Altered formation of leukotriene B4 in vitro by synovial fluid neutrophils in rheumatoid arthritis

The synthesis of products of the 5-lipoxygenase pathway by synovial fluid (SF) and peripheral blood neutrophils of 12 patients with rheumatoid arthritis was compared in 3 experimental conditions using high performance liquid chromatographic analysis. Major differences were observed when blood and SF neutrophils were incubated with ionophore A23187, the formation of all 5-lipoxygenase products being lower (p less than 0.0005) in the SF neutrophils. Addition of exogenous arachidonic acid to the A23187 stimulated cells partially overcame the difference in 5-lipoxygenase product synthesis between the 2 neutrophil populations. In contrast, upon incubation with arachidonic acid alone, SF neutrophils produced significantly larger amounts of 5-lipoxygenase products. The increased reactivity of the SF neutrophil 5-lipoxygenase to arachidonic acid and the decreased 5-lipoxygenase product synthesis upon A23187 stimulation may be the consequence of the previous activation of the cells and 5-lipoxygenase product synthesis in situ.     

Differential induction of BLT receptor expression on human endothelial cells by lipopolysaccharide, cytokines, and leukotriene B4

Leukotriene (LT) B4 is a powerful chemotactic and immune modulating agent that signals via two receptors denoted BLT1 and BLT2. Here we report that BLT1 and BLT2 are expressed at low levels in an apparently silent state in human umbilical vein endothelial cells (HUVEC). However, treatment with LPS leads to a >10 fold increase in the levels of BLT1 mRNA without any significant effects on BLT2 mRNA. In parallel, LPS also increases the amounts of BLT1 protein. Tumor necrosis factor-alpha (TNF-alpha) increases the expression of BLT2 mRNA approximately 6 times above basal levels with only a modest increase in BLT1 mRNA. Interleukin-1beta causes variable and parallel increases of both BLT1 and BLT2 mRNA. The natural ligand LTB4 also increases BLT1, but not BLT2, mRNA and protein expression. Along with the induction of BLT1 and/or BLT2, HUVEC acquire the capacity to respond to LTB4 with increased levels of intracellular calcium and these signals can be blocked by isotype selective BLT antagonists, CP-105696 and LY-255283. In addition, treatment of HUVEC with LTB4 causes increased release of both nitrite, presumably reflecting nitric oxide (NO), and monocyte chemoattractant protein-1. Our data indicate that expression of functional BLT receptors may occur at the surface of endothelial cells in response to LPS, cytokines, and ligand, which in turn may have functional consequences during the early vascular responses to inflammation. Moreover, the results point to BLT receptors as potential targets for pharmacological intervention in LT-dependent inflammatory diseases such as asthma, rheumatoid arthritis, and arteriosclerosis.     

Lower expression of histamine H4 receptor in synovial tissues from patients with rheumatoid arthritis compared to those with osteoarthritis

The aim of this study is to compare the expression level of histamine H(4) receptor (H(4)R) mRNA in synovial tissues of rheumatoid arthritis (RA) and osteoarthritis (OA) patients, and to study correlation of results with clinical characteristics of patients with RA. Synovial tissues were obtained from 7 RA and 7 OA patients undergoing artificial arthroplasty. Serum levels of erythrocyte sedimentation rate, C-reactive protein, matrix metalloproteinase-3 (MMP-3), rheumatoid factors, and cyclic citrullinated peptide antibodies were determined. The expression of H(4)R mRNA in synovial tissues was determined by real-time polymerase chain reaction. Expression of H(1)R and H(4)R mRNA were significantly lower in RA compared with OA patients (P < 0.005), while expression of H(2)R mRNA was comparable in both. While a significant negative correlation was found between H(4)R expression and serum MMP-3 concentration (r = -0.70, P < 0.05), no correlation was found between MMP-3 and H(1)R (r = -0.52) or H(2)R (r = 0.23). This study supports the supposition that H(4)R in synovial tissue may play a role in cartilage and bone destruction by influencing the secretion of MMP-3 in patients with RA.     

