The influence of synovial fibroblasts on the phagocytosis of Staphylococcus by polymorphonuclear cells

Summary It has been suggested that the reduced resistance of patients with rheumatoid arthritis (RA) to bacterial joint infections may be due in part to polymorphonuclear cell (PMN) function. To obtain further insight into the mechanis that contribute to the increased susceptiblity of RA patients to such infections we investigated the influence of different solid surfaces on the ingestion of various bacterial strains by PMN. Both in the presence and absence of serum, phagocytosis of bacteria by PMN was significantly lower on monolayers of synovial fibroblasts as compared to monolayers of endothelial cells and embryonic fibroblasts. It could be shown that the relative influence of the solid surfac on the results of the phagocytosis assay increased when decreasing concentrations of purified IgG were used. The results of this study sugpurified IgG were used. The results of this study suggested that the effect of synovial fibroblasts on PMB may lead to reduced clearance of bacteria from the joint.     

Effect of interleukin-1 on hyaluronate synthesis by synovial fibroblastic cells

Summary Interleukin-1 (IL-1) stimulates fibroblast-mediated hyaluronate (HA) synthesis in vitro. In the present study the degree of polymerization of such HA was studied using HPLC (high performance liquid chromatography) with a size exclusion column combined with¹²⁵I-HABP assay used to measure the HA concentration in various HA molecular weight fractions separated using HPLC. IL-1 stimulated HA was more polydisperse than that produced by resting fibroblasts with a molecular weight varying from more than 4×10⁶ daltons to less than 7.1×10³ daltons. This IL-1 effect may contribute to the low molecular weight HA produced by freshly explanted arthritic synovial tissue and to the low viscosity of arthritic synovial fluid in vivo.     

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.     

The effects of synovial fluid macrophages and interleukin-1 on hyaluronic acid synthesis by normal synovial fibroblasts

Summary The effects of peripheral blood monocyte and rheumatoid synovial fluid macrophage conditioned media were studied on hyaluronic acid (HA) metabolism of normal synovial fibroblasts. Both media stimulated HA synthesis about two-fold compared to controls (1% fetal calf serum). The activated mononuclear phagocyte conditioned media did not contain HA-degrading activity in these experiments. The effects of various concentrations of interleukin-1 (IL-1) on HA synthesis and proliferation of synovial fibroblasts were studied. Even at very low concentrations (0.1 IU IL-1/ml) HA synthesis was stimulated. With increasing concentrations HA synthesis did not increase but proliferation was stimulated. Stimulated fibroblasts synthesized mainly high molecular weight HA. Thus with IL-1-activation, normal synovial fibroblasts could not produce increased amounts of abnormal HA with decreased molecular weight.     

Comparison of the inhibitory effects of two types (90 kDa and 190 kDa) of hyaluronic acid on the expression of fibrinolytic factors in human synovial fibroblasts

 Hyaluronic acid (HA) has been shown to be clinically effective, and is currently used for the treatment of arthropathy. We previously reported that HA of molecular weight 90 kDa (90-HA) inhibits the fibrinolytic factors in human synovial fibroblasts. In the present study, we investigated the effect of high molecular weight (190 kDa) HA (190-HA) compared with 90-HA on the pericellular fibrinolytic system of human synovial fibroblasts in osteoarthritis (OA) and rheumatoid arthritis (RA). Human synovial fibroblasts were obtained from synovial tissues of OA and RA, and were cultured in the presence and absence of 90-HA and 190-HA. Antigens of urokinase-type plasminogen activator (u-PA) and PA inhibitor-1 (PAI-1) were measured by ELISA, and the u-PA activity of the cell surface fraction was evaluated by electrophoretic enzymography. The binding assay of u-PA and the immunocytochemical analysis of u-PA were performed to detect u-PA receptor (u-PAR). HA inhibited the secretion of both u-PA and PAI-1 antigens from the synovial fibroblasts of OA to their conditioned medium, and the degree of inhibition was more effective in OA than in RA. The u-RA binding assay to these cells showed that both 90-HA and 190-HA slightly decreased the maximal number of binding sites (Bmax) in OA. However, in RA, stimulation with 90-HA and 190-HA decreased Bmax by a half and a quarter, respectively. Immunohistochemical analysis showed that u-PAR was constitutively expressed in both synovial fibroblasts, but if these cells were treated with HA, the decrease in the staining of u-PAR was more pronounced in RA than in OA. Furthermore, the degree was more effective with 190-HA than with 90-HA. HA inhibited the pericellular fibrinolytic activity mediated by the u-PA/u-PAR system in synovial fibroblasts of OA and RA, and 190-HA inhibited it more effectively than 90-HA. Received: August 17, 2001 / Accepted: December 13, 2001     

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.     

