Production of PAF-acether by synovial fluid neutrophils in rheumatoid arthritis

PAF-acether (PAF) is a pro-inflammatory phospholipid molecule potentially involved in the pathogenesis of arthritis. PAF and related metabolites have been isolated in the synovial fluid from patients with arthritis.The aim of this study was to determine PAF production by neutrophils isolated from synovial fluid and blood in patients with rheumatoid arthritis. Blood neutrophils from normal donors were also studied for their capacity to form PAF. Neutrophils were stimulated with the calcium ionophore A23187 (2µM) for 1 to 60min. PAF released in the medium and PAF associated to cells were measured.In synovial fluid neutrophils, PAF production began as soon as 1 min of stimulation (16.1 ± 6.3 pmol per 1 × 10⁶ cells) and reached a maximum at 20min: 29.2 ± 2.8 pmol per 1 × 10⁶ cells (mean ± SEM, n = 5). The amount of PAF released in the supernatant increased with the length of stimulation and was maximal after 30min (33.5%, percentage of released over total PAF, n = 5). After A23187 stimulation, similar amounts of PAF were produced by blood neutrophils from RA and control patients. However, neutrophils isolated from the joint had a lower capacity to produce PAF than blood neutrophils from the same patients.The present results demonstrate the synthesis and release of PAF by synovial fluid neutrophils. They suggest that neutrophils may be the source of PAF locally present in the joint. Newly synthetised PAF could participate in the amplification of the local inflammatory reaction.     

Stimulation of osteoclast formation by inflammatory synovial fluid

Peri-articular bone resorption is a feature of arthritis due to crystal deposition and rheumatoid disease. Under these conditions, the synovial fluid contains numerous inflammatory cells that produce cytokines and growth factors which promote osteoclast formation. The aim of this study was to determine whether inflammatory synovial fluid stimulates the formation of osteoclasts. Synovial fluid from rheumatoid arthritis (RA), pyrophosphate arthropathy (PPA) and osteoarthritis (OA) patients was added to cultures (n=8) of human peripheral blood mononuclear cells (PBMCs) in the presence and absence of macrophage colony-stimulating factor (M-CSF) and the receptor activator of NF-κB ligand (RANKL). Osteoclast formation was assessed by the formation of cells positive for tartrate-resistant acid phosphatase (TRAP) and vitronectin receptor (VNR) and the extent of lacunar resorption. The addition of 10% OA, RA and PPA synovial fluid to PBMC cultures resulted in the formation of numerous multinucleated or mononuclear TRAP⁺ and VNR⁺ cells which were capable of lacunar resorption. In contrast to PBMC cultures incubated with OA synovial fluid, there was marked stimulation of osteoclast formation and resorption in cultures containing inflammatory RA and PPA synovial fluid which contained high levels of tumour necrosis factor alpha, a factor which is known to stimulate RANKL-induced osteoclast formation.     

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。     

Supravital staining of synovial fluid with Testsimplets.

We explored the use of Testsimplet (TS) in synovial fluid (SF) analysis. TS is a glass slide coated with a dry mixture of methylene blue and cresyl violet, which in contact with one drop of SF provides a stained fresh preparation. We applied the TS to the study of 159 SFs of patients with different rheumatic diseases. In those SFs of patients with crystal-associated diseases, the crystal search was performed both on unstained preparations and with TS. TS was as good as the Wright's and Papanicolaou stain in characterizing SF cells, lupus erythematosus cells, and detection of occasional bacteria. TS allowed a better visualization of Reiter's cells, cartilage fragments, synovial villi, fat droplets, and fibrin. Crystals were identified in every TS of those patients with crystal-associated diseases. TS is a rapid and reproducible method of SF supravital staining. Crystals are well preserved for simultaneous examination with compensated polarized light.     

Role of GADD45 beta in the regulation of synovial fluid T cell apoptosis in rheumatoid arthritis

Rheumatoid arthritis (RA) is characterized by persistent Th1 cell infiltration and production of inflammatory cytokines in the location of joint lesion. It is known that infiltrated Th1 cells in the synovial fluid (SF) of RA patients are resistant to apoptosis. Here we demonstrate that Th1 cells accumulated in patient SF expressed a high level of GADD45β (Growth Arrest and DNA Damage-inducible 45β) which further inhibited Th1 cell apoptosis. Interestingly, in vitro culture of T cells with SF from RA patients increased GADD45β expression in Th1 cells and inhibited their apoptosis. Silencing of GADD45β by RNAi abolished the anti-apoptotic effect of RA SF, which was accompanied by down-regulation of Bcl-2 and up-regulation of Bax. Further analysis showed that TNF-α and IL-12 in RA SF could stimulate GADD45β expression in Th1 cells and inhibit their apoptosis. Taken together, our results suggest a novel mechanism by which specific cytokines in the RA SF elevate GADD45β expression in local Th1 cells and subsequently leading to the enhanced T cell survival.     