Ia+ T cells in synovial fluid and tissues of patients with rheumatoid arthritis

Markedly elevated levels of T cells expressing Ia antigens were found in the synovial membranes and synovial fluids of patients with rheumatoid arthritis. The primary increase in expression of the Ia antigens was on the OKT 8+ (suppressor/cytotoxic) T cell subset. In addition, the total percentage of OKT 8+ T cells in intraarticular sites was usually greater than levels in peripheral blood. Small numbers of OKT 4+ (helper/inducer) cells bearing Ia antigens were also identified. The characteristic increase in the Ia+ T cells in peripheral blood was not encountered in most patients treated with D-penicillamine.     

Receptor expression in synovial fluid neutrophils from patients with rheumatoid arthritis.

OBJECTIVES--The aim of this study was to determine if neutrophils isolated from the blood and synovial fluid of patients with rheumatoid arthritis had patterns of receptor expression resembling those of blood neutrophils from controls which had been activated and primed in vitro. METHODS--Fluorescence activated cell sorting was used to measure receptor expression in paired blood and synovial fluid neutrophils from patients and in control neutrophils exposed to phorbol myristate acetate and granulocyte-macrophage colony stimulating factor. RESULTS--There was no significant difference in the patterns of receptor expression in blood neutrophils from patients and healthy controls, but neutrophils in the synovial fluid had been primed and activated within the joint. About 50% of rheumatoid synovial fluid neutrophil samples expressed Fc gamma RI, a high affinity receptor for monomeric IgG, which is only expressed in neutrophils exposed to cytokines. CONCLUSIONS--Synovial fluid neutrophils are activated and primed within the inflamed joint and hence their ability to respond to activating factors such as immune complexes will be modulated. As the expression of Fc gamma RI requires active biosynthesis, this work indicates that selective gene activation occurs when neutrophils are recruited into rheumatoid joints.     

尿激酶型纤溶酶原激活物受体在类风湿关节炎滑膜组织细胞的表达

目的观察尿激酶型纤溶酶原激活物受体（uPAR）在类风湿关节炎（an）滑膜组织细胞膜（muPAR）和细胞质（cuPAR）的表达及分布。方法分别采用间接免疫荧光和免疫组化等方法检测18份RA、10份骨关节炎（OA）和4份正常滑膜组织中uPAR的表达及分布。结果RA滑膜组织细胞中muPAR阳性表达率为（66．0±9．4）％,主要分布于滑膜组织中血管翳周围,如血管内皮细胞和间质细胞等;而cuPAR阳性表达率为（61．0±5．8）％,主要分布于滑膜衬里层和滑膜下层细胞,如滑膜下层巨噬细胞样细胞、滑膜衬里层细胞及单个核白细胞等。OA滑膜组织中uPAR也呈不同程度的阳性表达,但与RA相比阳性表达程度较弱,阳性细胞数量较少。相关性分析：muPAR、cuPAR与RA滑膜炎积分呈正相关（r分别为0．672、0．649,P〈0．01）。结论muPAR表达阳性的细胞可能主要参与RA血管翳的形成及滑膜组织的侵袭等病理过程;而cuPAR表达阳性的细胞则可能与炎性介质的释放和致炎作用有关;提示uPAR在RA的病理过程中起重要作用。     

Interleukin 1 activity in the synovial fluid of patients with rheumatoid arthritis

Summary The synovial fluids (SF) of patients with rheumatoid arthritis (RA) were investigated for their effects on thymocytes of C3H/HeJ mice. Of the 20 SF tested, 17 (85%) showed an augmentation of the phytohaemagglutinin (PHA) induced thymocyte stimulation. Out of 16 SF of patients with osteoarthrosis, such an activity was detected in only one (6.25%). Further characterisation of the amplification factor revealed that (1) the SF of RA patients augmented both the PHA and the Concanavalin A response of the thymocytes (2) in the absence of mitogens, SF-treated thymocytes showed an increased uptake of ³H-thymidine, (3) the SF did not propagate the growth of an interleukin 2 dependent ovalbumin specific T cell clone, but (4) the SF were found to be required for optimal interleukin 2 release by spleen cells stimulated with suboptimal doses of lectin. Based on these biological effects the factor in the SF of RA patients is suggested to represent an interleukin 1 (IL-1). IL-1 produced in cultures by activated macrophages has been shown to stimulate T and B cell functions and to induce the production of collagenase and prostaglandins by cultured synovial cells. Both properties of IL-1 could be relevant in the pathogenesis of RA.     

