青藤碱对类风湿关节炎树突状细胞趋化因子及受体的影响和机制
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摘要
研究背景
类风湿关节炎(RA)是一种累及全身多个组织和脏器的慢性、炎症性疾病。主要病理过程是滑膜受累导致滑膜炎症,引起骨和软骨的损伤,最后发展为关节畸形和关节强直。RA也可以产生全身性弥漫性炎症,包括肺间质的改变、心包、胸膜、巩膜的受累,以及常见的皮下组织的皮肤结节。虽然RA的发病机制尚未完全阐明,但自身免疫在RA发病机制、发生和发展中发挥的关键作用已经得到广泛的认可。RA在全世界的发病率约为1%,女性与男性的比例为3:1。所有年龄均可发病,最常见的发病年龄40岁到50岁。RA是主要的致残性疾病之一,造成关节活动功能的丧失,给社会和家庭带来巨大的压力和负担。
在RA的发病机制中,在免疫基因易感宿主的自身抗原诱导活化的T细胞发挥着重要的作用。T细胞活化后导致多种免疫效应,包括滑膜细胞的增殖,内皮细胞的活化。骨髓和外周血中前炎症细胞活化,巨噬细胞、成纤维样滑膜细胞、B细胞分泌大量的细胞因子和自身抗体。树突状细胞(DC)是体内功能最为强大的抗原提呈细胞,是唯一可以激活初始T细胞的抗原提呈细胞。DC起源于骨髓干细胞,平时寄居在组织和外周血中。外周组织中的DC识别抗原后在细胞因子的刺激下逐渐分化,高表达MHC分子和协同刺激分子,在向次级淋巴器官迁移的过程中逐渐成熟,而后在淋巴器官中与T细胞结合,刺激其活化或诱导其耐受。研究表明,RA患者滑膜和关节中浸润的T细胞周围可见成熟的DC。这些DC高表达MHCⅡ类分子和CD40、CD80、CD86以及DC-SIGN等粘附分子和趋化因子受体,对T细胞的活化和组织的损伤起着重要作用。
RA属祖国传统医学痹证范畴。痹证的分类,按病因可分为风痹、寒痹、湿痹、热痹、风湿热痹等;从病理特点可分为行痹、痛痹、着痹。痹证的发生主要是由于正气不足,感受风、寒、湿、热之邪所致。内因是基础,素体虚弱、正气不足、腠理不密、卫外不固,是引起痹证的内在因素,如再感受外邪,使肌肉、关节、经络痹阻而形成痹证。痹证的治疗以祛风通络为基本大法,首先应辨明寒热,治以祛风、除湿、舒筋活络,随其寒热而兼行清热或祛寒。对于病程日久,气血损伤,脏腑亏虚的痹证患者,应配合运用补益之法。
青藤碱是中药清风藤的单体提取物,用于治疗RA已经有2000年的历史。研究发现青藤碱有抗炎、抗风湿的药理作用。尽管青藤碱广泛的药理作用已经得到阐明,但其作用机制,尤其是免疫抑制的机理尚有待进一步研究。为了进一步阐明青藤碱的药理作用,探讨其免疫抑制和RA的机理,本课题观察了青藤碱对RA患者DC趋化因子及其受体表达的影响和机制。
目的:
1.观察RA患者DC表面趋化因子受体的表达,研究DC表面趋化因子受体与RA患者疾病活动的相关关系;
2.建立DC趋化因子及其受体的荧光定量PCR检测体系;
3.观察青藤碱对RA患者DC趋化因子及其受体表达的影响;
4.观察青藤碱对RA患者DC泛素和E3连接酶表达的影响;
5.揭示青藤碱免疫抑制的机理,为青藤碱治疗RA提供理论和实验依据。
方法:
1.人外周血DC体外培养体系的建立
根据课题组已经建立的方法,分离人外周血单核细胞,RPMI-1640完全培养基培养,采用GM-CSF和IL-4联合刺激,于培养第8天收获。
2.DC表面趋化因子受体与RA患者疾病活动性的关系
选取28名RA患者和10名正常健康人分为四组:正常组、低活动组、中活动组和高活动组。采用流式细胞仪检测RA患者DC表面CCR5和CCR7的表达,收集患者类风湿因子(RF)、C反应蛋白(CRP)和抗CCP抗体。采用直线相关分析CCR5、CCR7和RA患者疾病活动指标的相关关系。
3.青藤碱对RA患者外周血DC趋化因子及其受体表达的影响
采用1mM,2mM,5mM青藤碱溶液和空白对照溶液干预DC 12h。收集干预后的细胞提取DC总RNA并逆转录为cDNA。PCR扩增CCR5、CCR7、CXCL9、CXCL10、CXCL11和GAPDH cDNA片段,回收并纯化PCR产物。将CCR5、CCR7、CXCL9、CXCL10、CXCL11和GAPDH的PCR产物分别按10~8,10~7,10~6,10~5,10~4copies/μl梯度倍比稀释,荧光定量PCR仪检测并自动计算出样品的定量结果,描绘标准曲线。将各样本cDNA按照建立体系中的反应条件在荧光定量PCR仪上进行扩增,由电脑自动分析并计算出反应体系中待测样品cDNA达到设定阈值的循环数(Ct值)。以公式2~(-△△t)计算出相对于空白对照组的青藤碱干预各组的DC中CCR5、CCR7、CXCL9、CXCL10、CXCL11和GAPDH mRNA的表达水平。收集DC培养上清,检测经不同浓度青藤碱干预后DC培养上清中CXCL9、CXCL10、CXCL11的表达。
4.青藤碱对RA患者外周血DC中泛素和E3表达的影响
收集青藤碱干预后的RA患者外周血DC,提取DC总RNA和蛋白。Real timePCR检测DC中泛素和E3连接酶mRNA的表达,western-blotting检测青藤碱对RA患者DC中泛素和E3连接酶表达的影响。
5.统计学处理
采用SPSS13.0统计软件进行数据分析,结果以(?)±S表示,多组间均数比较采用One way-ANOVA,多个组分别与对照组比较采用Dunnett检验;连续变量间的直线相关采用Pearson相关分析,P<0.05为有统计学意义。
结果:
1.在GM-CSF和IL-4联合刺激的作用下,单核细胞成功的分化为DC。在光镜和电镜下观察可见其呈现典型的树突状形态,与对照组相比,RA低、中、高活动组患者CCR5(F=107.53,P=0.000)和CCR7(F=46.283,P=0.000)表达较高。
2.在低疾病活动组(RF,r=0.775,P=0.014;CRP,r=0.802,P=0.009)、中疾病活动组(RF,r=0.0854,P=0.002;CRP,r=0.818,P=0.004)、高疾病活动组(RF,r=0.773,P=0.015;CRP,r=0.699,P=0.036)表达在RA患者DC表面CCR5与RF和CRP存在直线相关关系,相关性有统计学意义;与CCR5相似,在低疾病活动组(RF,1=0.754,P=0.019;CRP,r=0.968,P=0.000)、中疾病活动组(RF,r=0.0854,P=0.002;CRP,r=0.949,P=0.000)、高疾病活动组(RF,r=0.938,P=0.000;CRP,r=0.968,P=0.000)表达在RA患者DC表面CCR7与RF和CRP存在直线相关关系,相关性有统计学意义。
3.流式细胞结果显示,青藤碱干预后DC表面CCR5(F=89.236,P=0.000)和CCR7(F=94.731,P=0.000)表达有显著差异,差异有统计学意义;与空白对照组相比,2mM、5mM sinomenine干预后DC表面CCR5和CCR7(P<0.05 or P<0.01)表达显著降低,差异有统计学意义;GAPDH、CCR5、CCR7、CXCL9、CXCL10、CXCL11阳性梯度定量模板数与Ct值关系对数拟合作图,得到不同梯度定量模板的对数值与循环数(Ct值)之间关系的标准曲线,分别有着非常好的相关关系(0.9943、0.9955、0.9926、0.9981、0.9938、0.9994)。荧光定量PCR检测结果显示,与空白对照相比,经2mM和5mM青藤碱干预后的DC中CCR5、CCR7、CXCL9、CXCL10、CXCL11 mRNA表达均有所有降低(P<0.05 or P<0.01);ELISA结果显示,与空白对照相比,经青藤碱干预后的DC培养上清中CXCL9、CXCL10、CXCL11差异有统计学意义。
4.荧光定量PCR结果显示,青藤碱4个剂量组之间RA患者外周血DC中泛素(F=41.222,P=0.000)、E3连接酶(F=59.657,P=0.000)mRNA表达有显著性差异;与空白对照组相比,经2mM和5mM青藤碱干预后DC中泛素和E3连接酶表达显著降低;Western-blot检测结果显示,经青藤碱干预后4组泛素和E3连接酶的表达均有所改变,差异有统计学意义。
结论:
1.建立了RA患者外周血树突状细胞趋化因子受体检测体系;
2.不同疾病活动情况下RA患者DC表面趋化因子受体的表达与RA患者疾病活动指标存在直线相关关系,DC表面CCR5和CCR7可能是监测RA患者疾病活动和治疗效果的有效指标;
3.青藤碱可有效抑制RA患者DC趋化因子及其受体的表达,青藤碱的免疫抑制机理可能与抑制DC的迁移有关,青藤碱可能通过抑制DC迁移到次级淋巴器官与T细胞结合,从而达到治疗RA的作用;
4.青藤碱可能抑制泛素-蛋白酶体中重要蛋白的表达,抑制了泛素-蛋白酶体的功能,降低了NF-(?)B途径的磷酸化,从而影响了DC的免疫功能,达到免疫抑制的效果。
Background
Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disorder that may affect many tissues and organs, but principally attacks the joints producing a inflammatory synovitis that often progresses to destruction of the articular cartilage and ankylosis of the joints. Rheumatoid arthritis can also produce diffuse inflammation in the lungs, pericardium, pleura, and sclera, and also nodular lesions, most common in subcutaneous tissue under the skin. Although the cause of rheumatoid arthritis is unknown, autoimmunity plays a pivotal role in its chronicity and progression. About 1% of the world's population is afflicted by rheumatoid arthritis, women three times more often than men. Onset is most frequent in 40 to 50 years, but no age is immune. It can be a disabling and painful condition, which can lead to substantial loss of functioning and mobility.
