The production of CXCR3-agonistic chemokines by synovial fibroblasts from patients with rheumatoid arthritis

The inflamed synovial tissue of rheumatoid arthritis (RA) is characterized by an infiltration with Th1 cells that predominantly express the chemokine receptors CXCR3 and CCR5. In this study, we investigated the production of the CXCR3-agonistic chemokines CXCL9, CXCL10, and CXCL11 by synovial tissue cells and synovial fibroblast-cell lines (fourth or fifth passage) from RA patients. Concentrations of all CXCR3 ligands in synovial fluids were markedly higher in RA patients than in osteoarthritis (OA) patients. Synovial tissue cells from RA patients more strongly expressed mRNAs for CXCR3 ligands and spontaneously secreted larger amounts of these chemokine proteins than the cells from OA patients. The mRNA expression of all CXCR3 ligands was induced in synovial fibroblasts from RA patients after stimulation with interferon gamma (IFN-), tumor necrosis factor alpha (TNF-), or interleukin-1 beta (IL-1). However, synovial fibroblasts significantly secreted CXCL9 and CXCL10 proteins, but not CXCL11 protein, after IFN- stimulation and secreted only CXCL10 protein after TNF- or IL-1 stimulation. When stimulated with a combination of IFN- and TNF-, these cells were able to secrete large amounts of all three chemokines. These results indicate that synovial fibroblasts may be involved in perpetuating the Th1 immune response by producing the Th1-associated CXCR3 ligands, and the synergistic effect of IFN- and TNF- may be important for their chemokine production in RA joints.     

Serum levels of soluble CD44 variant isoforms are elevated in rheumatoid arthritis

Serum levels of soluble CD44 variant proteins including sequences encoded by exon v5 and exon v6 (sCD44v5, sCD44v6) were determined in patients with inflammatory rheumatic diseases: 56 with rheumatoid arthritis (RA⁺) and 31 with miscellaneous inflammatory rheumatic diseases (MIRD). There were very significantly higher serum levels of sCD44v5 and sCD44v6 in patients with RA' than in those with MIRD (RA⁺ to MIRD: sCD44v5: 81 ± 54 ng/ml to 33 ± 13 ng/ml; sCD44v6: 237 ± 124 ng/ml to 166 ± 53 ng/ml; bothP0.001). In RA⁺ elevated serum levels of sCD44v5 were correlated with the inflammatory activity of disease. In 17 patients with RA⁺ three or four follow-up measurements of sCD44v5 were performed within 6 months. The development of sCD44v5 serum levels reflected the clinical course of disease in the patients investigated.     

Plasma and synovial fluid concentrations of sulphasalazine and two of its metabolites in rheumatoid arthritis

Summary A study was made of plasma and synovial fluid levels of sulphasalazine, one of its dissociation products — sulphapyridine and a metabolite of the latter — acetyl sulphapyridine in patients with rheumatoid arthritis (RA) who were in a steady state on sulphasalazine therapy. Combined sulphapyridine levels were significantly higher than those of sulphasalazine both in plasma and synovial fluid. Synovial fluid levels of both drugs correlated with their plasma levels and were generally slightly lower. Some patients accumulated sulphasalazine and sulphapyridine in the synovial fluid and the mean concentration of sulphasalazine was higher in the fluid than in the plasma. The explanation for this is uncertain. The concentration of combined sulphapyridine in synovial fluid was related to local joint inflammation and more active systemic disease. No consistent association was found between sulphasalazine levels and local or systemic activity. The higher sulphapyridine levels in synovial fluid found in this study suggest the possibility that this moiety could play a more active role in RA than it does in inflammatory bowel disease.     

Analyses of differential proteome of human synovial fibroblasts obtained from arthritis

There is mounting evidence indicating that the synovial fibroblasts (SFs) contribute to the pathogenesis of rheumatoid arthritis (RA). The present study showed the differential proteins expression pattern of SFs from patients with RA or osteoarthritis (OA) and healthy control. Cellular proteins of cultured SFs were subjected to 2-DE and visualized by silver nitrate staining. A total of 49 spots that were statistically and differentially overexpressed in RA or OA in comparison to healthy ones were identified by MALDI-TOF-MS, and 25 proteins were successfully identified. Western blot was used to further verify some of the differential proteins. These proteins included enzymatic and structural proteins, signal transduction proteins, calcium binding protein, etc. From all of the identified proteins, a number of proteins have been implicated that involved in the healthy or pathological SFs function (e.g., S100A4, S100A10, cathepsin D) or that have potential diagnostic and prognostic value for RA (α-enolase and TPI) or that may be the new therapeutic targets (Annexin, SOD, PRX). G.-P. Bo, L.-N. Zhou and W.-F. He contributed equally to this work.     

