Proliferation In Rheumatoid Arthritis Research Articles

Mir204 and Mir211 suppress synovial inflammation and proliferation in rheumatoid arthritis by targeting Ssrp1.

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease characterized by synovial hyperplasia. Mir204 and Mir211 are homologous miRNAs with the same gene targeting spectrum. It is known that Mir204/211 play an important role in protecting osteoarthritis development; however, the roles of Mir204/211 in RA disease have not been determined. In the present study, we investigated the effects and molecular mechanisms of Mir204/211 on synovial inflammation and hyperproliferation in RA. The effects of Mir204/211 on the inflammation and abnormal proliferation in primary fibroblast-like synoviocytes (FLSs) were examined by Mir204/211 gain-of-function and loss-of-function approaches in vitro and in vivo. We identified the structure-specific recognition protein 1 (Ssrp1) as a downstream target gene of Mir204/211 based on the bioinformatics analysis. We overexpressed Ssrp1and Mir204/211 in FLS to determine the relationship between Ssrp1 and Mir204/211 and their effects on synovial hyperplasia. We created a collagen-induced arthritis (CIA) model in wild-type as well as Mir204/211 double knockout (dKO) mice to induce RA phenotype and administered adeno-associated virus (AAV)-mediated Ssrp1-shRNA (AAV-shSsrp1) by intra-articular injection into Mir204/211 dKO mice. We found that Mir204/211 attenuated excessive cell proliferation and synovial inflammation in RA. Ssrp1 was the downstream target gene of Mir204/211. Mir204/211 affected synovial proliferation and decelerated RA progression by targeting Ssrp1. CIA mice with Mir204/211 deficiency displayed enhanced synovial hyperplasia and inflammation. RA phenotypes observed in Mir204/211 deficient mice were significantly ameliorated by intra-articular delivery of AAV-shSsrp1, confirming the involvement of Mir204/211-Ssrp1signaling during RA development. In this study, we demonstrated that Mir204/211 antagonize synovial hyperplasia and inflammation in RA by regulation of Ssrp1. Mir204/211 may serve as novel agents to treat RA disease.

A deep learning classification of metacarpophalangeal joints synovial proliferation in rheumatoid arthritis by ultrasound images.

To evaluate if an automatic classification of rheumatoid arthritis (RA) metacarpophalangeal joint conditions in ultrasound images is feasible by deep learning (DL) method, to provide a more objective, automated, and fast way of RA diagnosis in clinical setting. DenseNet-based DL model was used and both training and testing are implemented in TensorFlow 1.13.1 with Keras DL libraries. The area under curve (AUC), accuracy, sensitivity, and specificity values with 95% CIs were reported. The statistical analysis was performed by using scikit-learn libraries in Python 3.7. A total of 1337 RA ultrasound images were acquired from 208 patients, the number of images is 313, 657, 178, and 189 in OESS Grade L0, L1, L2, and L3, respectively. In Classification Scenario 1SP-no versus SP-yes, three experiments with region of interest of size 192 × 448 (Group 1), 96 × 224 (Group 2), and 96 × 224 stacked with pre-segmented annotated mask of SP area (Group 3) as input achieve an AUC of 0.863 (95% CI: 0.809, 0.917), 0.861 (95% CI: 0.805, 0.916), and 0.886 (95% CI: 0.836, 0.936), respectively. In Classification Scenario 2 Healthy versus Diseased, experiments in Group 1, Group 2 and Group 3 achieve an AUC of 0.848 (95% CI: 0.799, 0.896), 0.864 (95% CI: 0.819, 0.909), and 0.916 (95% CI: 0.883, 0.952), respectively. We combined DenseNet model with ultrasound images for RA condition assessment. The feasibility of using DL to create an automatic RA condition classification system was also demonstrated. The proposed method can be an alternative to the initial screening of RA patients.

Targeting synovial fibroblast proliferation in rheumatoid arthritis (TRAFIC): an open-label, dose-finding, phase 1b trial.

