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author:("Tan, wengen")
1.  Modulatory Effect of 1,25-Dihydroxyvitamin D3 on IL1β-Induced RANKL, OPG, TNFα, and IL-6 Expression in Human Rheumatoid Synoviocyte MH7A 
Receptor activator of nuclear factor κB ligand (RANKL) plays a crucial role in the bone erosion of rheumatoid arthritis (RA) by prompting osteoclastogenesis. Considering that 1,25(OH)2D3 has been suggested as a potent inducer of RANKL expression, it should clarify whether vitamin D supplement could result in RANKL overexpression and thereby facilitate excessive osteoclastogenesis and bone resorption in RA. Here, we investigated modulatory effect of 1,25(OH)2D3 on the expression of RANKL and its decoy receptor osteoprotegerin (OPG) in an inflammatory condition of human rheumatoid synoviocyte MH7A. MH7A cells were stimulated with IL1β and then treated with different concentrations of 1,25(OH)2D3 for 48 h. A significantly elevated OPG/RANKL ratio and markedly decreased levels of IL-6 and TNFβ mRNA expression in cells and IL-6 protein in supernatants were observed in IL1β-induced MH7A in the presence of 1,25(OH)2D3 compared with those in the absence of it. Osteoclast formation was obviously decreased when RAW264.7 cells were treated with both 1,25(OH)2D3 and IL1β. In summary, although it has a biological function to induce RANKL expression, 1,25(OH)2D3 could upregulate OPG/RANKL ratio and mediate anti-inflammatory action in an inflammatory milieu of synoviocyte, contributing to the inhibition of inflammation-induced osteoclastogenesis in RA.
PMCID: PMC3855937  PMID: 24348674
2.  IL-29 enhances Toll-like receptor-mediated IL-6 and IL-8 production by the synovial fibroblasts from rheumatoid arthritis patients 
Arthritis Research & Therapy  2013;15(5):R170.
We previously reported that IL-29, a newly described member of interferon (IFN) family, was overexpressed in blood and synovium of rheumatoid arthritis (RA) patients and triggered proinflammatory cytokine IL-6 and IL-8 mRNA expression in RA synovial fibroblasts (RA-FLS). This suggests that IL-29 has an important role in synovial inflammation. Toll-like receptors (TLRs) also activate RA-FLS to produce inflammatory mediators including tumor necrosis factor α (TNF-α) and IL-1β in RA-FLS. Since the TLR family plays an early role in the innate immune response and the subsequent induction of the adaptive immune response, we hypothesize that IL-29 interacts with TLRs in RA inflammation. This study aimed to investigate the effect of IL-29 on TLR-mediated proinflammatory cytokine production in RA-FLS.
The mRNA level of IL-29 receptors (IL-28Rα and IL-10R2) in RA-FLS was determined by semi-quantitative RT- PCR. IL-6 and IL-8 mRNA expressions in RA-FLS were evaluated by real-time PCR after pre-incubation with IL-29 and subsequent stimulation with peptidoglycan (PGN, TLR2 ligand), or polycytidylic acid (poly(I:C), TLR3 ligand), or lipopolysaccharide (LPS, TLR4 ligand) . The production of TLR2, 3, and 4 in RA-FLS after IL-29 stimulation was also assessed by real-time PCR and flow cytometry. IL-29 mRNA and protein expression in RA-FLS after stimulation with PGN, poly(I:C), or LPS were measured by real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively.
The IL-29 receptor complex (IL-28Rα and IL-10R2) was identified in RA-FLS. IL-29 enhanced TLR-mediated IL-6 and IL-8 expression in RA-FLS. IL-29 upregulated expression of TLR2, 3 and 4 in RA-FLS. Exposure to PGN, poly(I:C) or LPS triggered IL-29 production by RA-FLS.
We show for the first time that IL-29 enhances TLR-induced proinflammatory cytokine production in RA-FLS via upregulation of TLRs.
PMCID: PMC3978693  PMID: 24286242
3.  Correlation of Increased Blood Levels of GITR and GITRL with Disease Severity in Patients with Primary Sjögren's Syndrome 
Glucocorticoid-induced tumor necrosis factor receptor family-related protein (GITR) is a type I transmembrane protein belonging to the TNFR superfamily. After activated by its ligand GITRL, GITR could influence the activity of effector and regulatory T cells, participating in the development of several autoimmune and inflammatory diseases included rheumatoid arthritis and autoimmune thyroid disease. We previously reported that serum GITRL levels are increased in systemic lupus erythematosus (SLE) patients compared with healthy controls (HC). Here, we tested serum soluble GITR (sGITR) and GITRL levels in 41 primary Sjögren's syndrome (pSS) patients and 29 HC by ELISA and correlated sGITR and GITRL levels with clinical and laboratory variables. GITR and GITRL expression in labial salivary glands was detected by immunohistochemistry. pSS patients had significantly increased serum levels of sGITR and GITRL compared with controls (GITR: 5.66 ± 3.56 ng/mL versus 0.50 ± 0.31 ng/mL; P < 0.0001; GITRL: 6.17 ± 7.10 ng/mL versus 0.36 ± 0.28 ng/mL; P < 0.0001). Serum sGITR and GITRL levels were positively correlated with IgG (GITRL: r = 0.6084, P < 0.0001; sGITR: r = 0.6820, P < 0.0001) and ESR (GITRL: r = 0.8315, P < 0.0001; sGITR: r = 0.7448, P < 0.0001). Moreover, GITR and GITRL are readily detected in the lymphocytic foci and periductal areas of the LSGs. In contrast, the LSGs of HC subjects did not express GITR or GITRL. Our findings indicate the possible involvement of GITR-GITRL pathway in the pathogenesis of pSS. Further studies may facilitate the development of targeting this molecule pathway for the treatment of pSS.