Soluble interleukin-2 receptor in sera and synovial fluids of rheumatoid patients: correlations with disease activity

The measurement of serum soluble interleukin-2 receptor (sIL-2R), a sensitive marker of lymphocyte activation, has been proposed as an indicator of disease activity and outcome in patients with inflammatory diseases characterized by the activation of immune cells. Serum sIL-2R levels have been reported higher in rheumatoid patients than in controls. Using an enzyme-linked immunoabsorbent assay (ELISA), we evaluated soluble IL-2R levels in the serum of 34 patients with RA and in the synovial fluid of 25 of these patients and we compared it with levels found in the serum of 13 healthy controls. Serum sIL-2R levels were significantly elevated in RA patients compared with the healthy agematched control group (P<0.005). The mean level of soluble IL-2R in synovial fluids was significantly higher than the mean sera levels in RA patients (P<0.0001). Moreover, we examined the correlation between serum and synovial fluid sIL-2R levels and disease activity measures. Serum sIL-2R correlated only with ESR (P<0.04). The synovial fluid sIL-2R correlated with ESR (P<0.02) and a visual analogue scale (VAS) pain score (P<0.04). Both serum and synovial fluid sIL-2R levels correlated with the chronic arthritis systemic index (CASI; P<0.04 and P<0.005, respectively). Our data suggested that in RA the measurement of sIL-2R may certainly mirror the degree of chronic inflammation and the continuous activation of the immune cells in the joint, although the role of this molecule in the immune response is still unclear.     

Early lymphocyte activation in the synovial microenvironment in patients with osteoarthritis: comparison with rheumatoid arthritis patients and healthy controls

Osteoarthritis (OA) is largely considered to be a non-inflammatory disease, although there is compelling evidence that subclinical inflammation is a common event, even in the absence of acute inflammatory flares. In this study we analyze, by means of CD5 and CD69 expression, the infiltration and early activation of CD5+cells, mostly lymphocytes, in both synovial membrane and synovial fluid from advanced OA patients and compare them with samples from patients with rheumatoid arthritis and healthy controls. The number of infiltrating CD5+ cells in both synovial membrane and synovial fluid from patients with advanced OA was significantly reduced as compared with rheumatoid arthritis patients. However, synovial membrane and synovial fluid CD5+ cells on OA exhibited a phenotype with evidence of recent activation comparable to that observed in RA.     

Expression and citrullination of keratin in synovial tissue of rheumatoid arthritis

Keratin is the main component of cellular intermediate filaments, and its post-translational modification plays an important role in cell differentiation and apoptosis, as well as disease states. The conversion of peptidylarginine to citrulline, termed citrullination, is particularly involved in the pathogenesis of rheumatoid arthritis (RA). Immunohistochemistry using an antibody mixture that broadly recognized various keratin forms detected cytokeratin in many cells in the area lining the synovial membrane of RA. Furthermore, double immuno-florescent labeling showed that the cells expressing cytokeratin were also positive for citrulline when they were in the vicinity of extracellular deposits or approached the exterior of the synovial membrane. Western blot analysis demonstrated citrullination of keratin purified from RA synovial tissue by immuno-precipitation. The above results indicate the presence of citrullinated cytokeratin in synovial membranes in RA.     

Interleukin-6 levels in synovial fluids of patients with rheumatoid arthritis correlated with the infiltration of inflammatory cells in synovial membrane

To compare the histological appearance of synovial membrane and interleukin (IL)-6 levels in synovial fluids of patients with rheumatoid arthritis (RA). Synovial tissue and synovial fluids were obtained from 51 knee joints with RA undergoing synovectomy or joint replacements. A histological inflammation score was determined based on the hyperplasia of the synovial lining and infiltration of inflammatory cells. The concentrations of IL-6 in synovial fluids were measured by ELISA. The association between IL-6 levels and histological findings was evaluated. We found a positive correlation between the infiltration of inflammation cells in synovial tissues and the concentration of IL-6 in synovial fluids. The IL-6 level in synovial fluid partially reflects histological synovial inflammation.     

