Bacterial peptidoglycans but not CpG oligodeoxynucleotides activate synovial fibroblasts by toll-like receptor signaling.
OBJECTIVE
To test the hypothesis that bacterial products acting as adjuvants, such as CpG oligodeoxynucleotides (ODNs) and peptidoglycans (PGs), are able to activate synoviocytes, and to determine the involvement of Toll-like receptors (TLRs) in this activation process.


METHODS
Cultured synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) or osteoarthritis (OA) were stimulated with CpG ODNs or PGs. The expression of various integrins was determined by fluorescence-activated cell sorting. TLR and matrix metalloproteinase (MMP) messenger RNA (mRNA) was measured by real-time polymerase chain reaction. Additionally, levels of interleukin-6 (IL-6) and IL-8 in the culture supernatants were assessed by enzyme-linked immunosorbent assay. Blocking experiments were performed by adding anti-TLR-2 and anti-TLR-4 monoclonal antibodies to cultures stimulated with bacterial PGs.


RESULTS
Incubation of synovial fibroblasts with CpG ODNs resulted in neither up-regulation of the expression of integrins on the cell surface, up-regulation of MMP mRNA expression, nor IL-6 and IL-8 production. However, incubation of RA synovial fibroblasts as well as OA synovial fibroblasts with staphylococcal PGs led to an up-regulation of CD54 (ICAM-1) surface expression and to increased expression of MMP-1, MMP-3, and MMP-13 mRNA. Furthermore, production of the proinflammatory cytokines IL-6 and IL-8 was increased by treatment with PGs. We demonstrated that cultured synovial fibroblasts express low levels of TLR-2 and TLR-9 mRNA. TLR-2 was up-regulated after stimulation with PGs, whereas TLR-9 mRNA remained at baseline levels after stimulation with CpG ODNs. Anti-TLR-2 monoclonal antibodies significantly inhibited production of IL-6 and IL-8 induced by stimulation with PGs.


CONCLUSIONS
We demonstrate that bacterial PGs activate synovial fibroblasts, at least partially via TLR-2, to express integrins, MMPs, and proinflammatory cytokines. Inhibition of TLR signaling pathways might therefore have a beneficial effect on both joint inflammation and joint destruction.
Kyburz
Diego
D
Center of Experimental Rheumatology and Department of Rheumatolgy, University Hospital of Zurich, Zurich, Switzerland. diego.kyburz@ruz.usz.ch
Rethage
Janine
J
Seibl
Reinhart
R
Lauener
Roger
R
Gay
Renate E
RE
Carson
Dennis A
DA
Gay
Steffen
S
D001172:Arthritis, Rheumatoid / Q000378:metabolism / Q000473:pathology; D002478:Cells, Cultured; D018899:CpG Islands; D004269:DNA, Bacterial; D004305:Dose-Response Relationship, Drug; D029721:Drosophila Proteins; D004926:Escherichia coli; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D005434:Flow Cytometry; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis / Q000235:genetics; D007378:Interleukins / Q000096:biosynthesis; D008562:Membrane Glycoproteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000096:biosynthesis / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000378:metabolism / Q000494:pharmacology*; D010003:Osteoarthritis / Q000378:metabolism / Q000473:pathology; D010457:Peptidoglycan / Q000302:isolation & purification / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D013211:Staphylococcus aureus / Q000737:chemistry; D013583:Synovial Membrane / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D051195:Toll-Like Receptor 2; D051217:Toll-Like Receptor 9; D051193:Toll-Like Receptors
C408982:CPG-oligonucleotide; D004269:DNA, Bacterial; D029721:Drosophila Proteins; D007378:Interleukins; D008562:Membrane Glycoproteins; D009838:Oligodeoxyribonucleotides; D010457:Peptidoglycan; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; C495342:TLR2 protein, human; C495371:TLR9 protein, human; D051195:Toll-Like Receptor 2; D051217:Toll-Like Receptor 9; D051193:Toll-Like Receptors; D018799:Intercellular Adhesion Molecule-1; D008666:Metalloendopeptidases