The clock gene Bmal1 controls inflammatory mediators in rheumatoid arthritis fibroblast-like synoviocytes.

OBJECT: To clarify the involvement of clock genes in the production of inflammatory mediators from RA-FLS, we examined the role of Bmal1, one of the master clock genes. METHODS: RA-FLSs were stimulated with IL-1ß (0, 20 ng/mL), IL-6 (0, 20 ng/mL), IL-17 (0, 20 ng/mL), TNF-α (0, 20 ng/mL) or IFN-γ (0, 20 ng/mL) to examine the expression of Bmal1, MMP-3, CCL2, IL-6, IL-7 and IL-15 by qPCR and immunofluorescence staining. After silencing Bmal1, RA-FLSs were stimulated with IL-1ß (0, 20 ng/mL), TNF-α (0, 20 ng/mL) or IFN-γ (0, 20 ng/mL) to examine the expressions of inflammatory mediators; MMP-3, CCL2, IL-6 and IL-15 by qPCR, ELISA and immunofluorescence staining. RESULTS: Bmal1 expressions were increased by IL-1ß, TNF-α and IFN-γ stimulations. Under stimulations with TNF-α, IL-1ß, and IFN-γ, mRNA and protein expressions of MMP-3, CCL2 and IL-6 were suppressed by siBmal1. CONCLUSION: Results indicate that Bmal1 contributes the production of MMP-3, CCL2, and IL-6 from RA-FLS, implying Bmal1 is involved in the pathogenesis of RA by regulating the inflammation.

Tocilizumab suppresses NF-kappa B activation via toll-like receptor 9 signaling by reducing cell-free DNA in rheumatoid arthritis.

Endogenous DNA is released into the bloodstream as cell-free DNA (cfDNA) following cell death and is associated with various pathological conditions. However, their association with therapeutic drugs against rheumatoid arthritis (RA) remains unknown. Therefore, we investigated the significance of cfDNA in RA treated with tocilizumab and tumour necrosis factor inhibitor (TNF-I). Biological DMARDs (bDMARDs), including tocilizumab and TNF-I, were administered to 77 and 59 RA patients, respectively. Plasma cfDNA levels were measured at weeks 0, 4, and 12 by quantitative polymerase chain reaction. Disease activity was evaluated at the same time point using DAS28ESR. cfDNA levels from RA synovial cells treated with tocilizumab or etanercept for 24 h were measured. Human toll-like receptor 9 (hTLR9)-expressing HEK293 cells, which release secreted embryonic alkaline phosphatase (SEAP) upon NF-κB activation, were stimulated by cfDNA from RA patients, and subsequently, SEAP levels were determined. NF-κB translocation was evaluated by immunofluorescence staining with or without tocilizumab. The DAS28ESR significantly improved in both bDMARD groups at week 12. However, plasma cfDNA levels significantly decreased in the tocilizumab group at week 12 compared to that in week 0. cfDNA levels correlated with DAS28ESR in biological treatment-naïve patients administered tocilizumab. cfDNA levels in synovial cells were significantly suppressed by tocilizumab treatment and unaltered with etanercept. HEK293 cells released SEAP upon cfDNA stimulation, and the observed NF-κB nuclear translocation was suppressed by tocilizumab. Tocilizumab suppressed inflammation via the TLR9 pathway by decreasing cfDNA levels. Regulation of cfDNA may be a therapeutic target for RA.

TNF-α induces expression of the circadian clock gene Bmal1 via dual calcium-dependent pathways in rheumatoid synovial cells.

Tumor necrosis factor (TNF)-α is responsible for expressions of several clock genes and affects joint symptoms of rheumatoid arthritis (RA) with diurnal fluctuation. We tried to determine the mechanism involved in over-expression of Bmal1, induced by TNF-α, in primary cultured rheumatoid synovial cells. Cells were incubated with intra-cellular Ca2+ chelator BAPTA-AM, calcineurin inhibitor FK506 and p300/CBP (CREB binding protein) inhibitor C646, respectively, or transfected with p300 and CBP small interfering RNA (siRNA) before stimulation with TNF-α. Oscillation phase and amplitude of Bmal1, transcriptional activator Rorα, transcriptional repressor Rev-erbα, and histone acetyltransferases (p300 and Cbp) were evaluated by quantitative real-time PCR. As results, TNF-α did not influence the oscillation phase of Rev-erbα, while enhanced those of Rorα, resulting in over-expression of Bmal1. When Ca2+ influx was inhibited by BAPTA-AM, TNF-α-mediated up-regulation of Rorα was cancelled, however, that of Bmal1 was still apparent. When we further explored another pathway between TNF-α and Bmal1, TNF-α suppressed the expression of Rev-erbα in the absence of Ca2+ influx, as well as those of p300 and Cbp genes. Finally, actions of TNF-α, in increasing Bmal1/Rorα and decreasing Rev-erbα, were cancelled by C646 treatment or silencing of both p300 and Cbp. In conclusion, we determined a novel role of TNF-α in inducing Bmal1 via dual calcium dependent pathways; Rorα was up-regulated in the presence of Ca2+ influx and Rev-erbα was down-regulated in the absence of that. Results proposed that inhibition of p300/CBP could be new therapeutic targets for RA.

DcR3-TL1A signalling inhibits cytokine-induced proliferation of rheumatoid synovial fibroblasts.