PMCID: PMC3722791  PMID: 23935647
4.  Correlation of Circulating Glucocorticoid-Induced TNFR-Related Protein Ligand Levels with Disease Activity in Patients with Systemic Lupus Erythematosus 
The aim of this paper is to investigate the correlation of glucocorticoid-induced tumor necrosis factor receptor- (TNFR-) related protein ligand (GITRL) with disease activity and organ involvement in patients with systemic lupus erythematosus (SLE). Serum GITRL levels were measured in 58 patients with SLE and 30 healthy controls matched for age and sex. Patients were assessed for clinical and laboratory variables. Correlations of serum GITRL levels with SLEDAI, laboratory values, and clinical manifestations were assessed. Serum GITRL levels were determined by ELISA. Serum GITRL levels were markedly increased in patients with SLE compared with healthy controls (mean 401.3 ng/mL and 36.59 ng/mL, resp.; P < 0.0001). SLE patients with active disease showed higher serum GITRL levels compared to those with inactive disease (mean 403.3 ng/mL and 136.3 ng/mL, resp; P = 0.0043) as well as normal controls (36.59 ng/mL; P < 0.0001). Serum GITRL levels were positively correlated with SLEDAI, titers of anti-dsDNA antibody, erythrocyte sedimentation rate (ESR), and IgM and negatively correlated with complement3 (C3). Serum GITRL levels were higher in SLE patients with renal involvement and vasculitis compared with patients without the above-mentioned manifestations.
PMCID: PMC3521490  PMID: 23251213
5.  Interleukin-29 modulates proinflammatory cytokine production in synovial inflammation of rheumatoid arthritis 
Arthritis Research & Therapy  2012;14(5):R228.
The immunoregulatory function of interleukin (IL)-29 has recently been recognized. However, little is known about the involvement of IL-29 in the pathogenesis of rheumatoid arthritis (RA). This study aimed to examine the expression profiles of IL-29 in blood, synovial fluid (SF) and synovium in RA patients and investigate the effect of IL-29 on cytokines production in RA synovial fibroblasts.
The transcript levels of IL-29 and its specific receptor IL-28Rα in peripheral blood mononuclear cells (PBMC) and synovium were determined by real-time reverse transcription-polymerase chain reaction (real-time PCR). The concentrations of IL-29 in serum and synovial fluid (SF) were quantified by enzyme-linked immunoassay (ELISA), and the correlation of serum IL-29 levels with disease activity in RA patients was investigated. Furthermore, the expression of IL-29 in RA synovium was examined by immunohistochemistry and double immunofluorescence analysis. Finally, the expression of IL-6, IL-8, IL-10, IL-17 and matrix metalloproteinase-3 (MMP-3) in synovial fibroblasts upon IL-29 stimulation was determined by real-time PCR.
IL-29 and IL-28Rα mRNA expression in PBMC was significantly increased in patients with RA compared with healthy controls (HC). The serum levels of circulating IL-29 were higher in RA than those in HC. Increased IL-29 levels were detected in RA SF when compared with osteoarthritis (OA) SF. However, serum IL-29 levels showed no significant correlation with RA disease activity. IL-29 was mostly expressed in the lining region of RA synovium. Moreover, IL-29 was expressed predominately in synovial macrophages and fibroblasts. RA synovial fibroblasts exposed to IL-29 specifically upregulated IL-6, -8 and MMP-3 but downregulated IL-10.
The findings in the present study indicate, for the first time, that IL-29 is dysregulated in patients with RA, which may contribute to the RA pathogenesis via inducing the production of proinflammatory cytokines, chemokines or matrix metalloproteinases in synovial fibroblasts.
PMCID: PMC3580539  PMID: 23078630
6.  Association of PPP2CA polymorphisms with SLE susceptibility in multiple ethnic groups 
Arthritis and rheumatism  2011;63(9):2755-2763.
T cells from patients with SLE express increased amounts of PP2Ac which contribute to decreased production of IL-2. Because IL-2 is important in the regulation of several aspects of the immune response, it has been proposed that PP2Ac contributes to the expression of SLE. This study was designed to determine whether genetic variants of PPP2AC are linked to the expression of SLE and specific clinical manifestations and account for the increased expression of PP2Ac.