Effects of non-steroidal anti-inflammatory drugs and prednisolone on synovial fluid white cells, prostaglandin E2, leukotriene B4 and cyclic AMP in patients with rheumatoid arthritis

Altogether 53 patients (31 women, 22 men) with definite rheumatoid arthritis were randomly divided into groups of 5-6 patients and treated for one day only with one of the following non-steroidal anti-inflammatory drugs (NSAIDs): acetylsalicylic acid, carprofen, diclofenac, indomethacin, naproxen, proquazone, timegadine, tolfenamic acid or paracetamol, and with prednisolone, in recommended doses. Synovial fluid samples were collected before and after the treatment. White cell count and its differentiation as well as the concentrations of protein, cyclic adenosine-3',5'-monophosphate (cAMP), prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) were measured from the synovial fluid. Synovial fluid leukocyte counts correlated with PGE2 concentrations, but showed no correlation with LTB4 levels before treatment. Significant changes were seen in the form of lowered PGE2 values following treatment with the clinically and experimentally most potent NSAIDs, and as depressed LTB4 levels following prednisolone treatment. The other markers of inflammation are obviously more resistant, changing only slowly during prolonged treatment, and may thus be, at least in part, secondary to the changes in prostanoids.     

CD28-T细胞亚群在类风湿关节炎患者外周血和关节液中的表达

目的 探讨CD28-T细胞亚群在类风湿关节炎(RA)患者外周血和关节液中的变化和意义。方法 随机选择RA患者45例,取新鲜抗凝外周血单个核细胞(PBMC),其中15例同时提取关节液单个核细胞( SFMC),以流式细胞技术检测CD28-T细胞数量及其表面可诱导共刺激分子(ICOS)的表达。2 组间比较用独立样本t检验。结果 ①与PBMC相比,RA患者SFMC中CD4+CD28+ ICOS+、CD4+CD28-ICOS+、CD8+ CD28+、CD8+ CD28+ ICOS+T细胞明显升高[(36±19)％与(15±8)％,t=-4.234,P＜0.01;(2.1±2.2)％与(0.6±1.4)％,t=-3.143,P＜0.01;(62±15)％与(47±18)％,t=-2.885,P＜0.01;(9±9)％与(3±3)％,t=-2.131,P＜0.05];CD8+CD28-T细胞明显降低[(38±15)％与(54±18)％,t=2.975,P＜0.01];CD8+ CD28- ICOS+、C1D4+CD28+和CD4+CD28-T细胞无明显变化（P＞0.05）。②同一RA患者SFMC与PBMC相比,CD4+CD28+ICOS+、CD8+ CD28+T细胞明显升高[(38±18)％与(16±10)％,t=-4.065,P＜0.01;(61±16)％与(41±21)％,t=-2.883,P＜0.01];CD8+ CD28-T细胞明显降低[(39±16)％与(59±21)％,t=2.949,P＜0.01]。③缓解期与活动期RA患者相比,PBMC中CD4+CD28-、CD8+ CD28-、CD28-ICOS+T细胞无明显变化（P＞0.05）。结论 RA患者关节液中CD28-T细胞亚群失衡和ICOS分子表达异常,可能是导致RA关节损伤的重要机制。     

Platelets in the synovial fluid of patients with rheumatoid arthritis

In a study of synovial fluid from 110 patients with various forms of arthritis, platelets were identified in the synovial fluid of all the 50 rheumatoids, in 18 out of the 25 (72%) with osteoarthritis and in all 35 of those with other forms of inflammatory osteoarthrosis. Identification of platelets by light microscopy was confirmed by electron microscopy. Platelet counts were significantly higher in rheumatoid fluid (mean 14 988/mm3; range 1000-65 000/mm3) compared with fluid from patients with osteoarthrosis (mean 1 592/Mm3; 0-10 000/mm3). In addition, significantly higher platelet counts were found in the synovial fluid (SF) of inflamed joints. There was a positive correlation between the SF platelet count and the total white cell count, polymorph count, hydrogen ion concentration, knee score, acid phosphatase and 5-nucleotidase activity and a negative correlation with the glucose level. All these factors indicate joint activity. Finally, platelet numbers correlated with SF levels of immunoglobulin M, and seropositive patients had significantly higher platelet counts in the SF compared with seronegative patients. Rheumatoid patients with thrombocytosis also had higher SF platelet counts. The close relationship of the SF platelet count to other indices of inflammation supports the concept that platelets may directly contribute to synovial inflammation by a variety of pathways.     