SummaryBackgroundCurrent rheumatoid arthritis therapies target immune inflammation and are subject to ceiling effects. Seliciclib is an orally available cyclin-dependent kinase inhibitor that suppresses proliferation of synovial fibroblasts—cells not yet targeted in rheumatoid arthritis. Part 1 of this phase 1b/2a trial aimed to establish the maximum tolerated dose of seliciclib in patients with active rheumatoid arthritis despite ongoing treatment with TNF inhibitors, and to evaluate safety and pharmacokinetics.MethodsPhase 1b of the TRAFIC study was a non-randomised, open-label, dose-finding trial done in rheumatology departments in five UK National Health Service hospitals. Eligible patients (aged ≥18 years) fulfilled the 1987 American College of Rheumatology (ACR) or the 2010 ACR–European League Against Rheumatism classification criteria for rheumatoid arthritis and had moderate to severe disease activity (a Disease Activity Score for 28 joints [DAS28] of ≥3·2) despite stable treatment with anti-TNF therapy for at least 3 months before enrolment. Participants were recruited sequentially to a maximum of seven cohorts of three participants each, designated to receive seliciclib 200 mg, 400 mg, 600 mg, 800 mg, or 1000 mg administered in 200 mg oral capsules. Sequential cohorts received doses determined by a restricted, one-stage Bayesian continual reassessment model, which determined the maximum tolerated dose (the primary outcome) based on a target dose-limiting toxicity rate of 35%. Seliciclib maximum concentration (Cmax) and area under the plasma concentration time curve 0–6 h (AUC0–6) were measured. This study is registered with ISRCTN, ISRCTN36667085.FindingsBetween Oct 8, 2015, and Aug 15, 2017, 37 patients were screened and 15 were enrolled to five cohorts and received seliciclib, after which the trial steering committee and the data monitoring committee determined that the maximum tolerated dose could be defined. In addition to a TNF inhibitor, ten (67%) enrolled patients were taking conventional synthetic disease modifying antirheumatic drugs. The maximum tolerated dose of seliciclib was 400 mg, with an estimated dose-limiting toxicity probability of 0·35 (90% posterior probability interval 0·18–0·52). Two serious adverse events occurred (one acute kidney injury in a patient receiving the 600 mg dose and one drug-induced liver injury in a patient receiving the 400 mg dose), both considered to be related to seliciclib and consistent with its known safety profile. 65 non-serious adverse events occurred during the trial, 50 of which were considered to be treatment related. Most treatment-related adverse events were mild; 20 of the treatment-related non-serious adverse events contributed to dose-limiting toxicities. There were no deaths. Average Cmax and AUC0–6 were two-times higher in participants developing dose-limiting toxicities.InterpretationThe maximum tolerated dose of seliciclib has been defined for rheumatoid arthritis refractory to TNF blockade. No unexpected safety concerns were identified to preclude ongoing clinical evaluation in a formal efficacy trial.FundingUK Medical Research Council, Cyclacel, Research into Inflammatory Arthritis Centre (Versus Arthritis), and the National Institute of Health Research Newcastle and Birmingham Biomedical Research Centres and Clinical Research Facilities.

A Deep Learning Classification of Metacarpophalangeal Synovial Proliferation in Rheumatoid Arthritis by Ultrasound Images

Objective: To evaluate if an automatic classification of Rheumatoid arthritis (RA) metacarpophalangeal joint conditions in ultrasound images is feasible by deep learning (DL) method, to provide a more objective, automated and fast way of RA diagnosis in clinical setting. Materials and Methods: DenseNet-based DL model was used, both training and testing are implemented in TensorFlow 1.13.1 with Keras DL libraries. The area under curve (AUC), accuracy, sensitivity and specificity values with 95% CIs were reported. The statistical analysis was performed by using scikit-learn libraries in Python 3.7. Results: A total of 1337 RA ultrasound images were acquired from 208 patients, the number of images is 313, 657, 178 and 189 in OESS Grade L0, L1, L2, and L3 respectively. In Classification Scenario 1 S.P.-no vs. S.P.-yes, three experiments with region of interest of size 192*448 (Group 1), 96*224 (Group 2) and 96*224 stacked with pre-segmented annotated mask of S.P. area (Group 3) as input achieve an AUC of 0.863 (95% CI: 0.809, 0.917), 0.861 (95% CI: 0.805, 0.916) and 0.886 (95% CI: 0.836, 0.936), respectively. In Classification Scenario 2 Healthy vs. Diseased, experiments in Group 1, Group 2 and Group 3 achieve an AUC of 0.848 (95% CI: 0.799, 0.896), 0.864 (95% CI: 0.819, 0.909) and 0.916 (95% CI: 0.883, 0.952), respectively. Conclusion: We combined Dense Net model with ultrasound images for RA condition assessment. The feasibility of using DL to create an automatic RA condition classification system was also demonstrated. The proposed method can be an alternative to the initial screening of RA patients. Funding Statement: None. Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: The study was approved by the ethics committee of Shenzhen people’s Hospital.

