Virchows Archiv

, Volume 449, Issue 1, pp 69–77 | Cite as

Stimulation of osteoclast formation by inflammatory synovial fluid

  • Iannis E. Adamopoulos
  • Lynett Danks
  • Ichiro Itonaga
  • Rachel M. Locklin
  • Afsie Sabokbar
  • David J. P. Ferguson
  • Nicholas A. AthanasouEmail author
Original Article


Peri-articular bone resorption is a feature of arthritis due to crystal deposition and rheumatoid disease. Under these conditions, the synovial fluid contains numerous inflammatory cells that produce cytokines and growth factors which promote osteoclast formation. The aim of this study was to determine whether inflammatory synovial fluid stimulates the formation of osteoclasts. Synovial fluid from rheumatoid arthritis (RA), pyrophosphate arthropathy (PPA) and osteoarthritis (OA) patients was added to cultures (n=8) of human peripheral blood mononuclear cells (PBMCs) in the presence and absence of macrophage colony-stimulating factor (M-CSF) and the receptor activator of NF-κB ligand (RANKL). Osteoclast formation was assessed by the formation of cells positive for tartrate-resistant acid phosphatase (TRAP) and vitronectin receptor (VNR) and the extent of lacunar resorption. The addition of 10% OA, RA and PPA synovial fluid to PBMC cultures resulted in the formation of numerous multinucleated or mononuclear TRAP+ and VNR+ cells which were capable of lacunar resorption. In contrast to PBMC cultures incubated with OA synovial fluid, there was marked stimulation of osteoclast formation and resorption in cultures containing inflammatory RA and PPA synovial fluid which contained high levels of tumour necrosis factor alpha, a factor which is known to stimulate RANKL-induced osteoclast formation.


Synovial fluid Rheumatoid arthritis Crystal Pyrophosphate Osteoclast 



This study was approved by the Oxford Research Ethics Committee. The authors wish to thank all the volunteers who kindly donated blood samples for this study. We also extend our thanks to Dr. David Lacey, Amgen Inc., for providing the soluble RANKL. This work was funded by Action Research and the Frances and Augustus Newman Foundation. I.A. is the recipient of a Research into Ageing DPhil studentship.