The activation of T cells by as yet unknown antigens in the immunogenetically susceptible host is most probably the event that initiates the rheumatoid process. T cell activation subsequently leads to multiple effects including activation and proliferation of synovial lining and endothelial cells, recruitment and activation of additional proinflammatory cells from the bone marrow and circulation, secretion of cytokines and proteases by macrophages and fibroblast-like synovial cells, and autoantibody production. Dendritic cells are the most potent subset of antigen presenting cells. They are derived from bone marrow stem cells and reside in peripheral tissues or blood. Upon exposure to antigens and cytokines the peripheral DC s, express high amounts of peptide-MHC, and upregulate their costimulatory molecules, migrate to draining lymph nodes, and interact with T cells to stimulate or tolerize them. Dendritic cells have been found in synovium and joint fluid in rheumatoid arthritis, often at the center of a cluster of T cells. These DC s express MHCⅡ, the costimulatory molecules CD40, CD80, CD86, adhesion molecules such as DC-SIGN and chemokine receptors.
According to Traditional Chinese Medicine, this condition is called Bi Zheng, which is typically divided into four types: Wind-Cold Bi, Cold-Bi, Dampness-Bi and Heat-Bi. Through a thorough examination and consultation, including an assessment of the pulse and tongue, a diagnosis is made. Therapeutic principles For incipient bi-syndrome, expelling pathogenic factors should be the main therapeutic principle, including dispelling wind, dispersing cold, clearing away heat, eliminating dampness and dredging meridians and collaterals. For a weak patient suffering from bi-syndrome or a chronic case with deficiency of healthy qi, in addition to the therapy for expelling pathogenic factors, tonifying the spleen, liver, kidneys and blood should also be employed. For cases complicated by phlegm and blood stasis, activating blood circulation, dissipating blood stasis and masses and eliminating phlegm should be applied. Sinomenine, an alkaloid extracted from the Chinese medicinal plant, Sinomenium acutum, has been utilized to treat RA in China for over 2000 years. A wide range of pharmacological actions which includes anti-inflammatory and anti-rheumatic effects can be mediated by sinomenine. Although sinomenine exhibits a wide range of pharmacological activities, mechanistic study on sinomenine is very limited yet. In order to further illustrate the immunosuppressive action of sinomenine, we deeply investgated the effect and mechanism of sinomenine on chemokines and their receptors of DCs in patients with RA.
Objectives
1.To explore the relationship between disease activity and expression of chemokine receptors on the surface of dendritic cells in patients with RA;
2. To build up detection system of real time PCR for measuring chemokines and their receptors of dendritic cells;
3. To investigate the effect of sinomenine on expression of chemokines and chemokines receptors of dendritic cells in patients with RA;
4. To evaluate the effect of sinomenine on the expression of ubiquitin and ubiquitin-protein ligase of dendritic cells in patient with RA.
Methods
1. Culture of monocyte-derived DCs in patients with RA in vitro
Peripheral blood was collected and blood mononuclear cells were isolated. DCs were stimulalted with GM-CSF and IL-4 by the methods built in our past research.
2. Determining the relationship between expression of chemokine receptors of RA patients and disease activity
Tweenty eight patients and ten healthy control were chosen and disease activity score 28 were calculated. DCs from patients with RA were divided into four groups according to the disease activity of patients or healthy control. The expression of CCR5 and CCR7 were detected by flow cytometer. The level of serum rheumatoid factor, C reactive protein, anti-CCP antibody were observed. The correlation of expression of CCR5, CCR7 and disease activity of RA patients were analyzed.
3. Detecting the effect of sinomenine on the expression of chemokines and their receptors of DCs in patients with RA
DCs harvested from patients with RA were exposed to sinomenine 1mM, 2mM, 5mM sinomenine and medium for 12h, respectively. After treated with sinomenine, total RNA of DCs in patients with RA was isolated. cDNA of CCR5, CCR7, CXCL9, CXCL10, CXCL11 and GAPDH was amplified by PCR and products were purified. Templates were diluted in different concentration. Real time PCR was performed to build up standard curves for CCR5, CCR7, CXCL9, CXCL10, CXCL11 and GAPDH mRNA. Expression of CCR5, CCR7, CXCL9, CXCL10, CXCL11 and GAPDH mRNA for each sample was detected by real time PCR. The mRNA level of each sample for each gene was normalized to that of the GAPDH mRNA. The relative mRNA level was represented as 2~(-△△t) and expressed as the fold increase compared to the untreated cells. Then, expression of CXCL9, CXCL10, CXCL11 were detected by ELISA.
4. Observing the effect of sinomeine on expression ubiquitin and E3 ligase of DCs
After exposed to sinomenine, DCs were collected and total RNA and protein was extracted. Expression of ubiquitin and E3 were analyzed by real time PCR and western blotting, respectively.
5. Statistical analysis
All data are expressed as means±SD. Differences among one-way designed groups were analyzed by one-way ANOVA followed by Dunnett test. Linear relationship between two random variables were tested by Pearson correlation. A value of p<0.05 was considered statistically significant.
Result
1. Monocytes were cultured in the presence of GM-CSF and IL-4. Cells became nonadherent and clustered, exhibiting protruding veils typical of DCs. In contrast to healthy control, higher expression of CCR5 and CCR7 were observed in low, middle and high activity group.