MiR‑760 regulates skeletal muscle proliferation in rheumatoid arthritis by targeting Myo18b.

MicroRNAs serve an important role in the development of several diseases. Numerous genes regulate the skeletal muscle differentiation of C2C12 myoblasts. The role of miR-760 in rheumatoid arthritis (RA) has not been reported, to the best of our knowledge. Therefore, the aim of the present study was to examine the role of miR-760 in regulating skeletal muscle proliferation in RA. Potential genes functionally involved in the tarsal joint of a collagen-induced RA model were identified using Gene Expression Omnibus. Reverse transcription-quantitative PCR and western blot analyses were performed to determine the mRNA and protein expression levels. The proliferation, cell cycle progression and migration of C2C12 myoblasts were detected using Cell Counting Kit-8, flow cytometry and wound-healing assays, respectively. TargetScan was used to predict the potential target genes of miR-760, and this was verified using a dual-luciferase reporter assay. In the present study, myosin-18b (Myo18b) expression was determined to be downregulated in the RA model. Silencing Myo18b decreased the proliferation, abrogated the cell cycle progression, and reduced the migration and differentiation of C2C12 myoblasts. Expression levels of cyclin-dependent kinase 2, cyclin D1, matrix metalloproteinase (MMP)-2, MMP-9, myogenin and myosin heavy chain 6 were all decreased when Myo18b was silenced. Furthermore, overexpression of Myo18b induced opposing effects on C2C12 myoblasts. It was shown that Myo18b was a target gene of miRNA-760. Overexpression of miR-760 decreased proliferation, cell cycle progression, migration and differentiation in C2C12 myoblasts, and decreased the expression of Myo18b. The opposite results were observed when miR-760 was downregulated. In conclusion, miR-760 inhibited proliferation and differentiation by targeting Myo18b in C2C12 myoblasts. The results of the present study may contribute to understanding the mechanisms underlying RA skeletal muscle proliferation, and miR-760/Myo18b may serve as potential targets for treating patients with RA.

Intravoxel incoherent motion MRI for discrimination of synovial proliferation in the hand arthritis: A prospective proof-of-concept study.

Postcontrast-enhanced MRI is currently the reference standard for synovial proliferation in rheumatoid arthritis (RA). However, the technique is somewhat invasive due to the use of gadolinium contrast agents, which may cause severe adverse/side effects. Intravoxel incoherent motion (IVIM) simultaneously permits quantification of perfusion as well as diffusion using a single imaging scan. To test the capability of IVIM MRI for noninvasive discrimination of synovial proliferation in hand arthritis. Prospective. Seven suspected RA patients (three women and four men; mean age, 61 years; range, 26-74 years). 3 T/short tau inversion recovery (STIR), IVIM, postcontrast-enhanced MRI. Region of interest (ROI) was identified based on STIR. Contrast-enhanced MRI was evaluated using a 5-point grading scale of 0 (water) to 4 (synovial proliferation) according to the degree of contrast enhancement within the ROI. For each ROI, we calculated the apparent diffusion coefficient (ADC) and IVIM parameters (molecular diffusion coefficient [D], perfusion fraction [f], and perfusion-related diffusion coefficient [D*]). These parameters were subsequently compared with ROI contrast enhancement grades. Spearman's rank correlation test and a receiver operating characteristic (ROC) curve. A total of 90 ROIs of suspected synovial proliferation and/or joint effusion were identified. ROI grades were correlated with ADC and D values (r S = -0.385, P < 0.001, r S = -0.458, P < 0.0001, respectively), but not with the f and D* values (r S = -0.010, P = 0.936, r S = -0.084, P = 0.505, respectively). The area under the curves (AUCs) of D values (0.708-0.888, P = 0.002-0.0002) were slightly larger than those of ADC values (0.692-0.791, P = 0.013-0.001) when comparing low- vs. high-contrast enhancement grades. The IVIM parameter D and ADC may be useful for the noninvasive identification of synovial proliferation in hand arthritis. 2 Technical Efficacy Stage: 2 J. Magn. Reson. Imaging 2019;50:1199-1206.