We conducted a trans-ethnic study consisting of 8,695 SLE cases and 7,308 controls from four different ancestries. Eighteen single-nucleotide polymorphisms (SNPs) across the PPP2CA were genotyped using an Illumina custom array. PPP2CA expression in SLE and control T cells was analyzed by real-time PCR.
A 32-kb haplotype comprised of multiple SNPs of PPP2CA showed significant association with SLE in Hispanic Americans (HA), European Americans (EA) and Asians but not in African-Americans (AA). Conditional analyses revealed that SNP rs7704116 in intron 1 showed consistently strong association with SLE across Asian, EA and HA populations (pmeta=3.8×10−7, OR=1.3[1.14–1.31]). In EA, the largest ethnic dataset, the risk A allele of rs7704116 was associated with the presence of renal disease, anti-dsDNA and anti-RNP antibodies. PPP2CA expression was approximately 2-fold higher in SLE patients carrying the rs7704116 AG genotype than those carrying GG genotype (p = 0.008).
Our data provide the first evidence for an association between PPP2CA polymorphisms and elevated PP2Ac transcript levels in T cells, which implicates a new molecular pathway for SLE susceptibility in EA, HA and Asians.
PMCID: PMC3163110  PMID: 21590681
7.  A Functional RANKL Polymorphism Associated with Younger Age at Onset of Rheumatoid Arthritis 
Arthritis and rheumatism  2010;62(10):2864-2875.
We previously reported association of co-occurrence of HLA-DRB1 shared epitope (SE) and RANKL SNPs with younger age of RA onset in 182 rheumatoid factor positive (RF) European American (EA) early RA patients. Here, we fine-mapped the 48 kb RANKL region in the extended 210 EA RF-positive early RA cohort, sought replication of RA-associated SNPs in additional 501 EA and 298 African-Americans (AA) RA cohorts, and explored functional consequences of RA-associated SNPs.
SNP genotyping was conducted using pyrosequencing or TaqMan PCR assays. Associations of rs7984870 with RANKL expression in plasma, PBMC and isolated T cells were quantified using ELISA and RT-PCR. Site-directed mutagenesis of rs7984870 within the 2kb RANKL promoter was performed to drive the luciferase reporter gene in osteoblast and stromal cell lines. Interaction of DNA and protein was determined by electrophoretic mobility shift assay.
A single promoter SNP rs7984870 was consistently significantly associated with earlier age of RA onset in 3 independent seropositive (RF or anti-cyclic citrullinated peptide antibody positive) RA cohorts but not in seronegative RA patients. The risk C allele of rs7984870 conferred 2-fold higher plasma RANKL levels in RF-positive RA patients, significantly elevated RANKL mRNA expression in activated normal T cells, and increased promoter activity after stimulation in vitro via differential binding to transcription factor SOX5.
The RANKL promoter allele that increased transcriptional levels upon stimulation might promote interaction between activated T cells and dendritic cells, predisposing to younger RA onset in seropositive EA and/or AA individuals.
PMCID: PMC2944013  PMID: 20533289
8.  Altered Expression of Signaling Genes in Jurkat Cells upon FTY720 Induced Apoptosis 
FTY720, a novel immunosuppressant, has a marked activity in decreasing peripheral blood T lymphocytes upon oral administration. Recent investigations suggest that the action of FTY720 on lymphocytes may result from its ability to induce cell apoptosis. However, the cell signaling mechanism involved in the FTY720-induced cell apoptosis remains unclear. Here we examined the apoptotic signal pathways mediated by FTY720 in Jurkat cells using microarray analysis. The results showed that FTY720 can induce Jurkat cell apoptosis in a dose and time dependent manner as assessed by cell viability, Hoechst 33258 staining, Annexin V binding and DNA fragmentation tests. cDNA microarray analysis showed that 10 μM of FTY720 up-regulated 54 and down-regulated 10 genes in Jurkat cells among the 458 apoptotic genes examined following the 6 h incubation period. At least five-fold increased expression of modulator of apoptosis-1 (MOAP-1), vascular endothelial growth factor (VEGF), tumor necrosis factor receptor-associated factors (TRAF 6), Caspase 2 (CASP 2), E2F transcription factor 1 (E2F 1) and Casapse 5 (CASP 5) genes was observed in microarray analyses; these results were confirmed with reverse transcription polymerase chain reaction (RT-PCR) examination. Our findings suggest that the mitochondria related signaling pathways are the key pathways involved in the FTY720-induced apoptosis in Jurkat cells. And our results provide a new insight into the mechanism of FTY720, which allows us to draw the first simple diagram showing the potential pathways mediated by FTY720.
PMCID: PMC2956082  PMID: 20957081
FTY720; signal transduction; jurkat cell; apoptosis; cDNA microarray

Results 1-8 (8)