Hormone concentrations in synovial fluid of patients with rheumatoid arthritis

OBJECTIVE: Alterations in local concentrations of hormones, affecting directly synovial cells, could be involved in the modulation of the rheumatic inflammatory processes. The aim of present study was to investigate the levels of selected hormones (steroids, peptide and thyroid hormones) in synovial fluid of knee joint of patients with rheumatoid arthritis (RA) and control individuals with non-rheumatic exudate (with osteoarthrosis, OA). METHODS: Thirty-eight patients, 22 female and 16 males, with rheumatoid arthritis (RA) and 12 subjects with osteoarthrosis (OA, control group, 6 females and 6 males) participated in the study. Concentrations of cortisol (CS), 17-beta-estradiol (ES), dehydroepiandrosterone (DHEA), progesterone (PRG), aldosterone ALD), prolactin (PRL), insulin (INS), and C-peptide were determined by radioimmunoassay in synovial fluid. Insulin binding to isolated cell membrane of cells from synovial sediment was estimated by using radioiodine labeled insulin. In a group of patients (10 with RA and 4 with OS), the levels of free threeiodothyronine (FT3), TSH and growth hormone (GH) were also determined in synovial fluid. RESULTS: Increased levels of ES in synovial fluid of RA patients were observed, and higher differences were noted in men. TE concentrations were moderately elevated in synovial fluid of RA patients, however the ratio of ES/TE was significantly higher in male RA compared to OA patients. Higher levels of PRG, ALD and growth hormone were noted in synovial fluid of RA patients. Besides the steroid hormones the presence of insulin and C-peptide was noted in synovial fluid and the correlation between the levels of these two peptides was highly significant. The concentrations of INS and C-peptide in synovial fluid of patients from RA and OA group were not significantly different, however, highly significant increase of insulin binding to isolated membrane of synovial cells was found. Concentrations of cortisol, dehydroepiandosterone, prolactin, TSH and FT3 in synovial fluid were not significantly different in RA and OA groups. CONCLUSIONS: Besides the steroids also insulin, c-peptide, GH and FT3 were found in synovial fluid. The elevated ALD and GH levels in synovial fluid of RA patients and the presence of INS in synovial fluid with increase of INS binding to plasma membranes of cells from synovial fluid of RA patients suggest that besides the gonadal steroids also these hormones may affect the local inflammatory processes.     

Simultaneous evaluation of membrane bound and soluble interleukin 2 receptor expression in the blood and synovial fluid of patients with rheumatoid arthritis

We studied the levels of membrane-bound and soluble-form interleukin 2 (IL-2) receptors in forty patients with rheumatoid arthritis. Levels of IL-2 receptors in the sera and synovial fluid of patients with rheumatoid arthritis were elevated when compared to values observed in normal sera and synovial fluid derived from the osteoarthritic joint. Simultaneous elevation of IL-2 receptor expression in blood and synovial fluid lymphoid cells was also detected, but no correlation was found between the two parameters nor between serum IL-2 receptor levels and the hemosedimentation rate. We conclude that measurement of serum concentrations of soluble IL-2 receptors should be used with caution as an index of disease activity, but may be useful when used in conjunction with other parameters in the management of patients with rheumatoid arthritis.     

Polyamine levels in synovial tissues and synovial fluids of patients with rheumatoid arthritis.

We determined the polyamine contents of the synovial tissues from 11 patients with rheumatoid arthritis (RA), and the free putrescine levels in the synovial fluids (SF) from 10 patients with RA, 7 with osteoarthritis (OA), 5 with posttraumatic arthritis, and 3 with infectious arthritis. Putrescine levels in the synovial tissues correlated with serum C reactive protein concentration in patients with RA. Free putrescine levels in SF were significantly elevated in patients with infectious arthritis, compared with those found in RA, OA, and posttraumatic arthritis. Free putrescine levels in SF from patients with RA were significantly higher than in those with OA. Our findings suggest that polyamines may play an important role in RA.     