Methotrexate upregulates circadian transcriptional factors PAR bZIP to induce apoptosis on rheumatoid arthritis synovial fibroblasts

BackgroundEffects of methotrexate (MTX) on the proliferation of rheumatoid arthritis (RA) synovial fibroblasts are incompletely understood. We explored actions of MTX in view of circadian transcriptions of synovial fibroblasts.MethodsUnder treatment with MTX, expression of core circadian clock genes, circadian transcriptional factor proline and acidic amino acid-rich basic leucine zipper (PAR bZIP), and proapoptotic molecule Bcl-2 interacting killer (Bik) was examined by real-time polymerase chain reaction. Protein expression of circadian clock gene PERIOD2 (PER2) and CYTOCHROME C was also examined by western blotting and ELISA. Promoter activities of Per2 and Bik were measured by Luciferase assay. Expression of PER2, BIK, and CYTOCHROME C and morphological changes of the nucleus were observed by fluorescent immunostaining. Synovial fibroblasts were transfected with Per2/Bik small interfering RNA, and successively treated with MTX to determine cell viabilities. Finally, synovial fibroblasts were treated with MTX according to the oscillation of Per2/Bik expression.ResultsMTX (10 nM) significantly decreased cell viabilities, but increased messenger RNA expression of Per2, Bik, and PAR ZIP including D site of the albumin promoter binding protein (Dbp), hepatic leukemia factor (Hlf), and thyrotroph embryonic factor (Tef). MTX also increased protein expression of PER2 and CYTOCHROME C, and promoter activities of Per2 and Bik via D-box. Under fluorescent observations, expression of PER2, BIK, and CYTOCHROME C was increased in apoptotic cells. Cytotoxicity of MTX was attenuated by silencing of Per2 and/or Bik, and revealed that MTX was significantly effective in situations where Per2/Bik expression was high.ConclusionsWe present here novel unique action of MTX on synovial fibroblasts that upregulates PAR bZIP to transcribe Per2 and Bik, resulting in apoptosis induction. MTX is important in modulating circadian environments to understand a new aspect of pathogenesis of RA.

Ultrasound resistive index, power Doppler, and clinical parameters in established rheumatoid arthritis.

Ultrasonography (US) is a useful tool for the evaluation of sinovial vascularization and proliferation in rheumatoid arthritis (RA). Accordingly, resistive index (RI) on spectral Doppler (sD) US provides a quantitative analysis of vascular inflammation, but its utility in the evaluation of RA activity has not been established. Our objective was to determine the association of RI with other US parameters of synovitis and with clinical disease activity in established RA. Patients with positive power Doppler (pD) were included in a prospective cross-sectional study. Disease activity and disability were evaluated using the Disease Activity Score in 28-joints (DAS28) and Health Assessment Questionnaire (HAQ), respectively. Gray scale (GS) synovitis, pD, and sD analyses were performed by one of two examiners in wrists and the second and third metacarpophalangeal and proximal interphalangeal joints. The 10-joint GS and 10-joint pD scores and mean RI were then calculated. Weighted kappa (WK) values were employed to assess interobserver reability, and correlations were tested using the Spearman coefficient. Ninety-five RA patients (median duration of disease of 7years and mean DAS28 of 4.32±1.66) were included. WK values in real-time US were 0.77 for synovitis, 0.87 for pD, and 0.68 for RI. There were no significant correlations of RI with 10-joint GS, 10-joint pD, DAS28, joint counts, or HAQ (P>0.10 for all tests). Patients in remission had a mean RI similar to those with high disease activity (0.62±0.10, n=15 versus 0.63±0.13, n=34, respectively). The addition of the RI score did not seem to improve US performance in patients with established RA.

LBH Gene Transcription Regulation by the Interplay of an Enhancer Risk Allele and DNA Methylation in Rheumatoid Arthritis.