2. There are linear correlation relationship between CCR5 expressed on DCs and disease activity RF, CRP, anti-CCP antibody of RA patients in low activity(RF, r=0.775, P=0.014; CRP, r=0.802, P=0.009), middle activity(RF, r=0.0854, p=0.002; CRP, r=0.818, P=0.004) and high activity(RF, r=0.773, P=0.015; CRP, r=0.699, p=0.036) group, respectively. And the same relationship were also observed in low activity(RF, r=0.754, P=0.019; CRP, r=0.968, P=0.000), middle activity(RF, r=0.0854, P=0.002; CRP, r=0.949, P=0.000) and high activity(RF, r=0.938, P=0.000; CRP, r=0.968, P=0.000) group for CCR7.
3. Significant differences of expression of CCR5(F=89.236, P=0.000) and CCR7(F=94.731, P=0.000) were observed in DCs exposed to 1mM, 2mM, 5mM sinomenine or control. Compared with control, CCR5 and CCR7(P<0.05 or P<0.01) were down-regulated in DCs treated with sinomenine at dose of 2mM or 5mM. Standard curves were obtained by real time PCR. Perfect linear correlations between different multiproportion dilution template for quantitation and cycle number (0.9943 for GAPDH, 0.9955 for CCR5, 0.9926 for CCR7, 0.9981 for CXCL9, 0.9938 for CXCL10, 0.9994 for CXCL11) were found in each group. Sinomenine appeared to effectively inhibit the expression of CCR5, CCR7, CXCL9, CXCL10, CXCL11 mRNA in DCs. Compared with control, significant differences were found in DCs treated with 2mM and 5mM sinomenine (P0.05 or P<0.01). Significant down-regulation of CXCL9, CXCL10, CXCL11 secretion were noticed in DCs exposed to sinomenine comparing to that treated with medium alone.
4. There were significant differences at expression of ubiquitin(F=41.222, P=0.000) and E3 ligase(F=59.657, P=0.000) mRNA between control and sinomenine groups. In contrast to control, DCs treated with 2mM, 5mM sinomenine expressed lower ubiquitin and E3 ligase. The similar result was also observed in the expression of ubiquitin and E3 ligase in protein level detected by Western Blot assay.
Conclusion
1. Chemokine receptors are expressed on DCs in RA patients with different disease actitivy. Chemokine receptors can be used as a index to monitor the disease activiy of RA.
2. Sinomenine can effectively suppress the expression of chemokines and their receptors of DCs in patients with RA at mRNA and protein level. Sinomenine exhibits the effect of relieving RA through inhibiting the expression of chemokines and their receptors, which can lead to DCs migration.
3. DCs can effectively decrease the expression of ubiquitin and E3 ligase of DC. The influence of sinomenine on DCs may relate to inhibit the ubiquitin system, which account for the maturation and migration of DCs.
类风湿关节炎(RA)是一种累及全身多个组织和脏器的慢性、炎症性疾病。主要病理过程是滑膜受累导致滑膜炎症,引起骨和软骨的损伤,最后发展为关节畸形和关节强直。RA也可以产生全身性弥漫性炎症,包括肺间质的改变、心包、胸膜、巩膜的受累,以及常见的皮下组织的皮肤结节。虽然RA的发病机制尚未完全阐明,但自身免疫在RA发病机制、发生和发展中发挥的关键作用已经得到广泛的认可。RA在全世界的发病率约为1%,女性与男性的比例为3:1。所有年龄均可发病,最常见的发病年龄40岁到50岁。RA是主要的致残性疾病之一,造成关节活动功能的丧失,给社会和家庭带来巨大的压力和负担。
在RA的发病机制中,在免疫基因易感宿主的自身抗原诱导活化的T细胞发挥着重要的作用。T细胞活化后导致多种免疫效应,包括滑膜细胞的增殖,内皮细胞的活化。骨髓和外周血中前炎症细胞活化,巨噬细胞、成纤维样滑膜细胞、B细胞分泌大量的细胞因子和自身抗体。树突状细胞(DC)是体内功能最为强大的抗原提呈细胞,是唯一可以激活初始T细胞的抗原提呈细胞。DC起源于骨髓干细胞,平时寄居在组织和外周血中。外周组织中的DC识别抗原后在细胞因子的刺激下逐渐分化,高表达MHC分子和协同刺激分子,在向次级淋巴器官迁移的过程中逐渐成熟,而后在淋巴器官中与T细胞结合,刺激其活化或诱导其耐受。研究表明,RA患者滑膜和关节中浸润的T细胞周围可见成熟的DC。这些DC高表达MHCⅡ类分子和CD40、CD80、CD86以及DC-SIGN等粘附分子和趋化因子受体,对T细胞的活化和组织的损伤起着重要作用。
RA属祖国传统医学痹证范畴。痹证的分类,按病因可分为风痹、寒痹、湿痹、热痹、风湿热痹等;从病理特点可分为行痹、痛痹、着痹。痹证的发生主要是由于正气不足,感受风、寒、湿、热之邪所致。内因是基础,素体虚弱、正气不足、腠理不密、卫外不固,是引起痹证的内在因素,如再感受外邪,使肌肉、关节、经络痹阻而形成痹证。痹证的治疗以祛风通络为基本大法,首先应辨明寒热,治以祛风、除湿、舒筋活络,随其寒热而兼行清热或祛寒。对于病程日久,气血损伤,脏腑亏虚的痹证患者,应配合运用补益之法。
青藤碱是中药清风藤的单体提取物,用于治疗RA已经有2000年的历史。研究发现青藤碱有抗炎、抗风湿的药理作用。尽管青藤碱广泛的药理作用已经得到阐明,但其作用机制,尤其是免疫抑制的机理尚有待进一步研究。为了进一步阐明青藤碱的药理作用,探讨其免疫抑制和RA的机理,本课题观察了青藤碱对RA患者DC趋化因子及其受体表达的影响和机制。
目的:
1.观察RA患者DC表面趋化因子受体的表达,研究DC表面趋化因子受体与RA患者疾病活动的相关关系;
2.建立DC趋化因子及其受体的荧光定量PCR检测体系;
3.观察青藤碱对RA患者DC趋化因子及其受体表达的影响;
4.观察青藤碱对RA患者DC泛素和E3连接酶表达的影响;
5.揭示青藤碱免疫抑制的机理,为青藤碱治疗RA提供理论和实验依据。
方法:
1.人外周血DC体外培养体系的建立
根据课题组已经建立的方法,分离人外周血单核细胞,RPMI-1640完全培养基培养,采用GM-CSF和IL-4联合刺激,于培养第8天收获。
2.DC表面趋化因子受体与RA患者疾病活动性的关系
选取28名RA患者和10名正常健康人分为四组:正常组、低活动组、中活动组和高活动组。采用流式细胞仪检测RA患者DC表面CCR5和CCR7的表达,收集患者类风湿因子(RF)、C反应蛋白(CRP)和抗CCP抗体。采用直线相关分析CCR5、CCR7和RA患者疾病活动指标的相关关系。
3.青藤碱对RA患者外周血DC趋化因子及其受体表达的影响
采用1mM,2mM,5mM青藤碱溶液和空白对照溶液干预DC 12h。收集干预后的细胞提取DC总RNA并逆转录为cDNA。PCR扩增CCR5、CCR7、CXCL9、CXCL10、CXCL11和GAPDH cDNA片段,回收并纯化PCR产物。将CCR5、CCR7、CXCL9、CXCL10、CXCL11和GAPDH的PCR产物分别按10~8,10~7,10~6,10~5,10~4copies/μl梯度倍比稀释,荧光定量PCR仪检测并自动计算出样品的定量结果,描绘标准曲线。将各样本cDNA按照建立体系中的反应条件在荧光定量PCR仪上进行扩增,由电脑自动分析并计算出反应体系中待测样品cDNA达到设定阈值的循环数(Ct值)。以公式2~(-△△t)计算出相对于空白对照组的青藤碱干预各组的DC中CCR5、CCR7、CXCL9、CXCL10、CXCL11和GAPDH mRNA的表达水平。收集DC培养上清,检测经不同浓度青藤碱干预后DC培养上清中CXCL9、CXCL10、CXCL11的表达。
4.青藤碱对RA患者外周血DC中泛素和E3表达的影响
收集青藤碱干预后的RA患者外周血DC,提取DC总RNA和蛋白。Real timePCR检测DC中泛素和E3连接酶mRNA的表达,western-blotting检测青藤碱对RA患者DC中泛素和E3连接酶表达的影响。
5.统计学处理
采用SPSS13.0统计软件进行数据分析,结果以(?)±S表示,多组间均数比较采用One way-ANOVA,多个组分别与对照组比较采用Dunnett检验;连续变量间的直线相关采用Pearson相关分析,P<0.05为有统计学意义。
结果:
1.在GM-CSF和IL-4联合刺激的作用下,单核细胞成功的分化为DC。在光镜和电镜下观察可见其呈现典型的树突状形态,与对照组相比,RA低、中、高活动组患者CCR5(F=107.53,P=0.000)和CCR7(F=46.283,P=0.000)表达较高。
2.在低疾病活动组(RF,r=0.775,P=0.014;CRP,r=0.802,P=0.009)、中疾病活动组(RF,r=0.0854,P=0.002;CRP,r=0.818,P=0.004)、高疾病活动组(RF,r=0.773,P=0.015;CRP,r=0.699,P=0.036)表达在RA患者DC表面CCR5与RF和CRP存在直线相关关系,相关性有统计学意义;与CCR5相似,在低疾病活动组(RF,1=0.754,P=0.019;CRP,r=0.968,P=0.000)、中疾病活动组(RF,r=0.0854,P=0.002;CRP,r=0.949,P=0.000)、高疾病活动组(RF,r=0.938,P=0.000;CRP,r=0.968,P=0.000)表达在RA患者DC表面CCR7与RF和CRP存在直线相关关系,相关性有统计学意义。
