Induction of Fas-dependent apoptosis in synovial infiltrating cells in rheumatoid arthritis

Apoptosis is a feature of the synovium of rheumatoid arthritis(RA). We have recently shown that RA synoviocytes were susceptibleto anti-Fas mAb and undergo apoptosis in vitro. To investigatewhether infiltrating mononuclear cells also undergo Fas-dependentapoptosis, double-labeling techniques combined with immunohistochemicalexamination with anti-CD3 mAb and the TdT-mediated dUTP-biotinnick end labeling (TUNEL) method to detect apoptotic cells,or in situ RT assay to detect Fas mRNA, were performed usingfrozen tissue sections. We also examined the in vitro inductionof Fas-dependent apoptosis in freshly isolated synovium infiltratingmononuclear cells (SIM), synovial stromal cells (SSC) and peripheralblood lymphocytes (PBL) using tissues from nine patients withRA and three with osteoarthritis (OA). The results showed expressionof Fas antigen and apoptotic cells in a number of CD3-bearingcells in RA synovial tissues. In vitro treatment with anti-FasmAb produced a significant apoptosis of RA SIM and SSC, whilenone of PBL, and neither SIM nor SSC from OA exhibited apoptosis.Moreover, 50% of CD4⁺, CD3⁺ and CD45RO⁺ cells, and >90% ofFas-expressing cells of RA SIM underwent apoptosis in responseto anti-Fas mAb, as detected by flow cytometry. Our resultssuggest that RA synovial infiltrating lymphocytes acquire highsusceptibility to anti-Fas mAb and undergo apoptosis. Such aphenomenon of infiltrating T cells in RA synovium may play animportant pathophysiological role and suggest a possible therapeuticeffect for anti-Fas mAb in RA.     

Antikeratin antibodies in synovial fluid in rheumatoid arthritis

Serum and synovial fluid of 20 patients with classical or definite rheumatoid arthritis (RA) were tested for antikeratin antibodies (AKA) by indirect immunofluorescence using rat esophagus as antigen. AKA were found in 80% of the RA patients, in serum as well as in synovial fluid. None of the 54 serum control patients were AKA positive in serum. None of the 17 synovial fluid control patients were AKA positive in synovial fluid. F(ab)'2 fragments prepared from AKA positive RA serum retained antibody activity. AKA belonged to the IgG class of immunoglobulins. Corrected for the lower IgG content in synovial fluid, AKA constituted a higher percentage of the IgG in synovial fluid than in serum. This could imply a possibility of local production of AKA in the joint.     

Functional deficiency of antigen-presenting cells in the synovial fluid of rheumatoid arthritis.

In our study of rheumatoid arthritis (RA) patients, we observed a decrease of tetanus toxoid antigen-presenting capacity of synovial fluid (SF) adherent cells to autologous T cells of either SF or peripheral blood. Additionally, we found a higher capacity of adherent synovial cells to stimulate autologous T-lymphocytes. Our results suggest that antigen-presenting cells of the SF of RA patients have defects that may play a role in defective presentation of antigens in joints and may account for other abnormal functions important in the pathogenesis of RA.     

Induction of cyclooxygenase-1 in cultured synovial cells isolated from rheumatoid arthritis patients

Objective and design:The aim of this study was to confirm the involvement of cyclooxygenase (COX)-1 in rheumatoid arthritis (RA).Materials and subjects:Synovial cells isolated from arthritic patients were cultured primarily and consecutively for 8 passages.Treatment:The cultured synovial cells were incubated with 10 ng/ml of interleukin-1 (IL-1) for 6 h.Methods:The effects of either COX-1 or COX-2 selective inhibitor on prostaglandin E₂ (PGE₂) production was estimated by enzyme-linked immunosorbent assay (ELISA) and the expression of COX-1 and COX-2 were determined by Western blotting and immunocytochemistry.Results:IL-1-induced PGE2 production in synovial cells isolated from RA in primary culture was inhibited by mofezolac, a selective inhibitor of COX-1, as well as NS-398, a specific inhibitor of COX-2. The similar inhibitory patterns were obtained in the RA-derived synovial cells within 3 passages. However, COX activity in the RA-derived synovial cells after 5 passages was inhibited by NS-398, but not by mofezolac. In contrast, COX activity in primary and consecutively cultured synovial cells isolated from osteoarthritis (OA) or normal arthritis was inhibited by NS-398, but not by mofezolac. Western blot and immunocytochemical analyses of COX-1 and COX-2 in the synovial cells isolated from RA patients within 3 passages showed an induction in both COX-1 and COX-2 expression by IL-1. The induction of both COX-1 and COX-2 was inhibited by dexamethasone.Conclusions:These experiments demonstrate COX-1 induction in synovial cells isolated from RA patients, suggesting that COX-1 is involved in the progression of RA.Received 12 November 2003; returned for revision 3 December 2003; accepted by M. Katori 5 December 2003     