To identify nonobvious therapeutic targets for rheumatoid arthritis (RA), we performed an integrative analysis incorporating multiple "omics" data and the Encyclopedia of DNA Elements (ENCODE) database for potential regulatory regions. This analysis identified the limb bud and heart development (LBH) gene, which has risk alleles associated with RA/celiac disease and lupus, and can regulate cell proliferation in RA. We identified a novel LBH transcription enhancer with an RA risk allele (rs906868 G [Ref]/T) 6 kb upstream of the LBH gene with a differentially methylated locus. The confluence of 3 regulatory elements, rs906868, an RA differentially methylated locus, and a putative enhancer, led us to investigate their effects on LBH regulation in fibroblast-like synoviocytes (FLS). We cloned the 1.4-kb putative enhancer with either the rs906868 Ref allele or single-nucleotide polymorphism (SNP) variant into reporter constructs. The constructs were methylated in vitro and transfected into cultured FLS by nucleofection. We found that both variants increased transcription, thereby confirming the region's enhancer function. Unexpectedly, the transcriptional activity of the Ref risk allele was significantly lower than that of the SNP variant and is consistent with low LBH levels as a risk factor for aggressive FLS behavior. Using RA FLS lines with a homozygous Ref or SNP allele, we confirmed that homozygous Ref lines expressed lower LBH messenger RNA levels than did the SNP lines. Methylation significantly reduced enhancer activity for both alleles, indicating that enhancer function is dependent on its methylation status. This study shows how the interplay between genetics and epigenetics can affect expression of LBH in RA.

The Effect of SHH-Gli Signaling Pathway on the Synovial Fibroblast Proliferation in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by chronic synovitis. This study aims to investigate the role of sonic hedgehog (SHH)-Gli signaling pathway in synovial fibroblast proliferation in rheumatoid arthritis. The expression of serum SHH in RA patients group was significantly increased compared with the systemic lupus erythematosus (SLE), ankylosing spondylitis (AS), and healthy subject (healthy control, HC) groups, respectively; serum SHH expression of RA patients was positively correlated with rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (anti-CCP Ab), while there was no significant correlation between SHH expression and erythrocyte sedimentation rate (ESR). SHH, Ptch, Smo, and Gli molecules were highly expressed in rat RA-synovial fibroblast (RA-SF); after blocking the SHH-Gli signaling pathway with a Gli specific inhibitor, Gli-antagonist 61 (GANT61), RA-SF proliferation was inhibited in a dose-dependent manner and the apoptosis rate of RA-SF was increased as well; the expression levels of fibroblast growth factor receptor 1 (FGFR1) and FGFR3 declined in SF cells after GANT61 treatment. Our results suggest that SHH-Gli pathway is involved in the pathogenesis of RA, and blocking SHH-Gli pathway inhibits RA-SF cell proliferation and increases cell apoptosis, which may shed light on developing new ideas for RA treatment.

Role and mechanism of NKp44+NK cells in the proliferation and inflammation of synovium of rheumatoid arthritis patients

To explore the effects of NKp44+NK cells from rheumatoid arthritis (RA) patients on the proliferation and monocyte chemotactic protein 1 production of fibroblast-like synoviocytes (FLS). The proportions of natural killer (NK) p44 NK cells in peripheral blood (PB) of 50 RA patients and 50 healthy individuals were detected by flow cytometry. Synovial fluid (SF) samples from 30 RA patients were also detected. NKp44+NK cells in RA SF were sorted by flow cytometry for 5 times. The supernatant level of interleukin (IL)-22 was measured by enzyme-linked immunosorbent assay (ELISA). The proliferation of FLS after an addition of culture supernatant of NKp44+NK cells was detected by methyl thiazolyl tetrazolium (MTT) at 24, 48 and 72 h. Monocyte chemotactic protein (MCP)-1 production of RA FLS after an addition of rhIL-22 was detected by ELISA. The proportion of NKp44+NK cells in PB of RA patients was significantly higher than that of normal controls while the proportion of NKp44+NK cells in SF of RA patients was higher than that in PB of matched RA patients (1.270% vs 0, 15.190% vs 2.425%, P < 0.01). The supernatant level of IL-22 in NKp44+NK cell culture was (1 603 ± 332) ng/L. Rapid proliferation of RA FLS was observed at 24, 48, 72 h after an addition of culture supernatant (P < 0.01). IL-22 antibody obviously inhibited the proliferation of RA FLS induced by NKp44+NK cells. MCP-1 production of RA FLS was detected at 72 h after an addition of rhIL-22 (P < 0.01). NKp44+NK cells can promote the proliferation and MCP-1 production of RA FLS through the production of IL-22 so as to play an important role in the synovial proliferation and inflammation of RA.