3.流式细胞结果显示,青藤碱干预后DC表面CCR5(F=89.236,P=0.000)和CCR7(F=94.731,P=0.000)表达有显著差异,差异有统计学意义;与空白对照组相比,2mM、5mM sinomenine干预后DC表面CCR5和CCR7(P<0.05 or P<0.01)表达显著降低,差异有统计学意义;GAPDH、CCR5、CCR7、CXCL9、CXCL10、CXCL11阳性梯度定量模板数与Ct值关系对数拟合作图,得到不同梯度定量模板的对数值与循环数(Ct值)之间关系的标准曲线,分别有着非常好的相关关系(0.9943、0.9955、0.9926、0.9981、0.9938、0.9994)。荧光定量PCR检测结果显示,与空白对照相比,经2mM和5mM青藤碱干预后的DC中CCR5、CCR7、CXCL9、CXCL10、CXCL11 mRNA表达均有所有降低(P<0.05 or P<0.01);ELISA结果显示,与空白对照相比,经青藤碱干预后的DC培养上清中CXCL9、CXCL10、CXCL11差异有统计学意义。
4.荧光定量PCR结果显示,青藤碱4个剂量组之间RA患者外周血DC中泛素(F=41.222,P=0.000)、E3连接酶(F=59.657,P=0.000)mRNA表达有显著性差异;与空白对照组相比,经2mM和5mM青藤碱干预后DC中泛素和E3连接酶表达显著降低;Western-blot检测结果显示,经青藤碱干预后4组泛素和E3连接酶的表达均有所改变,差异有统计学意义。
结论:
1.建立了RA患者外周血树突状细胞趋化因子受体检测体系;
2.不同疾病活动情况下RA患者DC表面趋化因子受体的表达与RA患者疾病活动指标存在直线相关关系,DC表面CCR5和CCR7可能是监测RA患者疾病活动和治疗效果的有效指标;
3.青藤碱可有效抑制RA患者DC趋化因子及其受体的表达,青藤碱的免疫抑制机理可能与抑制DC的迁移有关,青藤碱可能通过抑制DC迁移到次级淋巴器官与T细胞结合,从而达到治疗RA的作用;
4.青藤碱可能抑制泛素-蛋白酶体中重要蛋白的表达,抑制了泛素-蛋白酶体的功能,降低了NF-(?)B途径的磷酸化,从而影响了DC的免疫功能,达到免疫抑制的效果。
Background
Rheumatoid arthritis (RA) is a chronic, systemic inflammatory disorder that may affect many tissues and organs, but principally attacks the joints producing a inflammatory synovitis that often progresses to destruction of the articular cartilage and ankylosis of the joints. Rheumatoid arthritis can also produce diffuse inflammation in the lungs, pericardium, pleura, and sclera, and also nodular lesions, most common in subcutaneous tissue under the skin. Although the cause of rheumatoid arthritis is unknown, autoimmunity plays a pivotal role in its chronicity and progression. About 1% of the world's population is afflicted by rheumatoid arthritis, women three times more often than men. Onset is most frequent in 40 to 50 years, but no age is immune. It can be a disabling and painful condition, which can lead to substantial loss of functioning and mobility.
The activation of T cells by as yet unknown antigens in the immunogenetically susceptible host is most probably the event that initiates the rheumatoid process. T cell activation subsequently leads to multiple effects including activation and proliferation of synovial lining and endothelial cells, recruitment and activation of additional proinflammatory cells from the bone marrow and circulation, secretion of cytokines and proteases by macrophages and fibroblast-like synovial cells, and autoantibody production. Dendritic cells are the most potent subset of antigen presenting cells. They are derived from bone marrow stem cells and reside in peripheral tissues or blood. Upon exposure to antigens and cytokines the peripheral DC s, express high amounts of peptide-MHC, and upregulate their costimulatory molecules, migrate to draining lymph nodes, and interact with T cells to stimulate or tolerize them. Dendritic cells have been found in synovium and joint fluid in rheumatoid arthritis, often at the center of a cluster of T cells. These DC s express MHCⅡ, the costimulatory molecules CD40, CD80, CD86, adhesion molecules such as DC-SIGN and chemokine receptors.
According to Traditional Chinese Medicine, this condition is called Bi Zheng, which is typically divided into four types: Wind-Cold Bi, Cold-Bi, Dampness-Bi and Heat-Bi. Through a thorough examination and consultation, including an assessment of the pulse and tongue, a diagnosis is made. Therapeutic principles For incipient bi-syndrome, expelling pathogenic factors should be the main therapeutic principle, including dispelling wind, dispersing cold, clearing away heat, eliminating dampness and dredging meridians and collaterals. For a weak patient suffering from bi-syndrome or a chronic case with deficiency of healthy qi, in addition to the therapy for expelling pathogenic factors, tonifying the spleen, liver, kidneys and blood should also be employed. For cases complicated by phlegm and blood stasis, activating blood circulation, dissipating blood stasis and masses and eliminating phlegm should be applied. Sinomenine, an alkaloid extracted from the Chinese medicinal plant, Sinomenium acutum, has been utilized to treat RA in China for over 2000 years. A wide range of pharmacological actions which includes anti-inflammatory and anti-rheumatic effects can be mediated by sinomenine. Although sinomenine exhibits a wide range of pharmacological activities, mechanistic study on sinomenine is very limited yet. In order to further illustrate the immunosuppressive action of sinomenine, we deeply investgated the effect and mechanism of sinomenine on chemokines and their receptors of DCs in patients with RA.
Objectives
1.To explore the relationship between disease activity and expression of chemokine receptors on the surface of dendritic cells in patients with RA;
2. To build up detection system of real time PCR for measuring chemokines and their receptors of dendritic cells;
3. To investigate the effect of sinomenine on expression of chemokines and chemokines receptors of dendritic cells in patients with RA;
4. To evaluate the effect of sinomenine on the expression of ubiquitin and ubiquitin-protein ligase of dendritic cells in patient with RA.
Methods
1. Culture of monocyte-derived DCs in patients with RA in vitro
Peripheral blood was collected and blood mononuclear cells were isolated. DCs were stimulalted with GM-CSF and IL-4 by the methods built in our past research.