Interleukin-6 localisation in the synovial membrane in rheumatoid arthritis

Summary Polyclonal antibodies were raised in rabbits against Interleukin-6 (IL-6) by immunisation with a synthetic peptide of identical sequence to the amino terminal 12 amino acids of human IL-6. These antibodies reacted with recombinant IL-6 by ELISA and stained the cytoplasm of the IL-6 secreting bladder tumour cell line T24. Staining was abolished by prior incubation of the antibody with the IL-6 peptide. F(ab)₂ fragments made by pepsin digestion of the IgG were immunopurified, labelled with biotin and retained activity in the biochemical and histological assays. Sections of synovial membrane from patients with rheumatoid arthritis (RA) were stained with these antibodies, using an immunoperoxidase technique, and cells containing IL-6 were domonstrated in the thickened synovial lining layer and also in a perivascular distribution in the deeper synovium. In osteoarthritis there were fewer cells in the lining layer and hence localisation appeared similar in both the interstitial area and lining layer. Double-staining techniques with mouse monoclonal antibodies against cell subset markers in five RA synovial membranes showed that up to 13% of T-cells and 19% of antibody-producing cells stained or IL-6. However, up to 70% of the macrophages contained IL-6 and these were found in close proximity to lg-producing plasma cells. This study showed that Anacrophages were the major cells of the immune system in which IL-6 could be localised in RA, and suggests a cole for locally produced IL-6 in the stimulation of theumatoid factor production.     

Characterization of IL-2 responsive synovial T lymphocytes in rheumatoid arthritis

Summary Lymphocytes from peripheral blood (PBL) and synovial fluid (SFL) were obtained from patients with rheumatoid arthritis (RA) and cloned under limiting-dilution conditions without prior activation but in the presence of exogenous interleukin (IL)-2. The precursor frequencies of such in vivo activated IL-2-responsive cells were higher in RA SFL (1/83) than in RA PBL (1/201) or normal PBL (1/377). These HLA-Dr/Ia-positive clones expressed T-cell markers CD3 and T101 and were either CD4- or CD8-positive but lacked NK markers CD11, CD16, and HNK-1. All such clones were cytotoxic for NK-sensitive K562 targets and NK-insensitive Raji cell targets. These cells, which most closely resemble non-major histocompatibility complex (MHC) restricted cytotoxic T (CTL) cells, are present with increased frequency in RA synovial fluids.     

类风湿关节炎滑膜细胞的增殖及细胞周期的研究

目的　研究类风湿关节炎 (RA )患者滑膜细胞增殖变化及其机制。方法　应用MTS/PMS比色法和流式细胞分析技术分别测定RA患者滑膜细胞的细胞增殖水平和细胞周期 ,以骨关节炎 (OA)病人及因外伤截肢的正常人作对照。结果　RA患者滑膜细胞接种后第 4天增殖水平 (0 .5 3 63± 0 .0 0 7)显著高于对照组 (0 .4187± 0 .0 0 8,P <0 .0 5 ) ,细胞周期分析显示RA患者滑膜细胞G1期的比例 (61.92 % )比对照组明显降低 (74.2 8% ) ,P <0 .0 5 ,而S、G2期的细胞比例比对照组则明显增加 ;加入RA患者滑液能以剂量依赖性明显提高滑膜细胞的增殖水平 ,并使G1期细胞进一步降低 ,S、G2期细胞进一步增加。结论　含有多种免疫调节因子的滑液使RA患者滑膜细胞在G2、S期的滞留 ,是滑膜细胞过度增殖 ,从而导致RA发病的重要因素。     

Ultrasonographic evaluation of synovial effusion and synovial proliferation pattern in the knee joints of patients with rheumatoid arthritis

In the present study, 49 knee joints of 26 patients with rheumatoid arthritis and 17 knee joints of 17 healthy subjects were ultrasonographically examined. Lateral, superior, and medial aspects of the patella were scanned using an ultrasonograph with a 7.5-MHz annular array transducer to evaluate the thickness of synovial effusion and the synovial proliferation pattern. The overall mean thickness of synovial effusion (mean of all three sites) in the knee joints was 4.9 ± 3.4 mm for rheumatoid arthritis patients and 1.4 ± 0.5 mm for healthy subjects. In rheumatoid arthritis patients, the mean thickness of synovial effusion at the superior aspect of the patella (6.5 ± 4.1 mm) was significantly greater than that at the lateral aspect (4.5 ± 4.8 mm) (P < 0.05) and the medial aspect (4.0 ± 3.1 mm) (P < 0.01). Patients with the villonodular pattern of synovial proliferation had a shorter duration of disease than those with uniform thickening or an overlapping pattern. Received: May 1, 2001 / Accepted: August 6, 2001     