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。     

Retinoic acid inhibition of hyaluronate synthesis in cultured human skin fibroblasts

The effects of all-trans retinoic acid on glycosaminoglycan (GAG) accumulation were determined in cultured primary human skin fibroblasts. Confluent cultures treated with retinoic acid accumulated less [3H]GAG than those without the compound, an effect with an apparent threshold of 10 nM which was dose dependent in the concentration range tested (0-10 microM). At 10 microM, the inhibition was 54%. Greater than 80% of the labeled macromolecular material was streptomyces hyaluronidase digestible in cultures labeled with [3H]acetate. The incorporation of H2[35S]O4 into chondroitin sulfate and dermatan sulfate was unaffected, as was total protein synthesis. Retinol also inhibited accumulation of [3H]GAG, but was far less potent. T3 and dexamethasone can inhibit [3H]hyaluronate synthesis. When retinoic acid was added to cultures treated with either of these hormones at concentrations that maximally inhibit [3H] GAG accumulation, there was a further decrease in the rate of macromolecular accumulation. The retinoic acid effect evolved over 24-48 h after addition to the culture medium. A pulse-chase study failed to demonstrate any effect on [3H]GAG degradation.     

Induction and activation of procollagenase in rabbit synovial fibroblasts after treatment with active oxygen released by xanthine/xanthine oxidase

Treatment of rabbit synovial fibroblasts with active oxygen (AO) released by xanthine/xanthine oxidase resulted in an induction of procollagenase in these cells in concentrations ranging from 12.5 g/ml xanthine plus 0.0025 U/ml xanthine oxidase to 50 g/ml xanthine plus 0.01 U/ml xanthine oxidase. Preceding this there was an accumulation of poly(ADP-ribose) for the same concentration range of xanthine/xanthine oxidase. Furthermore, it was found that AO caused activation of the latent procollagenase to the active enzyme in concentrations ranging from 0.1 g/ml xanthine plus 0.00002 U/ml xanthine oxidase to 1 g/ml xanthine plus 0.0002 U/ml xanthine oxidase. It is suggested that poly(ADP-ribosyl)ation participates in the induction of procollagenase by relaxing chromatin. Furthermore, it is proposed that AO activates latent procollagenase under physiological conditions.     

Diverse morphological responses of normal human synovial fibroblasts to mononuclear leukocyte products: relationship to prostaglandin production and plasminogen activator activities,and comparison with the effects of purified interleukin 1

Summary Supernatant media from cultured human mononuclear blood leukocytes (MCCM) induced morphological changes in normal human synovial fibroblasts in culture, including the formation of cells with a dendritic or stellate morphology and, less frequently, cells with a striking fenestrated appearance. These changes were fully reversed within 1 h of removing the MCCM. They were inhibited by indomethacin, the glucocorticoids hydrocortisone, prednisolone and dexamethasone, and by colcemid, but not by actinomycin D and only weakly by cycloheximide. The morphological responses to MCCM could be reproduced by MCCM fractions containing interleukin 1-like activity and by purified forms of human interleukin 1 (IL-1), including monocyte-derived IL-1 and recombinant IL-1. These responses were also inhibited by indomethacin, indicating a link with prostanoid production. However, the morphological responses were not related to the stimulation of plasminogen activator activity due to MCCM, MCCM fractions, or IL-1.     

Suppression of hyaluronic acid synthesis in synovial organ cultures by corticosteroid suspensions

The effects of sparingly soluble corticosteroid suspensions prepared for intraarticular therapy, of their vehicles, and of hydrocortisone on synovial hyaluronic acid (HA) synthesis were compared in organ cultures of normal canine villous synovium. Both hydrocortisone and the corticosteroid suspensions suppressed HA synthesis, as did 2 vehicle components, polysorbate 80 and myristyl-gamma-picrolinium chloride. Cultures of synovium from joints of dogs which, 1 day previously, had been injected with methylprednisolone acetate suspension synthesized less HA than did control cultures from noninjected joints of the same animals. The results indicate that suppression of HA synthesis is a mechanism by which these drugs can act to reduce joint HA content and promote resolution of synovial effusions.     

