Blocking ERK-1/2 reduces tumor necrosis factor Α–induced interleukin-18 bioactivity in rheumatoid arthritis synovial fibroblasts by induction of interleukin-18 binding protein A

Objective To examine the mechanism of regulation of interleukin-18 (IL-18) bioactivity by IL-18 binding protein (IL-18BP) induction. Methods Levels of IL-18 and IL-18BPa in synovial fluid samples from patients with osteoarthritis (OA) or rheumatoid arthritis (RA) were determined by enzyme-linked immunosorbent assays (ELISAs), followed by calculation of free IL-18. IL-18 and IL-18BPa synthesis in RA synovial fibroblasts that had been treated with proinflammatory and antiinflammatory cytokines were assessed by quantitative real-time polymerase chain reaction and ELISA, respectively, followed by IL-18 bioactivity determination using KG-1 cells. Chemical signaling inhibitors were used for determination of the signal transduction pathways involved in IL-18BPa/IL-18 regulation. Tumor necrosis factor Α (TNFΑ)–induced caspase 1 activity was determined by a colorimetric assay. Results IL-18BPa was lower in RA synovial fluid than in OA synovial fluid ( P < 0.05; n = 8), and free IL-18 was higher in RA synovial fluid than in OA synovial fluid. TNFΑ induced RA synovial fibroblast IL-18BPa and IL-18 in a time-dependent manner ( P < 0.05). Evaluation of signaling pathways suggested that TNFΑ induced IL-18 production through the ERK-1/2, protein kinase CΔ (PKCΔ), and Src pathways, whereas IL-18BPa synthesis was mediated through the NFΚB, PKC, Src, and JNK pathways. Furthermore, addition of exogenous IL-18BPa-Fc reduced the RA synovial fibroblast phosphorylation of ERK-1/2 induced by TNFΑ. Conclusion These results suggest that IL-18BPa reduces IL-18 bioactivity induced by TNFΑ, by regulating the ERK-1/2 pathway in RA synovial fibroblasts. Targeting IL-18 bioactivity by induction or addition of IL-18BPa may provide another therapeutic option in the management of RA.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/69190/1/27269_ftp.pd

Triggering of the dsRNA Sensors TLR3, MDA5, and RIG-I Induces CD55 Expression in Synovial Fibroblasts

Background: CD55 (decay-accelerating factor) is a complement-regulatory protein highly expressed on fibroblast-like synoviocytes (FLS). CD55 is also a ligand for CD97, an adhesion-type G protein-coupled receptor abundantly present on leukocytes. Little is known regarding the regulation of CD55 expression in FLS. Methods: FLS isolated from arthritis patients were stimulated with pro-inflammatory cytokines and Toll-like receptor (TLR) ligands. Transfection with polyinosinic-polycytidylic acid (poly(I:C)) and 5'-triphosphate RNA were used to activate the cytoplasmic double-stranded (ds)RNA sensors melanoma differentiation-associated gene 5 (MDA5) and retinoic acid-inducible gene-I (RIG-I). CD55 expression, cell viability, and binding of CD97-loaded beads were quantified by flow cytometry. Results: CD55 was expressed at equal levels on FLS isolated from patients with rheumatoid arthritis (RA), osteoarthritis, psoriatic arthritis and spondyloarthritis. CD55 expression in RA FLS was significantly induced by IL-1 beta and especially by the TLR3 ligand poly(I:C). Activation of MDA5 and RIG-I also enhanced CD55 expression. Notably, activation of MDA5 dose-dependently induced cell death, while triggering of TLR3 or RIG-I had a minor effect on viability. Upregulation of CD55 enhanced the binding capacity of FLS to CD97-loaded beads, which could be blocked by antibodies against CD55. Conclusions: Activation of dsRNA sensors enhances the expression of CD55 in cultured FLS, which increases the binding to CD97. Our findings suggest that dsRNA promotes the interaction between FLS and CD97-expressing leukocyte