title: RNA released from necrotic synovial fluid cells activates rheumatoid arthritis synovial fibroblasts via Toll-like receptor 3.
abstract: OBJECTIVE
  To assess the expression of Toll-like receptor 3 (TLR-3) protein in synovial tissues and cultured synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to investigate the consequences of stimulation of cultured synovial fibroblasts with TLR-3 ligands.
  
  
  METHODS
  TLR-3 expression in synovial tissues was determined by immunohistochemistry and immunofluorescence, and expression in cultured RA synovial fibroblasts (RASFs) was determined by fluorescence-activated cell sorting and real-time polymerase chain reaction techniques. TLR-3 signaling was assessed by incubating RASFs with poly(I-C), lipopolysaccharide, palmitoyl-3-cysteine-serine-lysine-4, or necrotic synovial fluid cells from RA patients in the presence or absence of hydroxychloroquine or Benzonase. Subsequent determination of interferon-beta (IFNbeta), CXCL10, CCL5, and interleukin-6 (IL-6) protein production in the culture supernatants was performed by enzyme-linked immunosorbent assays.
  
  
  RESULTS
  TLR-3 protein expression was found to be higher in RA synovial tissues than in OA synovial tissues. TLR-3 expression was localized predominantly in the synovial lining, with a majority of the TLR-3-expressing cells coexpressing fibroblast markers. Stimulation of cultured RASFs with the TLR-3 ligand poly(I-C) resulted in the production of high levels of IFNbeta, CXCL10, CCL5, and IL-6 protein. Similarly, coincubation of RASFs with necrotic synovial fluid cells from patients with RA resulted in up-regulation of these cytokines and chemokines in a TLR-3-dependent manner.
  
  
  CONCLUSIONS
  Our findings demonstrate the expression of TLR-3 in RA synovial tissue and the activation of RASFs in vitro by the TLR-3 ligand poly(I-C) as well as by necrotic RA synovial fluid cells, and indicate that RNA released from necrotic cells might act as an endogenous TLR-3 ligand for the stimulation of proinflammatory gene expression in RASFs.
authors.affiliation: University Hospital, Zurich, Switzerland.
authors.forename: Fabia
authors.initials: F
authors.lastname: Brentano
authors.forename: Olivier
authors.initials: O
authors.lastname: Schorr
authors.forename: Renate E
authors.initials: RE
authors.lastname: Gay
authors.forename: Steffen
authors.initials: S
authors.lastname: Gay
authors.forename: Diego
authors.initials: D
authors.lastname: Kyburz
mesh_terms: D001172:Arthritis, Rheumatoid / Q000378:metabolism* / Q000473:pathology; D002478:Cells, Cultured; D018925:Chemokines / Q000378:metabolism; D004338:Drug Combinations; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007124:Immunoenzyme Techniques; D007369:Interferon Inducers / Q000494:pharmacology; D016899:Interferon-beta / Q000235:genetics / Q000378:metabolism; D008024:Ligands; D008562:Membrane Glycoproteins / Q000378:metabolism*; D009336:Necrosis; D010003:Osteoarthritis / Q000378:metabolism / Q000473:pathology; D012313:RNA / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013582:Synovial Fluid / Q000166:cytology / Q000378:metabolism*; D051196:Toll-Like Receptor 3; D051193:Toll-Like Receptors; D015854:Up-Regulation
chemical_list: D018925:Chemokines; D004338:Drug Combinations; D007369:Interferon Inducers; D008024:Ligands; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; C495344:TLR3 protein, human; D051196:Toll-Like Receptor 3; D051193:Toll-Like Receptors; D012313:RNA; D016899:Interferon-beta