2. Determining the relationship between expression of chemokine receptors of RA patients and disease activity
Tweenty eight patients and ten healthy control were chosen and disease activity score 28 were calculated. DCs from patients with RA were divided into four groups according to the disease activity of patients or healthy control. The expression of CCR5 and CCR7 were detected by flow cytometer. The level of serum rheumatoid factor, C reactive protein, anti-CCP antibody were observed. The correlation of expression of CCR5, CCR7 and disease activity of RA patients were analyzed.
3. Detecting the effect of sinomenine on the expression of chemokines and their receptors of DCs in patients with RA
DCs harvested from patients with RA were exposed to sinomenine 1mM, 2mM, 5mM sinomenine and medium for 12h, respectively. After treated with sinomenine, total RNA of DCs in patients with RA was isolated. cDNA of CCR5, CCR7, CXCL9, CXCL10, CXCL11 and GAPDH was amplified by PCR and products were purified. Templates were diluted in different concentration. Real time PCR was performed to build up standard curves for CCR5, CCR7, CXCL9, CXCL10, CXCL11 and GAPDH mRNA. Expression of CCR5, CCR7, CXCL9, CXCL10, CXCL11 and GAPDH mRNA for each sample was detected by real time PCR. The mRNA level of each sample for each gene was normalized to that of the GAPDH mRNA. The relative mRNA level was represented as 2~(-△△t) and expressed as the fold increase compared to the untreated cells. Then, expression of CXCL9, CXCL10, CXCL11 were detected by ELISA.
4. Observing the effect of sinomeine on expression ubiquitin and E3 ligase of DCs
After exposed to sinomenine, DCs were collected and total RNA and protein was extracted. Expression of ubiquitin and E3 were analyzed by real time PCR and western blotting, respectively.
5. Statistical analysis
All data are expressed as means±SD. Differences among one-way designed groups were analyzed by one-way ANOVA followed by Dunnett test. Linear relationship between two random variables were tested by Pearson correlation. A value of p<0.05 was considered statistically significant.
Result
1. Monocytes were cultured in the presence of GM-CSF and IL-4. Cells became nonadherent and clustered, exhibiting protruding veils typical of DCs. In contrast to healthy control, higher expression of CCR5 and CCR7 were observed in low, middle and high activity group.
2. There are linear correlation relationship between CCR5 expressed on DCs and disease activity RF, CRP, anti-CCP antibody of RA patients in low activity(RF, r=0.775, P=0.014; CRP, r=0.802, P=0.009), middle activity(RF, r=0.0854, p=0.002; CRP, r=0.818, P=0.004) and high activity(RF, r=0.773, P=0.015; CRP, r=0.699, p=0.036) group, respectively. And the same relationship were also observed in low activity(RF, r=0.754, P=0.019; CRP, r=0.968, P=0.000), middle activity(RF, r=0.0854, P=0.002; CRP, r=0.949, P=0.000) and high activity(RF, r=0.938, P=0.000; CRP, r=0.968, P=0.000) group for CCR7.
3. Significant differences of expression of CCR5(F=89.236, P=0.000) and CCR7(F=94.731, P=0.000) were observed in DCs exposed to 1mM, 2mM, 5mM sinomenine or control. Compared with control, CCR5 and CCR7(P<0.05 or P<0.01) were down-regulated in DCs treated with sinomenine at dose of 2mM or 5mM. Standard curves were obtained by real time PCR. Perfect linear correlations between different multiproportion dilution template for quantitation and cycle number (0.9943 for GAPDH, 0.9955 for CCR5, 0.9926 for CCR7, 0.9981 for CXCL9, 0.9938 for CXCL10, 0.9994 for CXCL11) were found in each group. Sinomenine appeared to effectively inhibit the expression of CCR5, CCR7, CXCL9, CXCL10, CXCL11 mRNA in DCs. Compared with control, significant differences were found in DCs treated with 2mM and 5mM sinomenine (P0.05 or P<0.01). Significant down-regulation of CXCL9, CXCL10, CXCL11 secretion were noticed in DCs exposed to sinomenine comparing to that treated with medium alone.
4. There were significant differences at expression of ubiquitin(F=41.222, P=0.000) and E3 ligase(F=59.657, P=0.000) mRNA between control and sinomenine groups. In contrast to control, DCs treated with 2mM, 5mM sinomenine expressed lower ubiquitin and E3 ligase. The similar result was also observed in the expression of ubiquitin and E3 ligase in protein level detected by Western Blot assay.
Conclusion
1. Chemokine receptors are expressed on DCs in RA patients with different disease actitivy. Chemokine receptors can be used as a index to monitor the disease activiy of RA.
2. Sinomenine can effectively suppress the expression of chemokines and their receptors of DCs in patients with RA at mRNA and protein level. Sinomenine exhibits the effect of relieving RA through inhibiting the expression of chemokines and their receptors, which can lead to DCs migration.
3. DCs can effectively decrease the expression of ubiquitin and E3 ligase of DC. The influence of sinomenine on DCs may relate to inhibit the ubiquitin system, which account for the maturation and migration of DCs.
引文
[1]Valesini G,Barone F,Bompane D,et al.Advances in immunology and rheumatoid arthritis pathogenesis.Reumatismo.2004;56(1):9-20.
[2]Drosos AA.Epidemiology of rheumatoid arthritis.Autoimmun Rev.2004;3 Suppl 1:S20-22.
[3]Terlman H,Pagliari LJ,Liu H,et al.Rheumatoid arthritis synovial macrophages express the Fas-associated death domain-like interleukin-1β-converting enzyme inhibitory protein and are refractory to Fas-mediated apoptosis.Arthritis rheum.2001 ,44:21-30.
[4]Yocum DE.T cells:pathogenic cells and therapeutic targets in rheumatoid arthritis.Semin Arthritis Rheum.1999;29(1):27-35.
[5]Hang HG,Wang YM,Xie JF,et al.Regulation of tumor necrosis factora-mediated apoptosis of rheumatoid arthritis synovial fibroblasts by the protein kinase Akt.Arthritis rheum.2001;44:1555-1567.
[6]Black RA,Rauch CT,Kozlosky CJ,et al.A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells.Nature.1997;385(6618):729-733.
[7]Nawroth PP,Bank I,Handley D,et al.Tumor necrosis factor/cachectin interacts with endothelial cell receptors to induce release of interleukin 1.J Exp Med.1986;163(6):1363-1375.
[8]Chin JE,Winterrowd GE,Krzesicki RF,et al.Role of cytokines in inflammatory synovitis:the coordinate regulation of intercellular adhesion molecule 1 and HLA class Ⅰ and class Ⅱ antigens in rheumatoid synovial fibroblasts.Arthritis Rheum.1990;33(12):1776-1786.
[9]Butler DM,Maini RN,Feldmann M,et al.Modulation of proinflammatory cytokine release in rheumatoid synovial membrane cell cultures.Comparison of monoclonal anti TNF-alpha antibody with the interleukin-1 receptor antagonist.Eur Cytokine Netw.1995;6(4):225-230.
[10]Keffer J,Probert L,Cazlaris H,et al.Transgenic mice expressing human turnout necrosis factor:a predictive genetic model of arthritis.EMBO J.1991;10(13):4025-4031.
[11]Wooley PH,Dutcher J,Widmer MB,et al.Influence of a recombinant human soluble tumor necrosis factor receptor Fc fusion protein on type Ⅱ collagen-induced arthritis in mice.J Immunol.1993;151(11):6602-6607.
[12]Williams RO,Feldmann M,Maini RN.Anti-tumor necrosis factor ameliorates joint disease in marine collagen-induced arthritis.Proc Natl Acad Sci USA.1992;89(20):9784-9788.
[13]Koch AE,Kunkel SL,Strieter RaM.Cytokines in rheumatoid arthritis.J Invest Med.1995;43(1):28-38.
[14]Arend WP,Dayer JM.Inhibition of the production and effects of interleukin-1 and tumor necrosis factor alpha in rheumatoid arthritis.Arthritis Rheum.1995;38(2):151-160.