Pathological significance of elevated soluble CD14 production in rheumatoid arthritis: in the presence of soluble CD14, lipopolysaccharides at low concentrations activate RA synovial fibroblasts

In order to establish what contributes to elevated levels of soluble CD14 (sCD14) in rheumatoid arthritis (RA) plasma, levels of sCD14 were compared in RA-paired plasma and synovial fluids and, further, in the culture supernatants of monocyte-rich fractions from patients with RA and healthy donors, and macrophage-rich fractions from RA synovial tissues. The results showed elevated sCD14 in RA synovial fluid in 9 of 16 paired samples and in RA macrophage-rich fractions, suggesting that elevated sCD14 in RA plasma might be due to the sCD14 production by RA synovial macrophages. From the molecular analysis of elevated sCD14, the proteolytic cleavage of membranous CD14 (mCD14) was important in accelerated sCD14 production. Lipopolysaccharides (LPS) at low concentrations and sCD14 increased the ICAM-1 expression on RA synovial fibroblasts. This result implies that in vivo RA synovial fibroblasts may be sensitive to LPS in the presence of sCD14 and LPS-binding protein (LBP). Received: 4 October 1997 / Accepted: 20 January 1998     

透明质酸钠治疗老年膝骨性关节炎对患者关节滑液白介素-1β的影响分析

目的分析透明质酸钠（Sodium hyaluronate,SH）治疗老年膝骨性关节炎（Knee Osteoarthritis,KOA）对患者关节滑液白介素-1β（Interleukin-1β,IL-1β）的影响,探讨其临床治疗机制。方法选取我院2013年1月~2013年8月收治的136例老年KOA患者,按照随机数字表分为观察组及对照组,在常规治疗基础上分别实施SH及生理盐水注射治疗,比较两组患者临床疗效及治疗前后关节滑液IL变化。结果观察组治疗5周后ISOA评分显著降低,且显著低于对照组（P〈0.05）,对照组治疗5周后ISOA评分无显著变化（P〉0.05）;观察组临床改善32例,显效23例,有效9例,总有效率94.1%,对照组临床改善7例,显效10例,有效18例,总有效率51.5%,观察组总有效率显著高于对照组;观察组治疗1周后即见关节滑液IL-1β显著降低,且治疗期间呈显著降低趋势,治疗3个月后IL-1β仍显著低于治疗前含量（P〈0.05）;对照组治疗期间关节滑液IL-1β水平无明显变化（P〉0.05）。结论 SH可有效降低老年KOA患者关节滑液IL-1β水平,在降低关节软骨破坏、促进关节软骨修复、减轻炎性介质水平方面具有良好效果,可有效改善患者临床症状,保证其生活质量,可作为无手术指征的老年KOA患者的有效保守治疗方案加以推广。     

Urocortin对心肌成纤维细胞增殖和胶原合成作用的影响

目的 :探讨 urocortin对大鼠心肌成纤维细胞 （CFs）增殖和胶原合成作用的影响。方法 :消化法培养新生Sprague-Dawley （SD）大鼠的 CFs,采用四氮唑盐 （MTT）比色法测定细胞数目 ,流式细胞分析仪 （FCM）检测细胞周期 ,EL ISA法检测 型胶原合成 ,分别观察不同浓度 urocortin对 CFs增殖和胶原合成的影响。结果 :1随着 uro-cortin浓度的增高 ,CFs MTT比色法 A490 值呈明显的递增趋势 ,其中 10 - 10 、10 - 9、10 - 8、10 - 7和 10 - 6 mol/L组的 A490值分别为 0 .183± 0 .0 0 4、0 .2 2 2± 0 .0 0 4、0 .2 51± 0 .0 0 7、0 .2 80± 0 .0 0 6和 0 .3 17± 0 .0 0 2 ,均较对照组 （A490 值 0 .167± 0 .0 0 4）显著升高 （P均 <0 .0 1）。 2 FCM细胞周期分析结果表明 ,10 - 7mol/L urocortin作用 48h,CFs的 G0 /G1期细胞百分率均较对照组显著降低 （P<0 .0 1） ,S期细胞百分率、G2 /M期细胞百分率和增殖指数则较对照组显著增高 （均 P<0 .0 1）。 3 CFs的 型胶原分泌随着 urocortin作用浓度的增高呈明显的递增趋势 ,其中 10 - 10、10 - 9、10 - 8、10 - 7和 10 - 6 m ol/L urocortin组的 型胶原 A值分别为 0 .576± 0 .0 10、0 .614± 0 .0 0 8、0 .771± 0 .0 47、0 .83 9±0 .0 54和 0 .90 8± 0 .0 0 6,均较对照组     