共刺激分子HVEM在类风湿关节炎滑膜组织内的表达及分布研究

目的探讨TNF家族共刺激分子HVEM(herpesvirus entry mediator)在类风湿关节炎(RA)患者滑膜组织内的表达及分布。方法使用免疫组化法检测RA患者滑膜组织HVEM的表达;并使用免疫荧光法检测HVEM的细胞定位及分布。结果免疫组化结果证实,RA患者滑膜组织中有大量的HVEM阳性细胞,形态观察提示这些阳性的细胞主要是毛细血管、滑膜细胞、局部淋巴结T细胞及巨噬细胞;免疫荧光分析进一步表明这些HVEM+细胞主要为CD3+T细胞,CD68+巨噬细胞,少数CD31+内皮细胞也表达HVEM,但其不表达于CK-18+上皮细胞。对比其他B7家族共刺激分子在滑膜组织中的分布,免疫荧光发现HVEM共表达于B7-H3+血管,但不表达于B7-H1+、B7-DC+、B7-H4+及Z39Ig+细胞。结论关节炎滑膜组织内有大量HVEM阳性细胞,提示HVEM有可能参与并调节了关节炎的病理进程。     

Cytologic examination of synovial fluid

Cytologic study of synovial fluid has been relatively neglected both by cytologists and rheumatologists. Currently identified implications of cytologic findings include definitive diagnoses of a variety of joint diseases such as those due to malignancies, infections, or pathogenic crystals. Diagnoses can also be strongly suggested, for example, by visualization of lupus erythematosus cells formed in vivo. Immunocytochemistry is being explored for diagnostic and pathogenetic implications. We propose that complementary techniques used in our cytology and rheumatology laboratories may provide new insights into the clinical significance of cellular changes in joint fluids.     

IL-1ra ELISA: reduction and alkylation of synovial fluid eliminates interference by IgM rheumatoid factors

IL-1 and a specific receptor antagonist of IL-1, IL-1ra, may play important roles in the pathophysiology of rheumatoid arthritis and in other types of inflammatory synovitis. Measurement of IL-1ra in synovial fluids and in other body fluids may lead to a greater understanding of its possible activity as a modulator of the immune and inflammatory systems in vivo. Therefore, a modified sandwich ELISA was developed to measure IL-1ra protein concentration in synovial fluids. The antibodies used in this ELISA were polyclonal and derived from rabbits hyperimmunized with human recombinant IL-1ra. IgM rheumatoid factors within synovial fluid resulted in false elevation of determined IL-1ra by the sandwich ELISA through binding of the primary and secondary antibodies. Reduction and alkylation of synovial fluid samples before application to the ELISA plate eliminated the interference caused by > 2000 μg/ml IgM rheumatoid factor (latex agglutination titer of 1/5,120). This ELISA was specific for IL-1ra; there was no detection of IL-1, IL-1β, or lysozyme. The sensitivity of this ELISA was <200 pg/ml, making it a useful assay for the accurate measurement of synovial fluid IL-1ra protein concentration.     

Cartilage extracellular matrix metabolism differs in serum and synovial fluid

Most cartilage explant culture studies assume conventional serum-supplemented growth media are biologically equivalent to the natural synovial fluid which baths cartilage in vivo. Few studies have systematically compared the effects of serum versus synovial fluid in culture. To address this assumption we conducted a series of studies to determine if cartilage matrix synthesis is significantly different in serum-based versus synovial fluid-based media. Normal bovine cartilage explants were cultured in DMEM either alone or supplemented with bovine serum or bovine synovial fluid. Matrix synthesis was measured with radiolabeling techniques. We then compared responses to insulin-like growth factor I (IGF-I, a stimulator of matrix synthesis), and interleukin-1 (IL-1, an inhibitor of matrix synthesis). We observed significantly lower matrix synthesis activity in synovial fluid versus serum. Caution shoud be used in extrapolating studies of cartilage grown in media supplemented with serum rather than synovial fluid.     