[15]MacNaul KL,Chartrain N,Lark M,et al.Discoordinate expression of stromelysin,collagenase,and tissue inhibitor of metalloproteinases-1 in rheumatoid human synovial fibroblasts:synergistic effects of interleukin-1 and tumor necrosis factor-alpha on stromelysin expression.J Biol Chem.1990;265(28):17238-17245.
[16]Strand V,Kavanaugh AF.The role of interleukin- 1 in bone resorption in rheumatoid arthritis.Rheumatology.2004;43(Suppl-3):Ⅲ10-Ⅲ16.
[17]Dayer J M.The pivotal role of interleukin- 1 in the clinical manifestations of rheumatoid arthritis.Rheumatology(Oxford).2003;42(Suppl 2):3-10.
[18]Mosmann TR,Sad S.The expanding universe of T-cell subsets:Th1,Th2 and more.Immunol Today.1996;17(3):138-146.
[19]Aarvak T,Chabaud M,Miossec P,et al.IL-17 is produced by some proinflammatory Thl/ThO cells but not by Th2 cells.J Immunol.1999;162(3):1246-1251.
[20]Romagnani S.Regulation of the development of type 2 T-helper cells in allergy.Curr Opin Immunol.1994;6(6):838-846.
[21]Yudoh K,Matsuno H,Osada R,et al.Decreased cellular activity and replicative capacity of osteoblastic cells isolated from the periarticular bone of rheumatoid arthritis patients compared with osteoarthritis patients. Arthritis Rheum. 2000;43(10):2178-2188.
[22] Shimoyama Y, Nagafuchi H, Suzuki N, et al. Synovium infiltrating T cells induce excessive synovial cell function through CD28/B7 pathway in patients with rheumatoid arthritis. J Rheumatol. 1999;26(10):2094-2101.
[23] 张利宁,曹英林,赵文璞,等.CD137在SLE和RA病人外周血T细胞上的表达特点及意义.中华微生物学和免疫学杂志.2000;20(2):126-127.
[24] El Ewau TD, De Keyser F, De Wever N, et al. A comparative phenotypical analysis of rheumatoid nodules and rheumatoid synovium with special reference to adhesion molecules and activation markers. Ann Rheum Dis. 1998; 57(8): 480-486.
[25] Miltenburg AM, van Laar JM, de Kuiper R, et al. T cells cloned from human rheumatoid synovial membrance functionally represent the Th1 subset. Scand J Immunol. 1992;35(5):603-610.
[26] Quayle AJ, Chomarat P, Miossec P, et al. Rheumatoid inflammatory T-cell clones express mostly Thl but also Th2 and mixed (Th0-like) cytokine patterns. Scand J Immunol. 1993;38(1):75-82.
[27] Davis LS, Cush JJ, Schulze-Koops H, et al. Rheumatoid synovial CD4+T cells exhibit a reduced capacity to differentiate into IL-4-producing T-helper-2 effector cells. Arthritis Res. 2001;3(1):54-64.
[28] Park W, Weyand CM, Schmidt D, et al. Co-stimulatory pathways controlling activation and perpheral tolerance of human CD4+CD28-T cell. Eur J Immunol. 1997;27(5):1082-1090.
[29] Josien R, Wong BR, Li HL, et al. TRANCE, a TNF family member, is differentially expressed on T cell subsets and induces cytokine production in dendritic cells. J Immunol. 1999;162(5):2562-2568.
[30] Ross FP. Interleukin 7 and estrogen-induced bone loss. Trends Endocrinol Metab. 2003;14(4):147-149.
[31] Banchereau J, Briere F, Caux C, et al. Immunobiology of dendritic cells. Annu Rev Immunol.2000;18:767-811.
[32]Steinman RM,Cohn ZA.Identification of a novel cell type in peripheral lymphoid organs of mice.I.Morphology,quantitation,tissue distribution.J Exp Med.1973;137(5):1142-1162.
[33]Banchereau J,Steinman RM.Dendritic cells and the control of immunity.Nature.1998;392:245-252.
[34]Kitamura H,Iwakabe K,Yahata T,et al.The natural killer T (NKT) cell ligand alpha-galactosylceramide demonstrates its immunopotentiating effect by inducing interleukin (IL)-12 production by dendritic cells and IL-12 receptor expression on NKT cells.J Exp Med.1999;189:1121-1128.
[35]Brocker T.Survival of mature CD4 T lymphocytes is dependent on major histocompatibility complex class Ⅱ-expressing dendritic cells.J Exp Med.1997;186:1223-1232.
[36]Lutzky V,Thomas R:Dendritic cells.Contemporary Targeted Therapies in Rheumatology.Edited by Smolen JS,Lipsky PE.London:Informa Healthcare;2007:63-78.
[37]Marquez C,Trigueros C,Fernandez E,et al.The development of T and non-T-cell lineages from CD34+human thymic precursors can be traced by the differential expression of CD44.J Exp Med.1995;181(2):475-483.
[38]O'Keeffe M,Hochrein H,Vremec D,et al.Dendritic cell precursor populations of mouse blood:identification of the murine homologues of human blood plasmacytoid pre-DC2 and CD11c+ DC1 precursors.Blood.2003;101(4):1453-1459.
[39]Mohty M,Isnardon D,Blaise D,et al.Identification of precursors of leukemic dendritic cells differentiated from patients with acute myeloid leukemia.Leukemia.2002;16(11):2267-2274.
[40]Itano AA,McSorley SJ,Reinhardt RL,et al.Distinct dendritic cell populations sequentially present antigen to CD4 T cells and stimulate different aspects of cell-mediated immunity.Immunity.2003;19( 1 ):47-57.
[41]Ardav(?)n C.Thymic dendritic cells.Immunol Today.1997;18(7):350-361.
[42]Dauer M,Obermaier B,Herten J,et al.Mature dendritic cells derived from human monocytes within 48 hours:a novel strategy for dendritic cell differentiation from blood precursors.J Immunol.2003;170(8):4069-4076.
[43]Merrick A,Errington F,Milward K,et al.Immunosuppressive effects of radiation on human dendritic cells:reduced IL-12 production on activation and impairment of naive T-cell priming.Br J Cancer.2005;92(8):1450-1458.
[44]Dustin ML,Cooper JA.The immunological synapse and the actin cytoskeleton:molecular hardware for T cell signaling.Nat.Immunol.2000;1(1):23-29.
[45]Pettit AR,MacDonald KPA,O'Sullivan B,et al.Differentiated dendritic cells expressing nuclear RelB are predominantly located in rheumatoid synovial tissue perivascular mononuclear cell aggregates.Arthritis Rheum.2000;43:791-800.
[46]Steinman RM,Swanson J.The endocytic activity of dendritic cells.J Exp Med.1995;182:283-288.
[47]Sallusto F,Palermo B,Lenig D,et al.Distinct patterns and kinetics of chemokine production regulate dendritic cell function.Eur J Immunol.1999.29:1617-1625.
[48]Sallusto F,Lanzavecchia A.Understanding dendritic cell and Tlymphocyte traffic through the analysis of chemokine receptor expression.Immunol Rev.2000.177:134-140.
[49]Hermans IF,Ritchie DS,Yang J,et al.CD8+ T cell-dependent elimination of dendritic cells in vivo limits the induction of antitumor immunity.J Immunol.2000.164:3095-3101.
[50]Kalinski P,Vieira P,Schuitemaker JH,et al.Generation of human type 1- and type 2-polarized dendritic cells from peripheral blood.Methods Mol Biol.2003.215:427-436.
[51]Steptoe RJ,Ritchie JM,Jones LK,et al.Autoimmune diabetes is suppressed by transfer of proinsulin-encoding Gr-1+ myeloid progenitor cells that differentiate in vivo into resting dendritic cells.Diabetes.2005;54:434-442.
[52]Sallusto F,Lanzavecchia A.Mobilizing dendritic cells for tolerance,priming and chronic inflammation.J Exp Med.1999;189:611-614.
