title: Crucial role of synovial lining macrophages in the promotion of transforming growth factor beta-mediated osteophyte formation.
abstract: OBJECTIVE
  To investigate in vivo and in vitro whether macrophages have an intermediate role in transforming growth factor beta (TGFbeta)-induced osteophyte formation.
  
  
  METHODS
  In vivo, synovial lining macrophages were selectively depleted by injection of clodronate-laden liposomes 7 days prior to injection of 20 ng or 200 ng of TGFbeta into murine knee joints 3 times, on alternate days. Total knee joint sections were obtained on day 7 after the last injection and stained with Safranin O. Production of bone morphogenetic protein 2 (BMP-2) and BMP-4 was determined by immunolocalization. The interaction between murine macrophages and mesenchymal cells (precursors with chondrogenic potential) was studied in vitro using a Transwell system in which RAW macrophages were cocultured with C3H10T1/2 mesenchymal cells. Spheroid neocartilage formation was quantified microscopically after staining with May-Grünwald-Giemsa.
  
  
  RESULTS
  Triple injections of 20 ng or 200 ng of TGFbeta into normal murine knee joints induced significant osteophyte formation at the lateral and medial sites of the patella and femur on day 7 after the last injection. Strikingly, removal of synovial lining macrophages prior to TGFbeta injection resulted in a drastic reduction of osteophyte formation (by 70% and 64% after injection of 20 ng and 200 ng of TGFbeta, respectively). Synovial lining cells produced BMP-2 and BMP-4 after TGFbeta stimulation, whereas BMP-2 and BMP-4 were absent in the synovial tissue after macrophage depletion. In vitro, clustering and spheroid formation of C3H10T1/2 was induced by TGFbeta concentrations of >1 ng/ml. However, in the Transwell system, in the presence of murine macrophages, 0.5 ng/ml of TGFbeta was very effective in generating large spheroids, suggestive of macrophage-derived (co)factors. In coculture supernatants, TGFbeta concentrations were not elevated in the presence of macrophages, indicating generation of other growth factors involved in spheroid formation.
  
  
  CONCLUSIONS
  These findings indicate that macrophages are crucial intermediate factors in osteophyte formation induced by TGFbeta, probably by inducing other chondrogenic signals.
authors.affiliation: University Medical Center, Nijmegen, The Netherlands. P.vanLent@reuma.umcn.nl
authors.forename: P L E M
authors.initials: PL
authors.lastname: van Lent
authors.forename: A B
authors.initials: AB
authors.lastname: Blom
authors.forename: P
authors.initials: P
authors.lastname: van der Kraan
authors.forename: A E M
authors.initials: AE
authors.lastname: Holthuysen
authors.forename: E
authors.initials: E
authors.lastname: Vitters
authors.forename: N
authors.initials: N
authors.lastname: van Rooijen
authors.forename: R L
authors.initials: RL
authors.lastname: Smeets
authors.forename: K C A M
authors.initials: KC
authors.lastname: Nabbe
authors.forename: W B
authors.initials: WB
authors.lastname: van den Berg
mesh_terms: D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000378:metabolism; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology* / Q000187:drug effects; D004002:Clodronic Acid / Q000494:pharmacology; D008081:Liposomes; D008264:Macrophages / Q000187:drug effects / Q000502:physiology*; D008648:Mesoderm / Q000166:cytology; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D010003:Osteoarthritis / Q000378:metabolism / Q000473:pathology; D010521:Periosteum / Q000378:metabolism / Q000473:pathology; D013234:Stem Cells / Q000166:cytology / Q000187:drug effects; D013583:Synovial Membrane / Q000166:cytology* / Q000276:immunology; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
chemical_list: D000963:Antimetabolites; C525719:Bmp2 protein, mouse; C525810:Bmp4 protein, mouse; D055396:Bone Morphogenetic Protein 2; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D008081:Liposomes; D016212:Transforming Growth Factor beta; D004002:Clodronic Acid