Inhibitory effects of interleukin-10 on synovial cells of rheumatoid arthritis.

This paper describes the immunoregulatory effects of interleukin-10 (IL-10) on synovial cells in vitro. Synovial cells were cultured with IL-10 in the presence or absence of various cytokines. Following incubation, the costimulatory molecule expression on synovial cells and cytokine production in culture supernatants were analysed by an indirect immunofluorescence method and enzyme-linked immunosorbent assay, respectively. We also examined the effect of IL-10 on the function of synovial cells as antigen-presenting cells (APC). Synovial cells spontaneously express several kinds of costimulatory molecule and produce various kinds of cytokines. Stimulation of synovial cells with interferon-gamma (IFN-gamma), IL-1 beta, or 12-O-tetradecanoyl phorbol 13-acetate (TPA) markedly enhanced the expression of costimulatory molecules and cytokine production of these cells. Both spontaneous and up-regulated costimulatory molecule expression and cytokine production were significantly suppressed by the addition of IL-10. Autologous T-cell proliferation was stimulated by purified protein derivative (PPD) in IFN-gamma-treated synovial cells and treatment of these synovial cells with IL-10 also suppressed T-cell proliferation. Our results suggest that IL-10 has an inhibitory effect on synovial cells and is an important immunoregulatory component of the cytokine network in rheumatoid arthritis.     

The potential aggressiveness of synovial tissue in rheumatoid arthritis

Operation specimens of articular tissue from about 8000 patients with clinically evident rheumatoid arthritis have been studied by light microscopy and in 583 cases by electron microscopy over a period of several years. We examined the tissue in closest contact with eroded cartilage. In approximately 70 per cent. of cases this was a fibrous pannus of compact collagenous connective tissue with few fibrocytes; in approximately 25 per cent. of cases a pannus, consisting of loose granulation tissue, moderately rich in collagen fibres, fibroblasts, and small blood vessels; in approximately 5 per cent. of cases, however, we found, that the zone in close contact consisted of uniform layers of immature-looking synovial cells with large nuclei. These immature-looking cells encroach on the cartilage from the junction of synovium with the articular cartilage. The development of these aggressive cell structures seemed to be preceded by a fibrinous inflammatory exudate. The cells contain lysosomal enzymes which are able to destroy prcteoglycans and collagen fibres and could therefore be the basis of erosion of cartilage. The cell structures seem to be short-lived and they are avascular at least initially. Most of the cells seem to die within a few days. The remaining cells assume the appearance of fibroblasts and form the later pannus. The cell content decreases as the pannus gets older. It is suggested that the destruction of articular cartilage takes place only during the short time in which these aggressive cell structures are present. The erosive synovial cells may however reappear during the intermittent course of the disease, after episodes of quiescence and acute inflammation.     

Lack of preferential Vβ usage in synovial T cells of rheumatoid arthritis patients

The T-cell receptor Vβ subfamily repertoires of synovial and peripheral T cells of 8 rheumatoid arthritis (RA) patients were determined using the polymerase chain reaction. Three normal controls were included. Some of the rheumatoid synovial samples did not express the complete range of Vβ families and lacked as many as 6 gene families. However, these patients showed considerable individual variation in expression. Overall, the data do not support preferential T-cell receptor Vβ usage in synovial T cells of RA patients either in comparison to their autochthonous peripheral T cells or to peripheral T cells of normal subjects.     