  1. 1.
    Adamopoulos IE, Sabokbar A, Wordsworth BP, Carr A, Ferguson DJ, Athanasou NA (2006) Synovial fluid macrophages are capable of osteoclast formation and resorption. J Pathol 208:35–43PubMedCrossRefGoogle Scholar
  2. 2.
    Arnett FC, Edworthy SM, Bloch DA, McShane DJ, Fries JF, Cooper NS, Healey LA, Kaplan SR, Liang MH, Luthra HS et al (1998) The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum 31:315–24CrossRefGoogle Scholar
  3. 3.
    Aruffo A, Stamenkovic I, Melnick M, Underhill CB, Seed B (1990) CD44 is the principal cell surface receptor for hyaluronate. Cell 61:1303–13PubMedCrossRefGoogle Scholar
  4. 4.
    Athanasou NA (1996) Cellular biology of bone-resorbing cells. J Bone Joint Surg Am 78:1096–112PubMedGoogle Scholar
  5. 5.
    Athanasou NA, Quinn J (1990) Immunophenotypic differences between osteoclasts and macrophage polykaryons: immunohistological distinction and implications for osteoclast ontogeny and function. J Clin Pathol 43:997–1003PubMedCrossRefGoogle Scholar
  6. 6.
    Athanasou NA, Quinn J, Ferguson DJ, McGee JO (1991) Bone resorption by macrophage polykaryons of giant cell tumour of tendon sheath. Br J Cancer 63:527–533PubMedGoogle Scholar
  7. 7.
    Athanasou NA, Quinn J, Bulstrode CJ (1992) Resorption of bone by inflammatory cells derived from the joint capsule of hip arthroplasties. J Bone Joint Surg Br 74:57–62PubMedGoogle Scholar
  8. 8.
    Azuma Y, Kaji K, Katogi R, Takeshita S, Kudo A (2000) Tumor necrosis factor-alpha induces differentiation of and bone resorption by osteoclasts. J Biol Chem 275:4858–4864PubMedCrossRefGoogle Scholar
  9. 9.
    Darling JM, Goldring SR, Harada Y, Handel ML, Glowacki J, Gravallese EM (1997) Multinucleated cells in pigmented villonodular synovitis and giant cell tumor of tendon sheath express features of osteoclasts. Am J Pathol 150:1383–1393PubMedGoogle Scholar
  10. 10.
    Davies J, Warwick J, Totty N, Philp R, Helfrich M, Horton M (1989) The osteoclast functional antigen, implicated in the regulation of bone resorption, is biochemically related to the vitronectin receptor. J Cell Biol 109:1817–1826PubMedCrossRefGoogle Scholar
  11. 11.
    Feldmann M, Brennan FM, Maini RN (1996) Role of cytokines in rheumatoid arthritis. Annu Rev Immunol 14:397–440PubMedCrossRefGoogle Scholar
  12. 12.
    Freemont A, Denton J (1991) Atlas of synovial fluid cytopathology. Kluwer Academic Publishers, BostonGoogle Scholar
  13. 13.
    Fujikawa Y, Quinn JM, Sabokbar A, McGee JO, Athanasou NA (1996) The human osteoclast precursor circulates in the monocyte fraction. Endocrinology 137:4058–4060PubMedCrossRefGoogle Scholar
  14. 14.
    Fujikawa Y, Sabokbar A, Neale S, Athanasou NA (1996) Human osteoclast formation and bone resorption by monocytes and synovial macrophages in rheumatoid arthritis. Ann Rheum Dis 55:816–822PubMedGoogle Scholar
  15. 15.
    Hofbauer LC, Lacey DL, Dunstan CR, Spelsberg TC, Riggs BL, Khosla S (1999) Interleukin-1beta and tumor necrosis factor-alpha, but not interleukin-6, stimulate osteoprotegerin ligand gene expression in human osteoblastic cells. Bone 25:255–259PubMedCrossRefGoogle Scholar
  16. 16.
    Hofbauer LC, Khosla S, Dunstan CR, Lacey DL, Boyle WJ, Riggs BL (2000) The roles of osteoprotegerin and osteoprotegerin ligand in the paracrine regulation of bone resorption. J Bone Miner Res 15:2–12PubMedCrossRefGoogle Scholar
  17. 17.
    Itonaga I, Fujikawa Y, Sabokbar A, Murray DW, Athanasou NA (2000) Rheumatoid arthritis synovial macrophage–osteoclast differentiation is osteoprotegerin ligand-dependent. J Pathol 192:97–104PubMedCrossRefGoogle Scholar
  18. 18.
    Itonaga I, Schulze E, Burge PD, Gibbons CL, Ferguson D, Athanasou NA (2002) Phenotypic characterization of mononuclear and multinucleated cells of giant cell reparative granuloma of small bones. J Pathol 198:30–36PubMedCrossRefGoogle Scholar
  19. 19.
    Kania JR, Kehat-Stadler T, Kupfer SR (1997) CD44 antibodies inhibit osteoclast formation. J Bone Miner Res 12:1155–1164PubMedCrossRefGoogle Scholar
  20. 20.
    Knapp W, Dorken B, Gilks WR, Rieber EP, Schmidt RE, Stein H, von dem Borne AEGK (1989) Leukocyte typing IV: white cell differentiation antigen. Oxford University Press, New YorkGoogle Scholar
  21. 21.
    Kobayashi K, Takahashi N, Jimi E, Udagawa N, Takami M, Kotake S, Nakagawa N, Kinosaki M, Yamaguchi K, Shima N, Yasuda H, Morinaga T, Higashio K, Martin TJ, Suda T (2000) Tumor necrosis factor {alpha} stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction. J Exp Med 191:275–286PubMedCrossRefGoogle Scholar
  22. 22.
    Kotake S, Udagawa N, Hakoda M, Mogi M, Yano K, Tsuda E, Takahashi K, Furuya T, Ishiyama S, Kim KJ, Saito S, Nishikawa T, Takahashi N, Togari A, Tomatsu T, Suda T, Kamatani N (2001) Activated human T cells directly induce osteoclastogenesis from human monocytes: possible role of T cells in bone destruction in rheumatoid arthritis patients. Arthritis Rheum 44:1003–1012PubMedCrossRefGoogle Scholar
  23. 23.
    Krenn V, Morawietz L, Haupl T, Neidel J, Petersen I, Konig A (2002) Grading of chronic synovitis—a histopathological grading system for molecular and diagnostic pathology. Pathol Res Pract 198(5):317–325PubMedCrossRefGoogle Scholar