Increased generation of pre-plasmacytoid dendritic cells in bone marrow of rheumatoid arthritis

Objective. Plasmacytoid dendritic cells (pDCs) have been found to be accumulated in synovial tissues in rheumatoid arthritis (RA). Since pDCs originate from bone marrow (BM), we explored the differentiation of pDC in BM in RA and osteoarthritis (OA).Methods. BM mononuclear cells (BMMNCs) of the posterior ileac crest from 25 RA patients and 22 OA patients were examined for the expression of BDCA2 and CD34 by flow cytometry. The degree of synovial proliferation was assessed on light microscopy in 10 of 25 RA patients.Results. There were no significant differences in percentages of CD34 + cells or BDCA2 + cells within BMMNC between RA and OA. However, RA BMMNC contained higher percentages of BDCA2 + CD34 + cells (pre-pDCs) than OA BMMNCs. Accordingly, percentages of BDCA2 + CD34+ cells within BM CD34 + cells were significantly higher in RA than in OA. Finally, the percentages of BDCA2 + CD34+ cells within BM CD34 + cells were significantly correlated with the degree of synovial proliferation in RA.Conclusion. These results indicate that the generation of pre-pDC from BM CD34 + cells is increased in RA compared with OA. Moreover, the data suggest that the increased output of pDC from BM might be involved in the synovial proliferation in RA.

THU0019 BLyS Expression in Synovial Fluids from Rheumatoid Arthritis Patients

BackgroundBLyS is a novel TNF family ligand, mainly secreted by myeloid cellsandsynovial fibroblasts that exerts profound effects on B cells leading to their maturation and increased survival. Local expression of...

Epidermal growth factor receptor (EGFR) as a therapeutic target in rheumatoid arthritis

The epidermal growth factor receptor (EGFR) has an important role in the hyperplastic growth of tumor. Similar to tumor growth, rheumatoid arthritis (RA) synovium is hyperplastic, invasive, and expresses EGFR and its ligands. Activation of EGFR signaling is responsible for synovial fibroblast proliferation in RA. Furthermore, in addition to its role in proliferation, EGFR and its ligands can induce cytokine production of synovial fibroblasts during the pathogenesis of RA. Agents that target EGFR have yielded promising results in animal experiments involving RA, pharmacologic modulations targeting EGFR, or its ligands may give rise to new therapeutic approaches for RA. In this review article, we will discuss the biological features of EGFR and summarize recent advances regarding the role of EGFR in the pathogenesis and treatment of RA.

A regulatory effect of IL-21 on T follicular helper-like cell and B cell in rheumatoid arthritis

IntroductionInterleukin (IL)-21 is a member of type I cytokine family. Recent studies indicate that IL-21 can promote T follicular helper (Tfh) cell differentiation and survival, a specialized T cell subset which provides help for B cell. It can also regulate the activation, proliferation and differentiation of human B cell and immunoglobulin (Ig) production as well as isotype switching of plasma cell. Rheumatoid arthritis (RA) is characterized by auto-antibodies overproduction such as rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) antibody, suggesting a pivotal role of Tfh cell and B cell in the pathogenesis of RA. This study aimed to investigate whether IL-21 had a regulatory effect on Tfh cell and B cell in RA.MethodsSerum IL-21 concentrations were measured by ELISA. The correlations between serum IL-21 levels and clinical features of RA patients were analyzed by Spearman's rank test. The percentages of Tfh-like cells, IL-21 receptor (R) expression on Tfh-like cells and B cells in peripheral blood (PB) were analyzed by flow cytometry. Peripheral blood mononuclear cells (PBMC) were stimulated by rIL-21 (100 ng/ml) in the presence or absence of anti-CD40 and/or anti-IgM, and changes of IL-21R, activation-associated surface markers (CD25, CD69 and CD40), the proliferation, apoptosis and differentiation of B cells were analyzed by flow cytometry. Production of IgG and IgM in the culture supernatants was determined by ELISA.ResultsThe results showed that the serum IL-21 levels in RA patients were significantly higher than that of healthy controls (HC). IL-21 concentrations were positively correlated with 28-joint count disease activity score (DAS28) and anti-CCP antibody in RA patients with high IL-21 levels. Furthermore, the frequencies of peripheral CXCR5+PD-1+CD4+ Tfh-like cells markedly increased in RA patients and the percentages of Tfh-like cells were positively correlated with DAS28 and anti-CCP antibody levels. Moreover, elevated IL-21 levels were also correlated with the frequencies of Tfh-like cells. IL-21R expression on both Tfh-like cells and B cells were significantly enhanced in RA patients. In cultures vitro, exogenous IL-21 upregulated IL-21R expression and activation-associated surface markers on B cells and promoted more B cell proliferation in RA than in HC. This IL-21-mediated effect could be reversed by IL-21R-specific neutralizing antibody. Importantly, IL-21 promoted more differentiation of B cell into plasmablast and higher levels of IgG and IgM production in RA than in HC.ConclusionsIncreased serum IL-21 levels in RA patients correlate with DAS28, anti-CCP antibody and frequencies of Tfh-like cells. IL-21 supports B cell activation, proliferation and antibody secretion via IL-21R pathway. Thus, IL-21 may be involved in the pathogenesis of RA and antagonizing IL-21 could be a novel strategy for the therapy of RA.