Structure of synovial lymphatic capillaries in rheumatoid arthritis and juvenile idiopathic arthritis

The structure of lymphatic capillaries (LC) of the synovial membrane (SM) from patients with rheumatoid arthritis and juvenile idiopathic arthritis obtained by synovectomy was investigated by transmission electron microscopy. This method allows comparison of the structure of the same vessel under light and electron microscope and clear differentiation between lymphatic and blood capillaries and venules. Synovial LC were localized in the subintimal connective tissue of the SM in the vicinity of venules. The shape of some LC was irregular, suggesting edema of the interstitium. Lymphatic endothelium has extremely attenuated cytoplasm with the exception of the perinuclear region. Many nuclei of endothelial cells had distinct nucleoli. The basal lamina was discontinuous. The walls of LC showed close connection with the interstitium represented by anchoring filaments that were attached to the endothelial cells and to the surrounding connective tissue. In some LC connective tissue appeared to be disconnected from endothelium and gaps between their walls and the interstitium were seen. Mononuclear cells were accumulated adjacent to some LC. Specialized interendothelial junctions (endothelial microvalves) were observed in the LC walls. Their structure and function in the migration of cells and debris from synovial interstitium into LC lumina in rheumatoid arthritic synovium deserves further investigation. In the lumina of some of the LC lymphocytes, monocytes, macrophages, cell debris and enlarged endothelium were observed. Accumulation of such material may cause obstruction of tiny LC. We suggest that reported alterations of the fine synovial lymphatic vessels can contribute to the progression of the inflammatory process to chronicity.     

类风湿关节炎模型大鼠滑膜组织中证实有髓样细胞诱发受体2的表达

背景：髓样细胞诱发受体2在类风湿关节炎活动期患者滑膜组织呈高表达,但其在类风湿关节炎发病机制中发挥的作用,目前并不清楚。 目的：观察髓样细胞诱发受体2在牛源性Ⅱ型胶原诱导的关节炎大鼠模型滑膜组织中的表达。 方法：制备胶原诱导关节炎模型大鼠,动态观察大鼠各项关节炎的活动指标和滑膜组织的病理学改变,RT-PCR法检测滑膜组织中髓样细胞诱发受体2、肿瘤坏死因子α、白细胞介素1β、白细胞介素10 mRNA的表达,Western blot及免疫组织化学法分别检测关节滑膜组织中髓样细胞诱发受体2的蛋白表达及定位。 结果与结论：模型组大鼠免疫13 d后出现足爪红肿,关节炎指数评分逐渐升高(P < 0.01),炎症高峰期在19-25 d,苏木精-伊红染色可见诱导性关节炎大鼠滑膜组织增生及炎性细胞浸润、软骨骨质破坏。与对照组相比,模型组大鼠关节滑膜髓样细胞诱发受体2的mRNA和蛋白的表达、肿瘤坏死因子α、白细胞介素1β mRNA表达均明显升高(P < 0.05或P < 0.01),白细胞介素10 mRNA表达显著降低(P < 0.05)。结果证实,髓样细胞诱发受体2可能是类风湿关节炎模型大鼠发病过程中一个重要的炎症调节递质,其反应性升高在类风湿关节炎模型大鼠发病中发挥着重要作用。 中国组织工程研究杂志出版内容重点：肾移植;肝移植;移植;心脏移植;组织移植;皮肤移植;皮瓣移植;血管移植;器官移植;组织工程全文链接：     

Clock gene expression in different synovial cells of patients with rheumatoid arthritis and osteoarthritis