硫氧还蛋白5在类风湿关节炎患者滑膜组织滑液及血液中表达的研究

目的 在蛋白和mRNA水平上进一步研究硫氧还蛋白5(TXNDC5)在类风湿关节炎(RA)患者滑膜和血液中的表达水平与临床指标的关系,探讨该基因对RA的病理作用.方法 用免疫组织化学法、免疫荧光定量分析、实时荧光定量聚合酶链反应(PCR)、蛋白免疫印迹定量分析TXNDC5在RA滑膜中的表达水平;以骨关节炎(OA)、系统性红斑狼疮(SEE)、强直性脊柱炎(AS)及健康人为对照,用夹心酶联免疫吸附法(Sandwich-ELISA)分析TXNDC5在RA患者血液和滑液中的表达水平.采用单因素方差分析,LSD检验、Spearman相关分析进行统计学处理.结果 免疫组织化学法和免疫荧光定量分析显示TXNDC5在RA滑膜组织中高表达(100%,40±9),在OA滑膜中无表达,在AS滑膜中表达量(200%.4±4)也较低.实时定量PCR和Western blotting均证实TXNDC5在RA滑膜中高表达(P均<0.01).SandwichELISA显示,TXNDC5在RA患者血液及滑液中高表达(A值=1.31±0.37),但在OA、SEE、AS以及健康人低表达或无表达(P均<0.05).RA血液中TXNDC5的水平(A值=0.8185±0.299)与抗环瓜氨酸肽(CCP)抗体水平存在正相关(r=0.350,P=0.027).结论 TXNDC5在RA滑膜和血液中表达量明显升高,可能通过刺激RA滑膜血管翳形成参与RA病理进程.     

Plasminogen activator in synovial fluid from patients with rheumatoid arthritis

The plasminogen activator in synovial fluid from patients with rheumatoid arthritis (RA) and osteoarthritis (OA) was analyzed on a molecular basis. The level of plasminogen activator in RA was found to be higher than in OA. The plaminogen activators of both RA and OA revealed 3 different molecular weights: 90,000, 55,000 and 33,000. RA demonstrated the 3 plasminogen activators in broadly comparable ratios, but OA had the 55,000 form dominantly. The 90,000 plasminogen activator was a tissue-type plasminogen activator, while the 55,000 and 33,000 plasminogen activators were of the urokinase-type. beta-Methasone suppressed the tissue-type plasminogen activator, and urinary trypsin inhibitor suppressed the urokinase-type plasminogen activators. When urinary trypsin inhibitor was injected clinically into the joint space of a patient with RA, the urokinase-type plasminogen inhibitor was suppressed as in the in vitro study, and the clinical signs and symptoms were markedly improved. Open trials of intraarticular injections of urinary trypsin inhibitor demonstrated improvement of the clinical signs and symptoms.     

Plasma and synovial fluid cAMP in patients with rheumatoid arthritis.

cAMP was measured in plasma and synovial fluid from 11 patients suffering from rheumatoid arthritis with a specific protein-binding assay. Plasma and synovial fluid values were 17.0 +/- 6.8 pmol/ml and 8.3 +/- 3.7 pmol/ml, range 5-28 pmol/ml and 5-16 pmol/ml, respectively. No correlation could be established between plasma and synovial fluid levels, plasma and disease activity, or synovial fluid and disease activity, as judged by Lansbury's index. It is concluded that it seems unlikely that synovial fluid cAMP is derived solely from plasma and that no simple relation exists between cAMP in plasma and synovial fluid and total disease activity.     

Occurrence of (E)-4-hydroxy-2-nonenal in plasma and synovial fluid of patients with rheumatoid arthritis and osteoarthritis.

(E)-4-Hydroxy-2-nonenal (HNE), a cytotoxic propagation product of lipid peroxidation, is present in the synovial fluid (0.54 (0.19) mumol/l; mean (SE), n = 9) and plasma (0.34 (0.09) mumol/l, n = 9) of patients with rheumatoid arthritis. This compound was also found in the synovial fluid (0.24 (0.19) mumol/l, n = 9) and plasma (0.09 (0.03) mumol/l, n = 9) of patients with osteoarthritis. The concentration of HNE in the plasma of patients with rheumatoid arthritis was significantly greater than in patients with osteoarthritis.     