  24. 24.
    Kudo O, Fujikawa Y, Itonaga I, Sabokbar A, Torisu T, Athanasou NA (2002) Proinflammatory cytokine (TNFalpha/IL-1alpha) induction of human osteoclast formation. J Pathol 198:220–227PubMedCrossRefGoogle Scholar
  25. 25.
    Kudo O, Sabokbar A, Pocock A, Itonaga I, Fujikawa Y, Athanasou NA (2003) Interleukin-6 and interleukin-11 support human osteoclast formation by a RANKL-independent mechanism. Bone 32:1–7PubMedCrossRefGoogle Scholar
  26. 26.
    Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero A, Elliott G, Scully S, Hsu H, Sullivan J, Hawkins N, Davy E, Capparelli C, Eli A, Qian YX, Kaufman S, Sarosi I, Shalhoub V, Senaldi G, Guo J, Delaney J, Boyle WJ (1998) Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93:165–176PubMedCrossRefGoogle Scholar
  27. 27.
    Lam J, Takeshita S, Barker JE, Kanagawa O, Ross FP, Teitelbaum SL (2000) TNF-[alpha] induces osteoclastogenesis by direct stimulation of macrophages exposed to permissive levels of RANK ligand. J Clin Invest 106:1481–1488PubMedCrossRefGoogle Scholar
  28. 28.
    Lettesjo H, Nordstrom E, Strom H, Nilsson B, Glinghammar B, Dahlstedt L, Moller E (1998) Synovial fluid cytokines in patients with rheumatoid arthritis or other arthritic lesions. Scand J Immunol 48:286–292PubMedCrossRefGoogle Scholar
  29. 29.
    McCarty D (1993) Synovial fluid. In: McCarty D, Koopman WJ (eds) Arthritis and allied conditions. Lea & Febiger, PhiladelphiaGoogle Scholar
  30. 30.
    Minkin C (1982) Bone acid phosphatase: tartrate-resistant acid phosphatase as a marker of osteoclast function. Calcif Tissue Int 34:285–290PubMedCrossRefGoogle Scholar
  31. 31.
    Neale SD, Kristelly R, Gundle R, Quinn JM, Athanasou NA (1997) Giant cells in pigmented villo nodular synovitis express an osteoclast phenotype. J Clin Pathol 50:605–608PubMedCrossRefGoogle Scholar
  32. 32.
    Partsch G, Steiner G, Leeb BF, Dunky A, Broll H, Smolen JS (1997) Highly increased levels of tumor necrosis factor-alpha and other proinflammatory cytokines in psoriatic arthritis synovial fluid. J Rheumatol 24:518–523PubMedGoogle Scholar
  33. 33.
    Quinn JM, Elliott J, Gillespie MT, Martin TJ (1998) A combination of osteoclast differentiation factor and macrophage-colony stimulating factor is sufficient for both human and mouse osteoclast formation in vitro. Endocrinology 139:4424–4427PubMedCrossRefGoogle Scholar
  34. 34.
    Quinn JM, Whitty GA, Byrne RJ, Gillespie MT, Hamilton JA (2002) The generation of highly enriched osteoclast-lineage cell populations. Bone 30:164–170PubMedCrossRefGoogle Scholar
  35. 35.
    Sabokbar A, Fujikawa Y, Neale S, Murray DW, Athanasou NA (1997) Human arthroplasty derived macrophages differentiate into osteoclastic bone resorbing cells. Ann Rheum Dis 56:414–420PubMedCrossRefGoogle Scholar
  36. 36.
    Shigeyama Y, Pap T, Kunzler P, Simmen BR, Gay RE, Gay S (2000) Expression of osteoclast differentiation factor in rheumatoid arthritis. Arthritis Rheum 43:2523–2530PubMedCrossRefGoogle Scholar
  37. 37.
    Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R, Nguyen HQ, Wooden S, Bennett L, Boone T, Shimamoto G, DeRose M, Elliott R, Colombero A, Tan HL, Trail G, Sullivan J, Davy E, Bucay N, Renshaw-Gegg L, Hughes TM, Hill D, Pattison W, Campbell P, Boyle WJ et al (1997) Osteoprotegerin: a novel secreted protein involved in the regulation of bone density. Cell 89:309–319PubMedCrossRefGoogle Scholar
  38. 38.
    Smith JB, Bocchieri MH, Smith JB Jr, Sherbin-Allen L, Abruzzo JL (1990) Colony stimulating factor occurs in both inflammatory and noninflammatory synovial fluids. Rheumatol Int 10:131–134PubMedCrossRefGoogle Scholar
  39. 39.
    Sterling H, Saginario C, Vignery A (1998) CD44 occupancy prevents macrophage multinucleation. J Cell Biol 143:837–847PubMedCrossRefGoogle Scholar
  40. 40.
    Takayanagi H, Iizuka H, Juji T, Nakagawa T, Yamamoto A, Miyazaki T, Koshihara Y, Oda H, Nakamura K, Tanaka S (2000) Involvement of receptor activator of nuclear factor kappaB ligand/osteoclast differentiation factor in osteoclastogenesis from synoviocytes in rheumatoid arthritis. Arthritis Rheum 43:259–269PubMedCrossRefGoogle Scholar
  41. 41.
    Vignery A (2000) Osteoclasts and giant cells: macrophage–macrophage fusion mechanism. Int J Exp Pathol 81:291–304PubMedCrossRefGoogle Scholar
  42. 42.
    Yanni G, Whelan A, Feighery C, Bresnihan B (1994) Synovial tissue macrophages and joint erosion in rheumatoid arthritis. Ann Rheum Dis 53:39–44PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Iannis E. Adamopoulos
    • 1
    • 4
  • Lynett Danks
    • 1
  • Ichiro Itonaga
    • 2
  • Rachel M. Locklin
    • 1
  • Afsie Sabokbar
    • 1
  • David J. P. Ferguson
    • 3
  • Nicholas A. Athanasou
    • 4
    Email author
  1. 1.Institute of Musculoskeletal Sciences, Botnar Research CentreUniversity of OxfordOxfordUK
  2. 2.Department of Orthopaedic SurgeryOita Medical UniversityOita-gunJapan
  3. 3.Nuffield Department of Clinical Laboratory ScienceJohn Radcliffe HospitalOxfordUK
  4. 4.Department of PathologyNuffield Orthopaedic CentreHeadingtonUK

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