Abundant expression of the IL-23 subunit p19, but low levels of bioactive IL-23 in the rheumatoid synovium

OBJECTIVE: IL-23, composed of a p19 and a p40 subunit, is suggested to play key roles in rheumatoid arthritis (RA), dependent on the promotion and proliferation of IL-17-producing Th17 T-cells. However, previous studies on IL-23 expression in human tissues were based on p19 only. We aimed to study the expression and regulation of both IL-23 subunits, p19 and p40, in RA compared to osteoarthritis (OA) patients. METHODS: The expression of p19 and p40 in synovial tissues was analyzed by in-situ hybridization and immunohistochemistry. IL-23 in RA and OA synovial fluids and sera was determined by ELISA. TLR-dependent induction of p19, p40 and bioactive IL-23 was determined in RASF, monocytes and MDDC by RT-PCR, Real-time PCR, Western-blot and functional-assays. RESULTS: The p19 subunit was abundantly expressed in RA but not in OA synovial tissues. p19 was most prominently expressed by RASF in the synovial lining layer and at the site of invasion, but no heterodimeric IL-23 was detected at these sites. Correspondingly, soluble IL-23 was not detectable or found at very low levels in synovial fluids and sera of patients with RA. By in-vitro experiments, we confirmed that TLR-activated RASF expressed p19 but not p40, in contrast to monocytes, which produced IL-23 following TLR stimulation. CONCLUSION: The TLR-dependent induction of p19 but not p40 in RASF and the abundant expression of p19 along with the low or undetectable levels of IL-23 in RA patients, gives strong evidence that p19 does not necessarily indicate the presence of IL-23 as it has been proposed so far

Overexpression of toll-like receptors 3 and 4 in synovial tissue from patients with early rheumatoid arthritis: Toll-like receptor expression in early and longstanding arthritis

OBJECTIVE: To analyze the expression, regulation, and biologic relevance of Toll-like receptors (TLRs) 1-10 in synovial and skin fibroblasts and to determine the expression levels of TLRs 2, 3, and 4 in synovial tissues from patients with early rheumatoid arthritis (RA), longstanding RA, and osteoarthritis (OA). METHODS: Expression of TLRs 1-10 in RA synovial fibroblasts (RASFs), OASFs, and skin fibroblasts was analyzed by real-time polymerase chain reaction (PCR). Fibroblasts were stimulated with tumor necrosis factor alpha, interleukin-1beta (IL-1beta), bacterial lipopeptide, poly(I-C), lipopolysaccharide, and flagellin. Production of IL-6 was determined by enzyme-linked immunosorbent assay and induction of TLRs 2-5, matrix metalloproteinases (MMPs) 3 and 13 messenger RNA by real-time PCR. Expression of TLRs 2-4 in synovial tissues was analyzed by immunohistochemistry. RESULTS: Synovial fibroblasts expressed TLRs 1-6, but not TLRs 7-10. Among the expressed TLRs, TLR-3 and TLR-4 were the most abundant in synovial fibroblasts, and stimulation of synovial fibroblasts with the TLR-3 ligand poly(I-C) led to the most pronounced increase in IL-6, MMP-3, and MMP-13. In contrast, skin fibroblasts did not up-regulate MMP-3 or MMP-13 after stimulation with any of the tested stimuli. In synovial tissues from patients with early RA, TLR-3 and TLR-4 were highly expressed and were comparable to the levels of patients with longstanding RA. These expression levels were elevated as compared with those in OA. CONCLUSION: Our findings of high expression of TLRs, particularly TLRs 3 and 4, at an early stage of RA and the reactivity of synovial fibroblasts in vitro to TLR ligands suggest that TLR signaling pathways resulting in persistent inflammation and joint destruction are activated early in the disease process