Patients with rheumatoid arthritis (RA) show modulated circadian rhythms of inflammatory cytokines and cortisol, which may be associated with a modified expression of clock genes. The expression of major clock genes was previously studied in synovial tissues and fibroblasts of patients with RA and osteoarthritis (OA). We therefore especially aimed to examine the localization of clock genes at the cellular level in synovial tissue. Furthermore we were interested in studying the expression of the D site of albumin promoter (albumin D-box) binding protein (DBP) at the immunohistochemical level in human samples. Methods used include the in situ expression of the clock genes Brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 (Bmal 1), Circadian Locomotor Output Cycles Kaput (Clock), Period 1 and 2 (Per 1 and Per 2), and DBP was examined by immunohistochemistry in synovial tissues of patients with RA or OA. Additionally, expression profiles of different clock genes were determined over 24 h by real time PCR in synovial fibroblasts (SFs) after a 2 h serum shock or TNF-α. Results show that all clock genes investigated were found to be expressed both in RA and OA synovial tissues. Double staining against cell specific markers revealed that clock proteins were especially seen in macrophages, SFs and B-lymphocytes. Cell counting showed that clock proteins were found in approximately 5–20% of cells. Additionally, preliminary cell culture experiments showed that TNF-α treatment resulted in differential 24 h expression profiles between RA and OA samples and also compared to the results obtained from the serum shock experiments. From our study we conclude that the major clock genes, including DBP, are expressed in samples from RA and OA patients, especially in macrophages and synovial fibroblasts, but also in B-lymphocytes. Preliminary experiments suggest that TNF-α seems to be able to modify clock gene expression in synovial fibroblasts.     

Functional characterization of SV40-transformed adherent synovial cells from rheumatoid arthritis.

A total of 14 transformed cell clones were obtained by micro-injecting origin-defective SV40 DNA into three types of cloned adherent synovial cells (ASC) (dendritic cells (DCs), macrophage-like cells (MCs), and fibroblast-like cells (FCs)) from two rheumatoid arthritis patients (five DC clones (SV40-DCs), five MC clones (SV40-MCs) and four FC clones (SV40-FCs)). All the transformed cell nuclei expressed SV40-specific T antigen. The cells which formed a colony had a few times shorter doubling time than the original cells. IL-1 alpha, IL-1 beta and prostaglandin E2 were detected in the culture supernatant from the unstimulated transformed cells like untransformed cells. The SV40-DCs showed the most potent accessory cell function in oxidative mitogenesis assay among the three types of SV40-ASCs. Granulocyte macrophage colony stimulatory factor (GM-CSF) was detected only in the culture supernatant from the SV40-MCs without stimulation. Extensive phenotypic analysis revealed relatively cell-specific markers. SV40-DCs were HLA-DP+ and glial fibrillary acidic protein positive. SV40-MCs stained positive for 5'-nucleotidase and nonspecific esterase. These transformed ASCs retained much of the original cellular physiology of rheumatoid arthritis (RA) ASCs and may be a useful tool for characterizing the role of ASCs in the pathogenesis of RA.     

类风湿关节炎滑膜血管内皮细胞中Smoothened的表达及其意义

 目的:初步观察Sonic Hedgehog信号通路分子Smoothened（Smo）在类风湿关节炎（rheumatoid arthritis, RA）滑膜血管内皮细胞的表达及其生物学意义。方法:收集4例病情中度活动的RA患者的滑膜组织,同时收集4例外伤或半月板损伤（无关节炎）者滑膜组织作为对照组,免疫组化检测滑膜组织Smo蛋白表达情况。采用人脐静脉内皮细胞系EA.hy926作为滑膜血管内皮细胞的模型,予不同浓度肿瘤坏死因子α（TNF-α）处理,Western blotting检测Smo蛋白表达;采用RNAi技术转染体外合成的特异性Smo-siRNA,应用Western blotting检测沉默效果;转染siRNA 24 h后,经TNF-α/放线菌素D（actinomycin D, ActD）诱导细胞凋亡,CCK-8法检测细胞存活率,流式细胞术检测细胞凋亡情况。结果:RA患者滑膜组织Smo表达高于对照组,以血管内皮细胞表达尤为明显。EA.hy926细胞经TNF-α刺激后,Smo蛋白表达上调（P<0.05）。RNA干扰EA.hy926细胞Smo表达后,细胞存活率为（24.30±0.45）%,低于阴性对照组的（36.86±0.62）%（P<0.05）,细胞凋亡率为（48.00±1.96）%,高于阴性对照组的（31.70±0.82）%（P<0.05）。结论: Smo可能参与了RA患者滑膜组织血管内皮细胞凋亡的调控。     

Characterization of the IL-2-receptor on rheumatoid arthritis synovial fluid T cells