Inflammatory immune cell responses and Toll-like receptor expression in synovial tissues in rheumatoid arthritis patients treated with biologics or DMARDs

Biologic antirheumatic drugs (BIO) have been reported to be potent therapeutic agents in the prevention of inflammatory joint destruction in rheumatoid arthritis (RA). The aim of this study was to investigate the immune-inflammatory cells, including Toll-like receptor (TLR)-equipped cells, in synovial tissue samples from RA patients on BIO compared to patients, who are only on conventional disease-modifying antirheumatic drug (DMARD). We analyzed immune-inflammatory cells in RA synovitis in patients of BIO group (n?=?20) or DMARD group (n?=?20). The grading scores of synovitis was 1.7 and 1.8 in each BIO and DMARD group and correlated best with the CD3⁺ T (r?=?0.71/0.70, p?<?0.05) and CD20⁺ B (r?=?0.80/0.84, p?<?0.05) cells in the both groups, but less well with the CD68⁺ macrophages and S-100⁺ dendritic cells (DCs). Interestingly, both T (116 vs. 242, p?<?0.05) and B (80 vs. 142, p?<?0.05) cell counts were lower in the BIO than in the DMARD group, whereas macrophage and DC counts did not differ. In contrast, the C-reactive protein (CRP) and disease activity score DAS28-CRP did not show clear-cut correlations with the inflammatory grade of the synovitis (r range, 0–0.35). Similar numbers of cells immunoreactive for TLR-1 to TLR-6 and TLR-9 were found in synovitis in both groups. Patients clinically responding to biologics might still have the potential of moderate/severe local joint inflammation, composed in particular of and possibly driven by the autoinflammatory TLR⁺ cells.     

IgE-containing immune complexes in synovial fluid of patients with rheumatoid arthritis

Summary The prevalence and composition of IgE-containing immune complexes in paired synovial fluid and serum of 42 patients with classical or definite rheumatoid arthritis were studied. IgE-containing immune complexes were found in 15/42 synovial fluids; 15 sera were also positive. The correlation between serum and synovial fluid complexed IgE levels was high (r=0.77). The mean ratio of synovial fluid/serum levels was 1.96, i.e. significantly higher than 0.33, the synovial fluid/serum ratio for alpha-2-macroglobulin (molecular weight 820 kD), which was taken as high molecular weight control protein (p<0.0001). Apart from IgE in immune complex form, monomeric IgE was also significantly higher in synovial fluid compared to serum (ratio = 2.94). Other constituents which could be found in the immune complexes, i.e. anti-IgE antibodies, rheumatoid factors and anticollagen antibodies, were also higher in synovial fluid than predicted. Our results suggest intra-articular production of IgE-containing complexes in the synovial fluid, in addition to possible exudation of the complexes from the serum. These findings provide further evidence for the role of IgE-containing immune complexes in rheumatoid synovitis.     

Trisomy 7 in synovial fluid cells of patients with rheumatoid arthritis

Objective Recent studies revealed trisomy 7 as a chromosomal abnormality in non-neoplastic disorders such as rheumatoid arthritis (RA). In the present study, we investigated the presence of trisomy 7 in the synovial fluid cells of patients with RA using fluorescence in situ hybridisation (FISH) analysis.Methods Synovial fluid from 15 patients with RA was collected from knee joints. The control group consisted of seven patients with traumatic synovial effusion in their knee joints. The arthrocenteses were performed under aseptic conditions. Dual-colour FISH analysis was performed using chromosome-7-specific LSI D7S522 (7q31) and chromosome-5-specific LSI EGR1 (5q31)/D5S721 (5p15.2) probes on the slides prepared from synovial fluid of RA patients and controls.Results The slides of our cases were analysed using two different DNA probes. When the slides hybridised with chromosome-5-specific probes were analysed, no trisomic or monosomic cells were revealed in both patients and controls. However, in eight of 15 patients, trisomy 7 occurred in variable percentages of cells (23% to 48%) of synovial fluid. No monosomic 7 cells were detected in these specimens. All control cases were disomic for chromosome 7.Conclusion The results of the present investigation suggest that trisomy 7 may play a role in the pathogenesis of synovial hyperproliferation in RA.