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Abstract
Objecti
The aim of this study was to clarify whether stimulation of recombinant IL-17, TLR2 and TLR4 by their specific ligands induces the production of RANKL and IL-6 in the fibroblast-like synoviocytes (FLSs) from RA patients.
Methods
FLSs were isolated from RA synovial tissues and they were stimulated with the IL-17, TLR2 ligand bacterial peptidoglycan (PGN) and TLR4 ligand lipopolysaccharide (LPS). The RANKL levels were assessed by RT-PCR and western blotting. The expressions of IL-17, TLR2, TLR4, RANKL and IL-6 in the RA synovium were quantified by immunohistochemistry and these values were compared with the values obtained in the osteoarthritis synovium. The increased IL-6 production in the culture supernatants of the RA FLSs was quantified by sandwich ELISA.
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Fig. 1.
RANKL mRNA expression by the RA-FLSs after stimulation with IL-17, PGN and LPS (A) The RANKL mRNA expression induced by IL-17, PGN and LPS. The FLSs were cultured with IL-17 (1, 5, 25 ng/ml), PGN (1, 5, 25 ng/ml) and LPS (1, 10 ng/ml) for 72 h. The expression of RANKL mRNA was evaluated by RT-PCR. The optical density was normalized to the band for beta-actin. (B) The RANKL mRNA expression induced by IL-17, PGN, LPS, IL-17 plus PGN, IL-17 plus LPS, PGN plus LPS, and IL-17 plus PGN plus LPS. The expression of RANKL mRNA was evaluated by RT-PCR. ∗p<0.05, ∗∗p<0.01 compared with Nil, †<0.05 compared with IL-17 5 ng/ml, ‡P<0.05 compared with PGN 1 ug/ml +IL-17 5 ng/ml.
Fig. 2.
The RANKL protein expression in the RA-FLSs increased significantly after treatment with IL-17, PGN and LPS (A) The RA-FLSs were cultured with or without IL-17 (1, 5, 25 ng/ml), PGN (1, 5, 25 ng/ml) or LPS (1, 10 ng/ml) for 72 h. The RANKL protein expression was measured in the cell lysates by western blot analysis using goat polyclonal anti-RANKL antibody. (B) The RANKL protein expression induced by IL-17, PGN, LPS, IL-17 plus PGN, IL-17 plus LPS, PGN plus LPS, and IL-17 plus PGN plus LPS. The results are expressed as the ratio of the densitometric intensity of the RANKL product to that of the beta-actin product. The results are presented as the mean±S.E.M., n=3. ∗p<0.05, ∗∗p<0.01 compared with Nil, †p<0.05 compared with IL-17 5 ng/ml, ‡p<0.05 compared with PGN 1 ug/ml +IL-17 5 ng/ml.
Fig. 3.
Quantitation of the IL-6 production by IL-17, PGN and LPS in the RA-FLSs. (A) The FLSs were cultured with IL-17 (1, 5, 25 ng/ml), PGN (1, 5, 25 ng/ml) or LPS (1, 10 ng/ml) for 72 h, and the IL-6 concentrations in the culture supernatants were determined by enzyme-linked immunosorbent assay. (B) The IL-6 production induced by IL-17, PGN, LPS, IL-17 plus PGN, IL-17 plus LPS, PGN plus LPS, and IL-17 plus PGN plus LPS. Values are the mean and SEM of triplicate cultures. ∗p<0.05, ∗∗p<0.01 compared with Nil, †p<0.05 compared with IL-17 5 ng/ml, ‡p<0.05 compared with PGN 1 ug/ml +IL-17 5 ng/ml.
Fig. 4.
The expressions of IL-17, TLR-2, TLR-4, RANKL and IL-6 was significantly greater in the RA synovium than those in the OA synovium. Immunohistochemical detection of IL-17, TLR-2, TLR-4, RANKL and IL-6 in the synovium of patients with RA and OA. All the tissues were counterstained with hematoxylin (original magnification, ×400).
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