, 1 Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang Higashio K, et al. Osteoclastogenesis inhibitory factor MS, Lüthy R, et al. Osteoprotegerin: a novel secreted pro- (OCIF) directly inhibits bone-resorbing activity of isolated tein involved in the regulation of bone density. Cell mature osteoclasts. Biochem Biophys Res Commun 1998; 1997;89:309–19. 251:796–801. 2 Morinaga T, Nakagawa N, Yasuda H, Tsuda E, Higashio K. 25 Fox S, Fuller K, Chambers TJ. Activation of osteoclasts by Cloning and characterization of the gene encoding human interleukin-1: divergent responsiveness in osteoclasts osteoprotegerin/osteoclastogenesis-inhibitory factor. Eur J formed in vivo and in vitro. J Cell Physiol 2000;184:334– Biochem 1998;254:685–91. 40. 3 Lacey DL, Tan HL, Lu J, Kaufman S, Van G, Qiu W, et al. 26 Akatsu T, Murakami T, Ono K, Nishikawa M, Tsuda E, Osteoprotegerin ligand modulates murine osteoclast sur- Mochzuki SI, et al. Osteoclastogenesis inhibitory factor vival in vitro and in vivo. Am J Pathol 2000;157:435–48. exhibits hypocalcemic eVects in normal mice and in hyper- 4 Kodaira K, Kodaira K, Mizuno A, Yasuda H, Shima N, calcemic nude mice carrying tumors associated with Murakami A, et al. Cloning and characterization of the humoral hypercalcemia of malignancy. Bone 1998;23: gene encoding mouse osteoclast diVerentiation factor. 495–8. Gene 1999;230:121–7. 27 Wise GE, Lumpkin SJ, Huang H, Zhang Q. Osteoprote- 5 Wong B, Josien R, Choi Y. TRANCE is a TNF family that gerin and osteoclast diVerentiation factor in tooth erup- regulates and osteoclast function. J Leukoc tion. J Dent Res 2000;79:1937–42. Biol 1999;65:715–24. 28 Bucay N, Sarosi I, Dunstan CR, Morony S, Tarpley J, Cap- 6 Anderson DM, Maraskovsky E, Billingsley WL, Dougall parelli C, et al. Osteoprotegerin-deficient mice develop WC, Tometsko ME, Roux ER, et al. A homologue of the early onset osteoporosis and arterial calcification. Genes TNF receptor and its ligand enhance T-cell growth and Dev 1998;12:1260–8. dendritic-cell function. Nature 1997;390:175–9. 29 Mizuno A, Amizuka N, Irie K, Murakami A, Fujise N, 7 Hsu H, Lacey DL, Dunstan CR, Solovyev I, Colombero A, Kanno T, et al. Severe osteoporosis in mice lacking Timms E, et al. Tumor necrosis factor receptor family osteoclastogenesis inhibitory factor/osteoprotegerin. Bio- member RANK mediates osteoclast diVerentiation and chem Biophys Res Commun 1998;247:610–15. activation induced by osteoprotegerin ligand. Proc Natl 30 Min H, Morony S, Sarosi I, Dunstan CR, Capparelli C, Acad Sci USA 1999;96:3540–5. 8 Kong YY, Yoshida H, Sarosi I, Tan HL, Timms E, Scully S, et al. Osteoprotegerin reverses osteoporosis by inhibiting endosteal osteoclasts and prevents vascular calci- Capparelli C, et al. OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph- fication by blocking a process resembling osteoclastogen- node organogenesis. Nature 1999;397:315–23. esis. J Exp Med 2000;192: 463–74. 9 Li J, Sarosi I, Yan XQ, Moroni S, Capparelli C, Tan HL, et 31 Dougall WC, Glaccum M, Charrier K, Rohrbach K, Brasel al. RANK is the intrinsic hematopoietic cell surface recep- K, De Smedt T, et al. RANK is essential for osteoclast and tor that controls osteoclastogenesis and regulation of bone lymph node development. Genes Dev 1999;13:2412–24. mass and calcium metabolism. Proc Natl Acad Sci USA 32 Capparelli C, Kostenuik PJ, Morony S, Starnes C, 2000;97:1566–71. Wiemann B, Van G, et al. Osteoprotegerin prevents and 10 Nakagawa N, Kinosaki M, Yamaguchi K, Shima N, Yasuda reverses hypercalcemia in a murine model of humoral H, Yano K, et al. RANK is the essential signaling receptor hypercalcemia of malignancy. Cancer Res 2000;60:783–7. for osteoclast diVerentiation factor in osteoclastogenesis. 