We studied the hypoproliferative response of synovial fluid (SF) T cells in rheumatoid arthritis (RA) using a mitogenic monoclonal antibody (MoAb) specific for the T-cell antigen receptor-associated CD3 complex. RASF T cells are defective in their proliferative response and in the induction of the Tac (p55) component of the IL-2-receptor (IL-2-R) when stimulated with anti-CD3 monoclonal antibody (MoAb). However, fresh RASF T cells bear demonstrable IL-2-R in cross-linking experiments which are not seen in unstimulated peripheral blood (PB). These receptors are functional since RASF T cells proliferate in response to recombinant IL-2 (rIL-2) better than fresh PB T cells from either normal or RA patients. Scatchard analysis indicates increased (4-fold) numbers of high affinity IL-2-R on (phytohaemagglutinin) PHA-activated RASF T cells as compared with comparably activated RAPB T cells. Phorbol myristate acetate (PMA) induces Tac antigen expression in RASF but does not lead to proliferation. The hyporesponsiveness of RASF T cells does not appear to result from lack of IL-2-R, lack of IL-2-R inducibility, or proliferative potential.     

Polyamine oxidase activity in rheumatoid arthritis synovial fluid.

Oxidation of polyamides by polyamine oxidases (PAO) leads to the generation of highly reactive aminoaldehydes which have been shown to have a variety of effects, including killing of pathogenic microorganisms and regulation of leucocyte functions. Data presented here show that PAO are present in synovial fluid from patients with rheumatoid arthritis. This finding may have important implications in the various properties attributed to synovial fluid which includes anti-inflammatory activity.     

Altered metabolic pathways regulate synovial inflammation in rheumatoid arthritis

Rheumatoid arthritis is characterized by synovial proliferation, neovascularization and leucocyte extravasation leading to joint destruction and functional disability. The blood vessels in the inflamed synovium are highly dysregulated, resulting in poor delivery of oxygen; this, along with the increased metabolic demand of infiltrating immune cells and inflamed resident cells, results in the lack of key nutrients at the site of inflammation. In these adverse conditions synovial cells must adapt to generate sufficient energy to support their proliferation and activation status, and thus switch their cell metabolism from a resting regulatory state to a highly metabolically active state. This alters redox-sensitive signalling pathways and also results in the accumulation of metabolic intermediates which, in turn, can act as signalling molecules that further exacerbate the inflammatory response. The RA synovium is a multi-cellular tissue, and while many cell types interact to promote the inflammatory response, their metabolic requirements differ. Thus, understanding the complex interplay between hypoxia-induced signalling pathways, metabolic pathways and the inflammatory response will provide better insight into the underlying mechanisms of disease pathogenesis.     

FoxP3 mRNA splice forms in synovial CD4+ T cells in rheumatoid arthritis and psoriatic arthritis

Our aim was to elucidate the relative amount of the different splice forms of FoxP3 mRNA in CD4+ T cells in peripheral blood (PB) compared to synovial fluid (SF) in RA and PsA patients. FoxP3 mRNA was measured using a quantitative real-time PCR method. CD4+ T cells were isolated from 17 paired samples of PB and SF from RA and PsA patients, and PB from 10 controls. FoxP3fl and FoxP3Δ2 mRNA was significantly increased (6.7 and 2.1-fold, respectively) in PB CD4+ T cells from RA patients compared to controls. FoxP3fl and Δ2 mRNA in SF CD4+ T cells was increased compared to controls in sero-negative RA and PsA, but not in sero-positive RA patients, who had a high FoxP3 expression in both PB and SF. The FoxP3Δ2Δ7 mRNA was barely detectable in patient samples, and not at all in healthy individuals. We provide evidence of an increased expression of FoxP3 splice forms in synovial CD4+ T cells from RA patients. A skewed, high expression profile of FoxP3, but not CTLA-4, in sero-negative RA and PsA, indicates that synovial CD4+ T cells may represent unique subsets of T cells which have been induced locally or selectively recruited to the joint.     

滑膜成纤维细胞在类风湿性关节炎发病中的作用

类风湿性关节炎是复杂的多系统疾病,近年来,越来越多的证据表明,滑膜成纤维细胞在类风湿性关节炎中过度增殖,介导炎症反应,造成关节结构破坏。滑膜成纤维细胞在类风湿性关节炎发病过程中具有重要的作用。