title: The expression of toll-like receptors 3 and 7 in rheumatoid arthritis synovium is increased and costimulation of toll-like receptors 3, 4, and 7/8 results in synergistic cytokine production by dendritic cells.
abstract: OBJECTIVE
  To evaluate the expression of Toll-like receptors (TLRs) 3 and 7 in synovium and to study potential differences in the maturation and cytokine production mediated by TLR-2, TLR-3, TLR-4, and TLR-7/8 by dendritic cells (DCs) from rheumatoid arthritis (RA) patients and DCs from healthy controls.
  
  
  METHODS
  Synovial expression of TLR-3 and TLR-7 in RA was studied using immunohistochemistry. Monocyte-derived DCs from RA patients and healthy controls were cultured for 6 days and subsequently stimulated for 48 hours via TLR-mediated pathways (lipoteichoic acid, Pam(3)Cys, and fibroblast-stimulating lipopeptide 1 for TLR-2, poly[I-C] for TLR-3, lipopolysaccharide and extra domain A for TLR-4, and R848 for TLR-7/8). Phenotypic DC maturation was measured using flow cytometry. The secretion of tumor necrosis factor alpha (TNFalpha), interleukin-6 (IL-6), IL-10, and IL-12 was measured using the Bio-Plex system. Cell lines expressing TLR-2 and TLR-4 were used for the detection of TLR-2 and TLR-4 ligands in serum and synovial fluid from RA patients.
  
  
  RESULTS
  TLR-3 and TLR-7 were highly expressed in RA synovium. All TLR ligands elicited phenotypic DC maturation equally between DCs from RA patients and those from healthy controls. TLR-2- and TLR-4-mediated stimulation of DCs from RA patients resulted in markedly higher production of inflammatory mediators (TNFalpha and IL-6) compared with DCs from healthy controls. In contrast, upon stimulation of TLR-3 and TLR-7/8, the level of cytokine production was equal between DCs from RA patients and those from healthy controls. Remarkably, both TLR-3 and TLR-7/8 stimulation resulted in a skewed balance toward IL-12. Intriguingly, the combined stimulation of TLR-4 and TLR-3-7/8 resulted in a marked synergy with respect to the production of inflammatory mediators. As a proof of concept, TLR-4 ligands were increased in the serum and synovial fluid of RA patients.
  
  
  CONCLUSIONS
  TLRs are involved in the regulation of DC activation and cytokine production. We hypothesize that various TLR ligands in the joint trigger multiple TLRs simultaneously, favoring the breakthrough of tolerance in RA.
authors.affiliation: Radboud University Nijmegen Medical Centre, The Netherlands.
authors.forename: M F
authors.initials: MF
authors.lastname: Roelofs
authors.forename: L A B
authors.initials: LA
authors.lastname: Joosten
authors.forename: S
authors.initials: S
authors.lastname: Abdollahi-Roodsaz
authors.forename: A W T
authors.initials: AW
authors.lastname: van Lieshout
authors.forename: T
authors.initials: T
authors.lastname: Sprong
authors.forename: F H
authors.initials: FH
authors.lastname: van den Hoogen
authors.forename: W B
authors.initials: WB
authors.lastname: van den Berg
authors.forename: T R D J
authors.initials: TR
authors.lastname: Radstake
mesh_terms: D001172:Arthritis, Rheumatoid / Q000097:blood / Q000378:metabolism*; D016022:Case-Control Studies; D002478:Cells, Cultured; D016922:Cellular Senescence; D016207:Cytokines / Q000096:biosynthesis*; D003713:Dendritic Cells / Q000378:metabolism; D004357:Drug Synergism; D006801:Humans; D007150:Immunohistochemistry; D008024:Ligands; D008562:Membrane Glycoproteins / Q000819:agonists* / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000819:agonists* / Q000378:metabolism*; D013268:Stimulation, Chemical; D013582:Synovial Fluid / Q000378:metabolism; D013583:Synovial Membrane / Q000378:metabolism*; D051195:Toll-Like Receptor 2; D051196:Toll-Like Receptor 3; D051197:Toll-Like Receptor 4; D051199:Toll-Like Receptor 7; D051193:Toll-Like Receptors
chemical_list: D016207:Cytokines; D008024:Ligands; D008562:Membrane Glycoproteins; D011956:Receptors, Cell Surface; C495342:TLR2 protein, human; C495344:TLR3 protein, human; C495345:TLR4 protein, human; C495348:TLR7 protein, human; D051195:Toll-Like Receptor 2; D051196:Toll-Like Receptor 3; D051197:Toll-Like Receptor 4; D051199:Toll-Like Receptor 7; D051193:Toll-Like Receptors