Overexpression of SPACIA1/SAAL1, a newly identified gene that is involved in synoviocyte proliferation, accelerates the progression of synovitis in mice and humans

To identify novel genes associated with dysregulated proliferation of activated synovial fibroblasts, which are involved in arthritic joint destruction. We performed transcriptome analysis to identify genes that were up-regulated in the foot joints of mice with collagen-induced arthritis (CIA). The effect of candidate genes on proliferation of synovial fibroblasts was screened using antisense oligodeoxynucleotides and small interfering RNAs (siRNAs). We characterized the expression and function of a novel gene, synoviocyte proliferation-associated in collagen-induced arthritis 1 (SPACIA1)/serum amyloid A-like 1 (SAAL1) using antibodies and siRNA and established transgenic mice to examine the effect of SPACIA1/SAAL1 overexpression in CIA. Human and mouse SPACIA1/SAAL1 encoded 474 amino acid proteins that shared 80% homology. SPACIA1/SAAL1 was primarily expressed in the nucleus of rheumatoid arthritis (RA) synovial fibroblasts and was highly expressed in the hyperplastic lining of inflamed synovium. In addition, its expression level in RA- or osteoarthritis (OA)-affected synovial tissue was positively correlated with the thickness of the synovial lining. Furthermore, SPACIA1/SAAL1 siRNA inhibited the proliferation of synovial fibroblasts, especially tumor necrosis factor α-induced synovial fibroblasts, by blocking entry into the S phase without inducing apoptosis. Finally, transgenic mice overexpressing SPACIA1/SAAL1 exhibited early onset and rapid progression of CIA. These results suggest that SPACIA1/SAAL1 is necessary for abnormal proliferation of synovial fibroblasts and its overexpression is associated with the progression of synovitis in mice and humans. Thus, therapy targeting SPACIA1/SAAL1 might have potential as an inhibitor of synovial proliferation in RA and/or OA.

Insulin-like growth factor binding protein-related protein 1 is expressed in rheumatoid synovium and regulates synovial fibroblast proliferation

Insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) is a secretory protein that shares a structural similarity with IGFBP. Studies have shown that IGFBP-rP1 synergistically increases fibroblast growth with insulin and stimulates angiogenesis in tumor tissues. In this report, we examined the expression and function of IGFBP-rP1 in rheumatoid arthritis (RA). IGFBP-rP1 expression in synovial tissues was examined by reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, and immunohistochemical analysis. In vitro, IGFBP-rP1 expression was examined in synovial fibroblasts established from rheumatoid synovium (RASFs) by RT-PCR, Western blot, and immunostaining. The effect of IGFBP-rP1 small interfering RNA (siRNA) on RASF proliferation was assessed by alamarBlue assay. IGFBP-rP1 mRNA was detected by RT-PCR in all synovial tissues from RA and OA patients. In immunohistochemical analysis, IGFBP-rP1 was mainly expressed in synovial cells in the lining layers and endothelial cells in the sublining layers of RA synovium. In vitro, constitutive expression of IGFBP-rP1 in RASFs was detected by RT-PCR, Western blot, and immunostaining. Treatment with IGFBP-rP1 siRNA induced a 26% decrease in RASF growth compared to control siRNA. A similar extent of growth-suppressive effect by IGFBP-rP1 siRNA was also observed when RASF proliferation was induced by TNF-α. Collectively, these data suggest that IGFBP-rP1 may regulate synovial fibroblast proliferation in RA.