Serum and synovial fluid leptin levels and markers of inflammation in rheumatoid arthritis

This study was designed to investigate the serum and synovial fluid leptin levels, and inflammatory markers in rheumatoid arthritis (RA) patients. Serum and synovial fluid leptin levels were significantly higher (P > 0.05) in RA patients than control group; RA patients with moderate disease activity (DAS < 2.7) having significantly higher leptin levels (P > 0.05) than those with low disease activity (DAS < 2.7). Leukocytes and erythrocyte sedimentation rate (ESR) were found to be significantly higher in moderate disease activity RA group compared to low activity group (P > 0.05, P < 0.001, respectively). Serum leptin level is found to be independent of age and inflammatory markers. ESR is positively correlated with DAS activity and CRP values. Our finding of no correlation between leptin and BMI shows that regulation of leptinemia is complex, and leptin levels cannot be used to assess RA activity.     

Kynurenic acid, an endogenous constituent of rheumatoid arthritis synovial fluid, inhibits proliferation of synoviocytes in vitro

Kynurenic acid is an antagonist of ionotropic glutamate receptors. It has been found that glutamate antagonists inhibit proliferation of different human tumor cells. Since the hyperplasia of synovial fibroblasts is one of the most striking features of inflammatory arthritis, the main goals of this study were detection and quantification of kynurenic acid in synovial fluid obtained from patients with rheumatoid arthritis, and determination of its effect on proliferation of synoviocytes in vitro. Presence of kynurenic acid was determined by HPLC in all 58 samples of synovial fluid. The mean concentration was 15.89 pmol/ml. Kynurenic acid inhibited synoviocyte proliferation with the IC₅₀ value of 5.9 mM. In subthreshold concentration of 0.3 mM it enhanced antiproliferative action of celecoxib and nimesulide. In conclusion, the presence of kynurenic acid in synovial fluid was documented in patients with rheumatoid arthritis. Its potential role as an endogenous substance, controlling synoviocyte proliferation can be suggested.     

Two-dimensional electrophoresis of synovial tissue,synovial fluid and serum in patients with rheumatoid arthritis and related diseases

Summary Using the technique of two-dimensional (2D) electrophoresis with consecutive silver staining, we investigated samples of serum, synovial fluid and synovial tissue obtained from 19 patients suffering from rheumatoid arthritis (RA) or non-RA arthritis. From these experiments we have drawn the following conclusions. 2D electrophoresis of serum, synovial fluid and synovial tissue extracts taken from patients suffering from joint diseases is a reproducible method. Repeated runs of the same sample reveal an essentially constant protein spot pattern. The time period between surgery and tissue preparation did not influence the number of protein spots when less than 15 h was involved. The protein spot number is always lower in synovial fluid than in either synovial tissue or serum in RA and non-RA patients. The mean value for the number of spots is 68 for the inflamed tissue irrespective of the cause of arthritis (RA and non-RA group taken together) and 47 for the control group. This difference is significant. We were able to definitely identify 7 spots in the tissue extract. We did not find RA-specific protein spots in either serum, synovial fluid or tissue extracts from the synovial membrane. The only significant difference between RA patients and either non-RA or control group patients concerning the protein spot pattern is the increased size of the immunoglobulin spot (mainly IgG) in RA. In addition, we discuss possible reasons for failure of the 2D electrophoresis technique to detect disease-specific protein patterns.     

Ameliorative effect of ozone on cytokine production in mice injected with human rheumatoid arthritis synovial fibroblast cells

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by invasion of hyperplastic synovial cells and progressive joint destruction. Ozone therapy has been proposed as an immunomodulator and cellular metabolic activator which shows long-term anti-inflammatory effects and serves to reduce further the proinflammatory factors. We purified RA synovial fibroblast cells (RA-SFc) from patients and avoided contaminating macrophages by flow cytometry, then treated them with ozone. Following the observable decreased production of proinflammatory factors TNF-, IL-1, and IL-6 from RA-SFc, we infused the cultured RA-SFc into joints of severe combined immunodeficiency mice. The mRNA and protein levels of the RA-SFc exposed to 3% and 5% ozone were the same. As a result, 3% and 5% ozone applied externally ameliorated the inflammatory reaction of RA without toxicity or serious side effects. Therefore, ozone injected into the knees of RA patients could become a valuable treatment, and we confirm the interactive mechanism between ozone and RA-SFc.     