[53]Yan M,Peng J,Jabbar IA,et al.Activation of dendritic cells by human papillomavirus-like particlesthrough TLR4 and NF-kappaB-mediated signalling,moderated by TGF-beta.Immunol Cell Biol.2005;83:83-91.
[54]Reise Sousa C.Dendritic cells in a mature age.Nat Rev Immunol.2006;6:476-483.
[55]Siebenlist U,Franzoso G,Brown K.Structure,regulation and function of NF-kappa B.Annu Rev Cell Biol.1994;10:405-455.
[56]O'Sullivan B,Thompson AG,Thomas R:NF-kappa B as a therapeutic target in autoimmune disease.Curr Opin Ther Targets 2007,11:111-122.
[57]Bondeson J,Foxwell B,Brennan F,et al.Defining therapeutic targets by using adenovirus:blocking NF-kappaB inhibits both inflammatory and destructive mechanisms in rheumatoid synovium but spares anti-inflammatory mediators.Proc Natl Acad Sci USA.1999;96:5668-5673.
[58]Koch AE,Burrows JC,Haines GK,et al.Immunolocalization of endothelial and leukocyte adhesion molecules in human rheumatoid and osteoarthritic synovial tissues.Lab Invest.1991;64:313-320.
[59]Lo JC,Basak S,James ES,et al.Coordination between NF-kappaB family members p50 and p52 is essential for mediating LTbetaR signals in the development and organization of secondary lymphoid tissues.Blood.2006;107:1048-1055.
[60]Banchereau J,Paczesny S,Blanco P,et al.Dendritic cells:controllers of the immune system and a new promise for immunotherapy.Ann N Y Acad Sci.2003.987:180-187.
[61]Thomas R,Davis LS,Lipsky PE.Rheumatoid synovium is enriched in mature antigen-presenting dendritic cells.J Immunol.1994;152(5):2613-2623.
[62]Waalen K,Thoen J,Forre O,et al.Rheumatoid synovial dendritic cells as stimulators in allogeneic and autologous mixed leukocyte reactions—Comparison with autologous monocytes as stimulator cells.Scand J Immunol.1986;23(2):233-241.
[63]Devereux D,RE OH,McGuire J,et al.HLA-DR4Dw4-restricted T cell recognition of self antigen(s) in the rheumatoid synovial compartment.Int Immunol.1991;3(7):635-640.
[64]Palmer DG.The anatomy of the rheumatoid lesion.Br Med Bull.1995;51(2):286-295.
[65]Chen E,Keystone EC,Fish EN.Restricted cytokine expression in rheumatoid arthritis.Arthr Rheum.1993;36(7):901-910.
[66]Piemonti L,Bernasconi S,Luini W,et al.IL-13 supports differentiation of dendritic cells from circulating precursors in concert with GM-CSF.Eur Cytokine Netw.1995;6(4):245-252.
[67]MacPherson GG,Jenkins CD,Stein MJ,et al.Endotoxin-mediated dendritic cell release from the intestine.Characterization of released dendritic cells and TNF dependence.J Immunol.1995;154(3):1317-1322.
[68]Santiago Schwarz F,Sullivan C,Rappa D,et al.Distinct alterations in lineage committed progenitor cells exist in the peripheral blood of patients with rheumatoid arthritis and primary Sjogren's syndrome.J Rheumatol.1996;23(3):439-446.
[69]Thomas R,Lipsky PE.Could endogenous self-peptides presented by dendritic cells initiate rheumatoid arthritis? Immunol Today.1996;17(12):559-564.
[70]MacDonald KP,Nishioka Y,Lipsky PE,et al.Functional CD40-ligand is expressed by T cells in rheumatoid arthritis.J Clin Invest.1997;100(9):2404-2414.
[71]Dittel BN,Visintin I,Merchant RM,et al.Presentation of the self antigen myelin basic protein by dendritic cells leads to experimental autoimmune encephalomyelitis.J Immunol.1999;163:32-39.
[72]Thomas R,Davis LS,Lipsky PE.Rheumatoid synovium is enriched in mature antigen-presenting dendritic cells.J Immunol.1994;152:2613-2623.
[73]Page G,Lebecque S,Miossec P.Anatomic localization of immature and mature dendritic cells in an ectopic lymphoid organ:correlation with selective chemokine expression in rheumatoid synovium.J Immunol.2002;168:5333-5341.
[74]Cavanagh LL,Boyce A,Smith L,et al.Rheumatoid arthritis synovium contains plasmacytoid dendritic cells.Arthritis Res Ther.2005;7:R230-R240.
[75]Steinman RM,Pack M,Inaba K.Dendritic cells in the T-cell areas of lymphoid organs.Immunol Rev.1997;156:25-37.
[76]Guermonprez P,Valladeau J,Zitvogel L,et al.Antigen presentation and T cell stimulation by dendritic cells.Annu Rev Immunol.2002;20:621-667.
[77]Liu YJ.Dendritic cell subsets and lineages,and their functions in innate and adaptive immunity.2001;106(3):259-62.
[78]Mc William AS,Napoli S,Marsh AM,et al.Dendritic cells are recruited into the airway epithelium during the inflammatory response to a broad spectrum of stimuli.J Exp Med.1996;184(6):2429-32.
[79]Cook DN,Prosser DM,Forster R? et al.CCR6 mediates dendritic cell localization,lymphocyte homeostasis,and immune responses in mucosal tissue.Immunity.2000;12(5):495-503.
[80]Steinman RM,Turley S,Mellman I,et al.The induction of tolerance by dendritic cells that have captured apoptotic cells.J Exp Med.2000 Feb 7;191(3):411-416.
[81]Shortman K,Liu YJ.Mouse and human dendritic cell subtypes.Nat Rev Immunol.2002 Mar;2(3):151-61.
[82]Kadowaki N,Ho S,Antonenko S,et al.Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens.J Exp Med.2001 Sep 17;194(6):863-9.
[83]Jahnsen FL,Lund-Johansen F,Dunne JF,et al.Experimentally induced recruitment of plasmacytoid (CD123high) dendritic cells in human nasal allergy.J Immunol.2000 Oct l;165(7):4062-8.
[84]Rossi D,Zlotnik A.The biology of chemokines and their receptors.Annu Rev Immunol.2000;18:217-42.
[85]Vanbervliet B,Homey B,Durand I,et al.Sequential involvement of CCR2 and CCR6 ligands for immature dendritic cell recruitment:possible role at inflamed epithelial surfaces.Eur J Immunol.2002;32(1):231-42.
[86]Dieu MC,Vanbervliet B,Vicari A,et al.Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites. J Exp Med. 1998;188(2):373-86.
[87] Gunn MD, Tangemann K, Tarn C, et al. A chemokine expressed in lymphoid high endothelial venules promotes the adhesion and chemotaxis of naive T lymphocytes. Proc Natl Acad Sci USA. 1998;95(1):258-63.
[88] Nakano H, Tamura T, Yoshimoto T, et al. Genetic defect in T lymphocyte-specific homing into peripheral lymph nodes. Eur J Immunol. 1997;27(1):215-21.
[89] Gunn MD, Kyuwa S, Tam C, et al. Mice lacking expression of secondary lymphoid organ chemokine have defects in lymphocyte homing and dendritic cell localization. J Exp Med. 1999;189(3):451-60.
[90] Mori S, Nakano H, Aritomi K, et al. Mice lacking expression of the chemokines CCL21-ser and CCL19 (pit mice) demonstrate delayed but enhanced T cell immune responses. J Exp Med. 2001;193(2):207-18.
[91] Forster R, Schubel A, Breitfeld D, et al. CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell. 1999;99(1):23-33.
[92] 李时珍.本草纲目.第1版,第18卷,草目部.北京:北京人民出版社,1977;31340.
[93] 刘东平.青藤碱的研究进展.安徽医药.2005;9(11):859-860.
[94] 王文君,王培训,李晓娟,等.青藤碱抗炎机制-青藤碱对人外周血单个核细胞环氧化酶活性及其基因表达的影响.中国中药杂志.2003;28(4):352-356.