Analysis of synovial fluid from clinically healthy Iranian fat-tailed sheep

In this study synovial fluid from the radiocarpal joints of 100 clinically healthy Iranian sheep (Lori-Bakhtiari) were analyzed. Total nucleated cell count (TNCC) of the synovial fluid was 178.9±75 cells/l (mean±SD). Lymphocytes were the predominant cell type composing 48.34±17.2% of the cells found in the synovial fluid, whereas monocytes, macrophages and neutrophils composed 36.52±3.5%, 12.75±5.9% and 2.28±1.18% of the cells found in the synovial fluid, respectively. The glucose concentration of synovial fluid was 44.9±9 mg/dl. The concentration of total protein, albumin and globulin of the synovial fluid were 2.31±0.55, 1.49±0.38, 0.81±0.28 g/dl, respectively. The albumin to globulin ratio (A/G) was 2.02±0.61. Age and sex had no significant effects on TNCC, percentage of lymphocytes, monocytes, macrophages and neutrophils, concentration of total protein, albumin, globulin, and A/G ratio of fluid from the radiocarpal joint. However, glucose concentration in radiocarpal fluid in sheep less than 1-year-old was significantly (P0.05) higher (48.27±1.4 mg/dl) than 1- to 2-years-old (42.1±1.4 mg/dl) and more than 2-years-old sheep (43.9±1.8 mg/dl). No significant differences were found between right and left limbs for any parameters evaluated in this study.     

Interleukin 2 (IL 2) inhibitor in rheumatoid synovial fluid: Correlation with prognosis and soluble IL 2 receptor levels

A soluble activity inhibiting over 50% of the CTLL-2 cell line response to recombinant human interleukin 2 (IL 2) was found in 17 of 29 (59%) rheumatoid synovial fluids. To study the prognosis value of this activity, 16 rheumatoid synovial fluids were collected before a radiation synovectomy of the knee with 7 mCi of⁹⁰Yt. Patients with a good clinical result after the synovectomy had a lower IL 2 inhibitory activity than those with a bad or uncomplete result (P<0.01). Levels of inhibitory activity and of soluble IL 2 receptors were correlated with each other and with the response of the synovitis to the radiation synovectomy. These results extend the clinical usefulness of soluble IL 2 receptor measurements and indicate a correlation between the immune activation of the rheumatoid synovitis and its clinical activity.     

Immortalized B-lymphocytes from rheumatoid synovial tissue show specificity for bacterial HSP 60

Several studies indicate a pathogenetic role of T-lymphocytes with specificity for heat shock proteins (HSP) in rheumatoid arthritis (RA). Surprisingly, there are no experimental data for B-lymphocytes with specificity for HSP. To investigate whether B-lymphocytes from rheumatoid synovial tissue show a specificity for HSP 60 we immortalized synovial tissue B-lymphocytes by the electrofusion technique and tested the specificity of the B-cell clones for HSP 60 by ELISA. Tissue samples from four patients with classic, active RA were used in this study. The isolated cells were electrofused in strongly hypo-osmolar medium with cells either of the mouse strain X63-Ag8-653 (Ag8) or the heteromyeloma strain HAB-1. Clones positive for IgG, the IgG fraction of the supernatant of the isolated synovial cells and the IgG of the serum of the patients were tested in an ELISA for reactivity to the recombinant HSP 60 of Yersinia enterocolitica, which shows great homology with mycobacterial HSP 65 and human HSP 60. The expression of this HSP 60 was studied in normal and rheumatoid synovial tissue using a polyclonal rabbit serum against HSP 60 from Y. enterocolitica (Ye HSP 60). In this way we investigate differences in the expression of HSP 60 and compared the pattern of this HSP60 with the pattern of mycobacterial HSP65 and human HSP 60 described by others. In three of four patients 10 IgG secreting B-cell clones showing a specificity for HSP 60 were detected. IgG specific for HSP 60 was also detected in the supernatant of the isolated synovial cells before fusion and in the serum of these patients. HSP 60 was demonstrated immunohistochemically within the rheumatoid synovial tissue and showed stronger expression with a different distribution when compared with the expression in normal synovial tissue. B-cell clones from rheumatoid synovial tissue thus exhibit a specificity for bacterial HSP 60, and a monospecific rabbit serum against this HSP shows strong reactivity within the rheumatoid synovial tissue. It may be postulated that a humoral HSP 60 response, initially directed against an infectious agent, could react with cross-reactive epitopes of rheumatoid synovial tissue or with self-HSP perpetuating the local inflammatory process.     