Lymphotoxin α stimulates proliferation and pro-inflammatory cytokine secretion of rheumatoid arthritis synovial fibroblasts

Objective TNFα plays a crucial role in rheumatoid arthritis (RA) by stimulating fibroblast-like synoviocytes (FLS). Lymphotoxin α (LTα) is a pro-inflammatory cytokine with significant homology to TNFα. We compared the effects of both cytokines on cultured RA FLS. Methods Receptor expression on RA FLS was analyzed by FACS. Cells were stimulated with LTα or TNFα and proliferation was measured by [3H]thymidine incorporation and secretion of inflammatory cytokines and metalloproteinase 3 by ELISA. Activation of MAP kinases and Akt was analyzed by Western blotting. Nuclear translocation of NFκB was visualized by immunofluorescence. Results 60–80% and 30–50% of the RA FLS tested expressed TNF receptors I and II, respectively, and 70–75% expressed HVEM. LTα induced RA FLS proliferation at the same level of TNFα, which was blocked by etanercept. Both LTα and TNFα induced activation of MAP kinases ERK1/2 and p38 as well as Akt. 95–98% of FLS showed nuclear translocation of NFκB after stimulation with either cytokines. LTα and TNFα were potent to induce secretion of IL-6, IL-8 and metalloproteinase 3 in FLS. Conclusion LTα is as effective as TNFα in stimulating RA FLS. Blocking both cytokines might allow a better control of inflammation and synovial proliferation in RA.

High mobility group box 1 induces synoviocyte proliferation in rheumatoid arthritis by activating the signal transducer and activator transcription signal pathway

Recently, it was discovered that high mobility group box chromosomal protein 1 (HMGB1) acts as a potent pro-inflammatory cytokine that is released into the extracellular milieu. Signal transducer and activator of transcription (STAT) proteins play an important role in cytokine signaling. Some investigators have reported preferential activation of STAT1, and others have reported preferential activation of STAT3 in response to endogenous interleukin-6 (IL-6) in patients with rheumatoid arthritis. Here, we show that expression of the HMGB1 protein is increased in the articular tissues of collagen-induced arthritis (CIA) rats, especially in cytoplasm and extracellular, following synoviocyte proliferation and STAT1 activation. In vitro, recombined human HMGB1 induced RSC-364 cell proliferation, activated STAT1 phosphorylation, increased the expression of cyclin D1 and CDK4 protein, and decreased the expression of p21 protein. In summary, our study suggests that HMGB1 plays an important role of cytokine in the pathogenesis of RA, which possibly induces synovial cell proliferation by activating the STAT1 signal pathway.

Insulin-like growth factor binding protein-related protein 1 is expressed in rheumatoid synovium and regulates synovial fibroblast proliferation

Insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) is a secretory protein that shares a structural similarity with IGFBP. Studies have shown that IGFBP-rP1 synergistically increases fibroblast growth with insulin and stimulates angiogenesis in tumor tissues. In this report, we examined the expression and function of IGFBP-rP1 in rheumatoid arthritis (RA). IGFBP-rP1 expression in synovial tissues was examined by reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, and immunohistochemical analysis. In vitro, IGFBP-rP1 expression was examined in synovial fibroblasts established from rheumatoid synovium (RASFs) by RT-PCR, Western blot, and immunostaining. The effect of IGFBP-rP1 small interfering RNA (siRNA) on RASF proliferation was assessed by alamarBlue assay. IGFBP-rP1 mRNA was detected by RT-PCR in all synovial tissues from RA and OA patients. In immunohistochemical analysis, IGFBP-rP1 was mainly expressed in synovial cells in the lining layers and endothelial cells in the sublining layers of RA synovium. In vitro, constitutive expression of IGFBP-rP1 in RASFs was detected by RT-PCR, Western blot, and immunostaining. Treatment with IGFBP-rP1 siRNA induced a 26% decrease in RASF growth compared to control siRNA. A similar extent of growth-suppressive effect by IGFBP-rP1 siRNA was also observed when RASF proliferation was induced by TNF-α. Collectively, these data suggest that IGFBP-rP1 may regulate synovial fibroblast proliferation in RA.