精氨酸加压素诱导大鼠心脏成纤维细胞胶原合成的作用

目的 :探讨精氨酸加压素 （AVP）对大鼠心脏成纤维细胞 （CFs）胶原合成的影响。方法 :采用胰酶消化法分离、培养新生 SD大鼠 CFs,3H-脯氨酸掺入法和羟脯氨酸比色法分别观察不同浓度 AVP及其 V1 受体拮抗剂 [d（CH2 ） 5 Tyr2 （Me） ]AVP对 CFs的影响。结果 :1CFs3H-脯氨酸掺入率随着 AVP浓度的增加而增高 ,其中 10 - 7mol/ L,10 - 6 mol/ L AVP组的 3H-脯氨酸掺入率分别为 （5 41± 96 ） cpm/ 5 0 0 0 cells和 （5 6 5± 72 ） cpm/ 5 0 0 0 cells,较对照组 （16 6± 31） cpm/ 5 0 0 0 cells明显增高 （均 P<0 .0 1） ;10 - 7m ol/ L AVP+ 10 - 7mol/ L[d（CH2 ） 5 Tyr2 （Me） ]AVP组3H -脯氨酸掺入率 （2 6 8± 6 4） cpm/ 5 0 0 0 cells较 10 - 7m ol/ L AVP组明显降低 （P<0 .0 1）。 2 CFs培养上清光吸收值（A值 ）随着 AVP浓度的增加而增高 ,其中 10 - 7m ol/ L ,10 - 6 m ol/ L AVP组的培养上清 A值分别为 0 .2 2± 0 .0 1和0 . 2 4± 0 .0 1,明显高于对照组 0 .2 0± 0 .0 1,有统计学意义 （均 P <0 .0 1） ;10 - 7m ol/ L AVP+ 10 - 7mol/ L [d（CH2 ） 5 Tyr2 （Me） ]AVP组的 A值为 0 .19± 0 .0 1,明显低于 10 - 7mol/ L AVP组 （P<0 .0 1）。结论 :AVP促进 SD大鼠 CFs胶原合成 ,其作用可能与 V1 受体介导有关     

A decrease in hyaluronic acid synthesis by aging human fibroblasts leading to heparan sulfate enrichment and growth reduction

Cultured normal human fibroblasts during in vitro aging exhibited increased proportions of heparan sulfate (HS; a glycosaminoglycan (GAG) species) in the cell-associated GAG pool, coincident with decreased cell growth activity. An analysis of GAG metabolism demonstrated that human fibroblasts during aging became relatively rich in HS due to an alteration in the profile of GAG synthesis. HS became relatively enriched and hyaluronic acid (HA) relatively depleted through a decrease in HA synthase activity. An experimental enrichment of human fibroblast cultures with exogenous HS brought about an arrest of the cells in the G0/G1 phase and a decrease in the rate of S phase entry, coincident with aged cell growth behaviour. These results suggest that the change in HA synthesis is responsible, at least to some extent, for the growth reduction during aging of normal human fibroblasts.     

辛伐他汀对血管升压素诱导鼠心脏成纤维细胞增殖和胶原合成的影响

目的探讨辛伐他汀（S imvastatin,S im）对血管升压素（AVP）诱导的新生SD大鼠心脏成纤维细胞（CF）增殖和胶原合成作用的影响,为防治高血压左室肥厚提供理论依据。方法以培养的新生SD大鼠CF为实验模型,采用胰酶消化、差速贴壁法培养CF,运用MTT比色法和3H-脯氨酸掺入法分别观察不同浓度S im对AVP诱导CF增殖和胶原合成的作用及甲羟戊酸（m evalonate,MVA）干预的影响。结果①CF的3H-脯氨酸掺入率随着S im干预浓度的增加而降低,其中1μmol/L和10μmol/L S im组的3H-脯氨酸掺入率分别为（3.67±0.39）mBq/cell和（2.35±0.36）mBq/cell,明显低于对照组（5.01±0.58）mBq/cell（P<0.01）;②MTT比色法A490值随S im浓度的增加而降低,其中1μmol/L和10μmol/L S im组的A490值分别为0.221±0.038和0.163±0.021,均较对照组A490值（0.395±0.039）显著降低（P<0.01）;③10μmol/L S im+1 mmol/L MVA组的3H-脯氨酸掺入率和MTT比色法A490值分别为（5.38±0.72）mBq/cell和0.419±0.051,均显著高于同组10μmol/L S im（P<0.01）。结论S im抑制AVP诱导的CF增殖和胶原合成,其机制可能通过MVA代谢途径实现。