MicroRNA-16对类风湿关节炎患者滑膜成纤维细胞增殖、侵袭及细胞因子分泌的影响

 目的:研究microRNA-16（miR-16）对类风湿关节炎（rheumatoid arthritis,RA）患者滑膜成纤维细胞（rheumatoid arthritis synovial fibroblasts,RASFs）增殖、侵袭及细胞因子分泌的影响。方法: 体外分离培养RASFs,脂质体转染化学合成的miR-16 mimic或miR-16抑制剂,分别采用MTT法、Transwell小室法和流式细胞术检测其对RASFs增殖、侵袭及凋亡的影响;RT-PCR和Western blotting检测miR-16对RASFs基质金属蛋白酶3/13（matrix metalloproteinase 3/13,MMP3/13）及白细胞介素1β（interleukin 1β,IL-1β）表达的影响。结果:增殖实验结果表明miR-16可显著抑制RASFs的增殖;细胞侵袭结果表明miR-16可显著抑制RASFs的侵袭;流式细胞术检测发现miR-16对RASFs凋亡无显著影响;miR-16可下调MMP3/13及IL-1β的表达水平。结论:  miR-16在RA的发生中起着重要作用,它可能通过下调MMP3/13及IL-1β的表达抑制RASFs增殖和侵袭。这为进一步研究miR-16在RA中的作用机制奠定了基础。     

Augmented plasma and tissue kallikrein like activity in synovial fluid of patients with inflammatory articular diseases

We studied some of the components of the kininogen-kallikrein-kinin system, simultaneously, in plasma and synovial effusions of patients with inflammatory articular diseases. Plasma and tissue kallikrein like activity and kininogen levels were evaluated. Active plasma and tissue kallikreins in plasma and synovial fluid were detected by their amidase activity upon specific chromogenic substrates. Kininogen levels were determined by a bioassay. Both specific amidase activity of plasma and tissue kallikreins were augmented in synovial effusions in relation to their own plasma activity. Kininogen levels in synovial fluid tended to be diminished in relation to plasma, however statistical significance was not reached. The consumption of kininogen is probably related to kinin production. This finding together with increased activities of plasma and tissue kallikreins reinforce the involvement of kinins in pathogenesis of inflammatory articular diseases.accepted by W. B. van den Berg     

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：     

Loss of microRNA-147 function alleviates synovial inflammation through ZNF148 in rheumatoid and experimental arthritis

MicroRNA-147 (miR-147) had been previously found induced in synoviocytes by inflammatory stimuli derived from T cells in experimental arthritis. This study was designed to verify whether loss of its function might alleviate inflammatory events in joints of experimental and rheumatoid arthritis (RA). Dark Agouti (DA) rats were injected intradermally with pristane to induce arthritis, and rno-miR-147 antagomir was locally administrated into individual ankle compared with negative control or rno-miR-155-5p antagomir (potential positive control). Arthritis onset, macroscopic severity, and pathological changes were monitored. While in vitro, gain or loss function of hsa-miR-147b-3p/hsa-miR-155-5p and ZNF148 was achieved in human synovial fibroblast cell line SW982 and RA synovial fibroblasts (RASF). The expression of miRNAs and mRNAs was detected by using RT-quantitative PCR, and protein expression was detected by using Western blotting. Anti-miR-147 therapy could alleviate the severity, especially for the synovitis and joint destruction in experimental arthritis. Gain of hsa-miR-147b-3p/hsa-miR-155-5p function in TNF-α stimulated SW982 and RASF cells could upregulate, in contrast, loss of hsa-miR-147b-3p/hsa-miR-155-5p function could downregulate the gene expression of TNF-α, IL-6, MMP3, and MMP13. Hence, such alteration could participate in synovial inflammation and joint destruction. RNAi of ZNF148, a miR-147's target, increased gene expression of TNF-α, IL-6, MMP3, and MMP13 in SW982 and RASF cells. Also, mRNA sequencing data showed that hsa-miR-147b-3p mimic and ZNF148 siRNA commonly regulated the gene expression of CCL3 and DEPTOR as well as some arthritis and inflammation-related pathways. Taken together, miR-147b-3p contributes to synovial inflammation through repressing ZNF148 in RA and experimental arthritis.     

关节滑液检测在关节假体周围感染诊断中的研究进展

关节假体周围感染(periprosthetic joint infection,PJI)是人工关节置换术后可能发生的一种严重并发症,其导致的严重后果,无论对于医生还是患者来说,都很难接受。目前,由于多种不确定因素的存在,PJI诊断的准确性较低。传统的血清学检查、影像学检查有一定的价值,但是易受全身情况的影响,导致特异性不高。为了正确诊断关节假体周围感染,不同学科的研究人员采用各种不同的方法进行了大量的诊断研究,并取得了丰富的成果。近年来,关节滑液炎性标志物检测、分子生物学方法等被研究证实具有较高的敏感性和特异性。因此,关节滑液CRP、-防御素、白细胞酯酶、PCR技术等被广泛研究,期望能从中找到诊断关节假体周围感染的特异性指标,提高临床诊断的准确性。