Decoy receptor 3 (DcR3), a member of the tumour necrosis factor receptor (TNFR) superfamily, lacks the transmembrane domain of conventional TNFRs in order to be a secreted protein. DcR3 competitively binds and inhibits members of the TNF family, including Fas ligand (FasL), LIGHT and TL1A. We previously reported that TNFα-induced DcR3 overexpression in rheumatoid synovial fibroblasts (RA-FLS) protects the cells from Fas-induced apoptosis and that DcR3 induces VLA-4 expression in THP-1 macrophages to inhibit cycloheximide-induced apoptosis. Meanwhile, recent studies have suggested that DcR3 acting as a ligand directly induces the differentiation of macrophages to osteoclasts. Therefore, in the present study, we analyzed the direct effects of DcR3 as a ligand in RA-FLS. The experiments showed that DcR3 binds to TL1A expressed in RA-FLS resulting in the negative regulation of cell proliferation induced by inflammatory cytokines. DcR3-TL1A signalling may be involved in the pathogenesis of rheumatoid arthritis (RA).

Decoy receptor 3 is highly expressed in patients with rheumatoid arthritis.

Decoy receptor 3 (DcR3), a member of the tumor necrosis factor receptor (TNFR) superfamily, is a soluble receptor that binds to the TNF family including Fas ligand (Fas-L), LIGHT, and TL1A. DcR3 is mostly expressed in tumor cells and competitively inhibits the TNF family. We previously demonstrated that overexpressed DcR3 in rheumatoid synovial cells protects the cells from apoptosis in vitro. The objective of the study was to investigate DcR3 expression in serum and joint fluids of patients with rheumatoid arthritis (RA) and osteoarthritis (OA), and to analyze the correlations with disease activities and TNFalpha expression. Sera and joint fluids were collected from patients with RA and OA. Expression of DcR3 in sera and joint fluids was measured by ELISA. The concentration of DcR3 in sera and joint fluids of RA patients was significantly higher than that in sera and joint fluids of OA patients. A correlation between serum DcR3 concentration and disease activity was not observed, but the serum DcR3 concentration was strongly correlated with the TNFalpha concentration. DcR3 was highly expressed in serum and joint fluids of RA patients.

DcR3 protects THP-1 macrophages from apoptosis by increasing integrin alpha4.

In order to analyze the function of DcR3 for the regulation of cell adhesion and apoptosis in macrophages, we investigated the expression of decoy receptor 3 (DcR3) in THP-1 monocytes/macrophages. DcR3 was expressed in THP-1 and increased by phorbol 12-myristate 13-acetate (PMA). The formation of macrophage aggregates was observed when THP-1 cells were differentiated by PMA or stimulated with DcR3-Fc. Undifferentiated THP-1 cells were also induced to form aggregates by DcR3-Fc. The expression of integrin alpha4 was significantly increased by DcR3-Fc. CHX-induced apoptosis in THP-1 was inhibited by DcR3-Fc, of which inhibition against CHX-induced apoptosis and aggregate formation were ameliorated by anti-VLA4 antibody. DcR3 may play a significant role in macrophages not only by a decoy receptor but also by increasing alpha4 integrin.

Decoy receptor 3 expressed in rheumatoid synovial fibroblasts protects the cells against Fas-induced apoptosis.

OBJECTIVE: Decoy receptor 3 (DcR3), a newly identified member of the tumor necrosis factor receptor (TNFR) superfamily, is a soluble receptor that binds to members of the TNF family, including FasL, LIGHT, and TNF-like molecule 1A. DcR3 is mostly expressed in tumor cells, and it competitively inhibits binding of TNF to TNFRs. The present study was undertaken to investigate DcR3 expression in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA) and osteoarthritis (OA), and to analyze the effects of DcR3 on Fas-induced apoptosis in RA FLS. METHODS: Expression of DcR3 in FLS was measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. FLS were incubated with DcR3-Fc chimera protein or transfected with DcR3 small interfering RNA (siRNA) using the lipofection method, before induction of apoptosis. Apoptosis induced by Fas in FLS was detected with TUNEL staining and Western blotting of caspase 8 and poly(ADP-ribose) polymerase. Finally, FLS were incubated with TNFalpha prior to Fas-induced apoptosis, expression of DcR3 was analyzed by quantitative RT-PCR, and apoptosis was measured. RESULTS: DcR3 was expressed in both RA FLS and OA FLS. DcR3-Fc protein inhibited Fas-induced apoptosis in FLS. Down-regulation of DcR3 in FLS by siRNA increased Fas-induced apoptosis. TNFalpha increased DcR3 expression and inhibited Fas-induced apoptosis in RA FLS, but not in OA FLS. CONCLUSION: DcR3 expressed in RA FLS is increased by TNFalpha and protects the cells against Fas-induced apoptosis. These findings indicate that DcR3 may be a possible therapeutic target in RA.

Bilateral painful snapping elbows triggered by daily dumbbell exercises: a case report.

We treated a 64-year-old female with bilateral painful snapping elbows due to synovial folds. Resection of the bilateral synovial folds resulted in relief from pain and snapping, and resulted in patient satisfaction. Daily dumbbell exercises of the bilateral elbows starting at an elderly age may have led to the bilateral snapping. Painful snapping elbow is a relatively rare condition, which usually occurs in a unilateral elbow of a younger patient. To our knowledge, bilateral painful snapping elbows triggered by daily dumbbell exercises in older patients have not been previously reported.

[Anesthetic management of a pediatric patient with a laryngeal foreign body].

Laryngeal foreign bodies in children are less common than bronchial foreign bodies, but are more dangerous. We report an 8-month-old boy who accidentally inhaled an open safety pin into his larynx. Anesthesia was induced and maintained with sevoflurane, oxygen. No muscle relaxant was administered during operation. The open safety pin was removed with Jackson's esophagoscope. No complications were observed during the operation.