33 Clohisy DR, Ramnaraine ML, Scully S, Qi M, Van G, Tan Biochem Biophys Res Commun 1998;253:395–400. HL, et al. Osteoprotegerin inhibits tumor-induced osteo- 11 Takahashi N, Udagawa N, Suda T. A new member of tumor clastogenesis and bone tumor growth in osteopetrotic necrosis factor ligand family, ODF/OPGL/TRANCE/ mice. J Orthop Res 2000;18:967–76. RANKL, regulates osteoclast diVerentiation and function. 34 Fata JE, Kong YY, Li J, Sasaki T, Irie-Sasaki J, Moorehead Biochem Biophys Res Commun 1999;256:449–55. RA, et al. The osteoclast diVerentiation factor 12 Hofbauer LC, Khosla S, Dunstan CR, Lacey DL, Boyle osteoprotegerin-ligand is essential for mammary gland http://ard.bmj.com/ WB, Riggs BL. The roles of osteoprotegerin and osteopro- development. Cell 2000;103:41–50. tegerin ligand in the paracrine regulation of bone 35 Kong YY, Feige U, Sarosi I, Bolon B, Tafuri A, Morony S, et resorption. J Bone Miner Res 2000;15:2–12. al. Activated T cells regulate bone loss and joint 13 Horwood NJ, Elliott J, Martin TJ, Gillespie MT. Osteo- destruction in adjuvant arthritis through osteoprotegerin tropic agents regulate the expression of osteoclast diVeren- ligand. Nature 1999;402:304–9. tiation factor and osteoprotegerin in osteoblastic stromal 36 Sansom DM. CD28, CTLA-4 and their ligands: who does cells. Endocrinology 1998;139:4743–6. what and to whom? Immunology 2000;101:169–77. 14 Lee SK, Lorenzo JA. Parathyroid hormone stimulates 37 Waterhouse P, Penninger JM, Timms E, Wakeham A, TRANCE and inhibits osteoprotegerin messenger ribonu- Shahinian A, Lee KP, et al. Lymphoproliferative disorders cleic acid expression in murine bone marrow cultures: cor- with early lethality in mice deficient in Ctla-4. Science relation with osteoclast-like cell formation. Endocrinology 1995;270:985–8. on September 28, 2021 by guest. Protected copyright. 1999;140:3552–61. 15 Morony S, Capparelli C, Lee R, Shimamoto G, Boone T, 38 Tivol EA, Borriello F, Schweitzer AN, Lynch WP, Bluestone Lacey DL, Dunstan CR. A chimeric form of osteoprote- JA, Sharpe AH. Loss of CTLA-4 leads to massive lympho- gerin inhibits hypercalcemia and bone resorption induced proliferation and fatal multiorgan tissue destruction, by IL-1beta, TNF-alpha, PTH, PTHrP, and revealing a critical negative regulatory role of CTLA-4. 1,25(OH)2D3. J Bone Miner Res 1999;14:1478–85. Immunity 1995;3:541–7. 16 Fuller K, Wong B, Fox S, Choi Y, Chambers TJ. TRANCE 39 Horwood NJ, Kartsogiannis V, Quinn JM, Romas E, Martin is necessary and suYcient for -mediated activa- TJ, Gillespie MT. Activated T lymphocytes support osteo- tion of bone resorption in osteoclasts. J Exp Med clast formation in vitro. Biochem Biophys Res Commun 1998;188:997–1001. 1999;265:144–50. 17 Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, 40 Weitzmann MN, Cenci S, Rifas L, Haug J, Dipersio J, Burgess T, et al. Osteoprotegerin ligand is a cytokine that Pacifici R. T cell activation induces human osteoclast regulates osteoclast diVerentiation and activation. Cell formation via receptor activator of nuclear factor kappaB 1998;93:165–76. ligand-dependent and -independent mechanisms. J Bone 18 Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, Miner Res 2001;16:328–37. Martin TJ. Modulation of osteoclast diVerentiation and 41 Feige U, Hu YL, Gasser J, Campagnuolo G, Munyakazi L, function by the new members of the tumor necrosis factor Bolon B. Anti-interleukin-1 and anti-tumor necrosis receptor and ligand families. Endocr Rev 1999;20:345–57. factor-alpha synergistically inhibit adjuvant arthritis in 19 Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki Lewis rats. Cell Mol Life Sci 2000;57:1457–70. M, Mochizuli SI, et al. Osteoclast diVerentiation factor is a 42 Bekker PJ, Holloway D, Nakanishi A, Arrighi M, Leese PT, ligand for osteoprotegerin/osteoclastogenesis-inhibitory Dunstan CR. The eVect of a single dose of osteoprotegerin factor and is identical to TRANCE/RANKL. Proc Natl in postmenopausal women. J Bone Miner Res 2001;16: Acad Sci USA 1998;95:3597–602. 348–60.

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