Serum and synovial fluid histidine: a comparison in rheumatoid arthritis and osteoarthritis

Summary The serum and synovial fluid (SF) histidine, sulphydryl, and protein concentrations were compared in simultaneous samples from 84 patients with rheumatoid arthritis (RA) and a control group comprising 29 patients with osteoarthritis (OA). The SF levels of histidine were higher than the serum levels in the RA patients but significantly lower than corresponding results in patients with OA (P<0.001). The latter had levels of serum and SF histidine which were equivalent and within the normal range. Greater quantities of protein were found in the SF of the patients with RA compared with the OA group. The serum and SF sulphydryl concentrations expressed as mol/g protein were low but in equilibrium in patients with RA. However the SF sulphydryl (mol/g protein) was depressed relative to serum levels in patients with OA.     

Identification of citrullinated cellular fibronectin in synovial fluid from patients with rheumatoid arthritis

Abstract

Objectives. Cellular fibronectin (cFn) has been implicated in the pathogenesis of rheumatoid arthritis (RA), and we previously demonstrated the presence of citrullinated cFn in rheumatoid synovial tissues. The present study aimed to investigate whether citrullinated cFn can be detected in the plasma or synovial fluid of RA patients.

Methods. Twenty-five rheumatoid arthritis synovial fluid (RASF), seven osteoarthritis synovial fluid (OASF) and 12 plasma samples from RA patients were examined. Citrullination of cFn was determined by immunoprecipitation (IP), western blotting and enzyme-linked immunosorbent assay (ELISA), in which peptidyl-citrulline within cFn was detected using a specific anti-cFn monoclonal antibody in combination with anti-modified citrulline antibody after chemical modification.

Results. Levels of citrullination associated with cFn, as determined by ELISA, were significantly higher in RASF than in OASF samples. IP and western blotting detected citrullinated cFn in RASF but not in plasma samples from RA patients. Levels of total cFn were elevated in RASF compared with OASF, and 24 out of 25 RASF samples were positive for anti-CCP antibody. However, no correlation was observed between levels of citrullinated cFn and those of total cFn or anti-CCP antibody in RASF. On the other hand, a significant positive correlation was observed between the levels of matrix metalloproteinase-3 (MMP-3) and cFn citrullination in RASF.

Conclusions. Citrullinated cFn appears to be produced within the affected joint and might be involved in the pathogenesis of rheumatoid synovitis.     

Functional analysis of choline transporters in rheumatoid arthritis synovial fibroblasts

Objectives: In this study, we examined the functional characteristics of choline uptake and sought to identify the transporters in rheumatoid arthritis synovial fibroblasts (RASFs).

Methods: The expression of choline transporters was evaluated by quantitative real-time PCR, western blotting, and immunocytochemistry. Time course, Na⁺-dependency, and kinetics of [³H]choline uptake were investigated. Effects of cationic drugs on the uptake of [³H]choline, cell viability, and caspase-3/7 activity were also examined. Finally, we investigated the influence of choline uptake inhibitor, hemicholinium-3 (HC-3), and choline deficiency on cell viability and caspase-3/7 activity.

Results: Choline transporter-like protein 1 (CTL1) and CTL2 mRNA and protein were highly expressed in RASFs and were localized to the plasma membrane. [³H]Choline uptake occurred via a Na⁺-independent and pH-dependent transport system. The cells have two different [³H]choline transport systems, high- and low-affinity. Various organic cations, HC-3 and choline deficiency inhibited both [³H]choline uptake and cell viability, and enhanced the caspase-3/7 activity. The functional inhibition of choline transporters could promote apoptotic cell death. In RASFs, [³H]choline uptake was significantly increased compared with that in OASFs without a change in gene expression.

Conclusions: These results suggest that CTL1 (high-affinity) and CTL2 (low-affinity) are highly expressed in RASFs and choline may be transported by a choline/H^+?antiport system. Identification of this CTL1- and CTL2-mediated choline transport system should provide a potential new target for RA therapy.