[95] 涂胜豪,胡永红,陆付耳.青藤碱对人淋巴细胞产生IL-2,IL-2R和IL-6的影响.中国实验临床免疫学杂志.1998;10(5):268-269.
[96] 王勇,方勇飞,周新,等.青藤碱对佐剂性关节炎大鼠腹腔巨噬细胞表达细胞因子的影响.中华风湿病杂志.2003;7(7):415-418.
[97] 刘良,李晓娟,王培训,等.青藤碱对人外周血单个核细胞IL-1β、IL-8细胞因子基因表达的影响.中国免疫学杂志.2002;18(4):241-244.
[98] 李晓娟,王培训,刘良,等.青藤碱对T淋巴细胞活化及TH1类细胞内细胞因子表达的影响.中国免疫学杂志.2004;20:249-252.
[99] 徐琳本,邱赛红.正清风痛宁对免疫作用影响的实验研究.湖南中医杂志.1996;12(2):47-48.
[100] 戴英波,黄循,罗志刚,等.青藤碱对大鼠肾移植排斥反应时细胞间黏附分子-1表达的抑制作用.现代中西医结合杂志.2003;12(13):1358-1361.
[101] 王毅,陈正,熊烈,等.青藤碱对肾移植大鼠急性排斥反应及T细胞增殖的影响.中华实验外科杂志.2004;21(5):573-574.
[102] 孔庆峰,李军.青藤碱研究新进展.中国中医杂志.2005;30(20):1573-1576.
[103] 罗烈,杨剑,宋金春,等.苯甲酰青藤碱的镇痛抗炎作用.中国医院药学杂志.2005;25(3):196-198.
[104] 王晓洪,邱赛红,董绍象.青藤碱片的药效学研究.中药药理与临床.1997;13(4):23.
[105] 刘继红,李卫东,藤慧玲,等.青藤碱治疗类风湿关节炎免疫作用和机制.药学学报.2005;40(2):127-131.
[106] 杨德森,刘芳,曾繁典,等.青藤碱对大鼠佐剂性关节炎治疗作用及机制的研究.中国中药杂志.2005;30(17):1361-1363.
[107] Pap T, Aupperle KR, Gay S, et al. Invasiveness of synovial fibroblasts is regulated by P53 in the SCID morse in vivo model of cartilage invasion. Arthritis Rheum. 2001; 44:676-681.
[108] 陈光星,刘良,赵诗哲,等.青藤碱对胶原诱导型关节大鼠滑膜细胞增殖及凋亡影响的研究.中华风湿病学杂志.2005;9(5):284-287.
[109] 易丹丹,蔡鸿生,罗顺德,等.藤碱凝胶对大鼠佐剂性关节炎的治疗作用.中国医院药学杂志.2002;22(8):465-468.
[110] 刘晓玲,陈光星,李晓娟,等.青藤碱对Ⅱ型胶原诱导关节炎大鼠滑膜炎症的抑制作用及其机理探讨.广州中医药大学学报.2002;19(3):214-217.
[111] Zhao Y, Li J, Yu K, et al. Sinomenine inhibits maturation of monocyte-derived dendritic cells through blocking activation of NF-kappa B. Int Immunopharmacol. 2007;7(5):637-645.
[112] 赵毅,余克强,李娟.青藤碱对类风湿关节炎病人树突状细胞免疫活性的影响.中国药学杂志.2006;41(20):1543-1545.
[113] 赵毅,余克强,李娟.青藤碱对类风湿关节炎树突状细胞核转录因子-кB活性的影响.广东医学.2006;27(1):55-57.
[114] 余克强,赵毅,李娟.青藤碱对类风湿关节炎树突状细胞CD80、CD86mRNA表达的影响.中国药房.2006;17(5):331-333.
[115] Robbiani DF, Finch RA, Jager D, Muller WA, Sartorelli AC, Randolph GJ. The leukotriene C (4) transporter MRP1 regulates CCL19 (MIP-3beta, ELC) -dependent mobilization of dendritic cells to lymph nodes. Cell. 2000; 103: 757-768.
[116] Baggiolini M. Chemokines and leukocyte traffic. Nature. 1998; 392: 565-568.
[117] Rossi D, Zlotnik A. The Biology of Chemokines and Their Receptors. Annual Reviews. 2000; 18: 217-243.
[118] Ardavin C. Thymic dendritic cells. Immunol Today. 1997; 18: 350-361.
[119] Kappler JW, Roehm N, Marrack P. T cell tolerance by clonal elimination in the thymus. Cell. 1987; 49:273-280.
[120] Sakaguchi N, Takahashi T, Hata H, et al. Altered thymic T-cell selection due to a mutation of the ZAP-70 gene causes autoimmune arthritis in mice. Nature. 2003; 426:454-460.
[121] Drakesmith H, O'Neil D, Schneider SC, et al. In vivo priming of T cells against cryptic determinants by dendritic cells exposed to interleukin 6 and native antigen. Proc Natl Acad Sci USA. 1998; 95:14903-14908.
[122] Kuhn KA, Kulik L, Tomooka B, et al. Antibodies against citrullinated proteins enhance tissue injury in experimental autoimmune arthritis. J Clin Invest 2006;l16:961-973.
[123] van Gaalen FA, van Aken J, Huizinga TW, et al. Association between HLA class Ⅱ genes and autoantibodies to cyclic citrullinated peptides (CCPs) influences the severity of rheumatoid arthritis. Arthritis Rheum. 2004; 50: 2113-2121.
[124] Hill JA, Southwood S, Sette A, et al. Cutting edge: the conversion of arginine to citrulline allows for a high-affinity peptide interaction with the rheumatoid arthritis-associated HLA-DRB1*0401 MHC class Ⅱ molecule.J Immunol.2003;171:538-541.
[125]Dessen A,Lawrence CM,Cupo S,et al.X-ray crystal structure of HLA-DR4 (DRA*0101,DRB1*0401) complexed with a peptide from human collagen Ⅱ.Immunity.1997;7:473-481.
[126]Michaelsson E,Holmdahl M,Engstrom A,et al.Macrophages,but not dendritic cells,present collagen to T cells.Eur J Immunol.1995;25:2234-2241.
[127]Yoshida M,Tsuji M,Kurosaka D,et al.Autoimmunity to citrullinated type Ⅱ collagen in rheumatoid arthritis.Mod Rheumatol.2006;16:276-281.
[128]Binstadt BA,Patel PR,Alencar H,et al.Particularities of the vasculature can promote the organ specificity of autoimmune attack.Nat Immunol.2006;7:284-292.
[129]Baeten D,Steenbakkers PG,Rijnders AM,et al.Detection of major histocompatibility complex/ human cartilage gp-39 complexes in rheumatoid arthritis synovitis as a specific and independent histologic marker.ArthritisRheum.2004;50:444-451.
[130]Thomas R,Quinn C.Functional differentiation of dendritic cells in rheumatoid arthritis:role of CD86 in the synovium.J Immunol.1996;156:3074-3086.
[131]Highton J,Kean A,Hessian PA,et al.Cells expressing dendritic cell markers are present in the rheumatoid nodule.J Rheumatol.2000;27:339-346.
[132]Summers KL,O'Donnell JL,Heiser A,et al.Synovial fluid transforming growth factor beta inhibits dendritic cell-T lymphocyte interactions in patients with chronic arthritis.Arthritis Rheum.1999;42:507-518.
[133]Fan L,Reilly CR,Luo Y,et al.Cutting edge:ectopic expression of the chemokine TCA4/SLC is sufficient to trigger lymphoid neogenesis.J Immunol.2000;164:3955-3959.
[134]Nanki T,Urasaki Y,Imai T,et al.Inhibition of fractalkine ameliorates murine collagen-induced arthritis.J Immunol.2004;173:7010-7016.
[135]van Lieshout AW,van der Voort R,le Blanc LM,et al.Novel insights in the regulation of CCL18 secretion by monocytes and dendritic cells via cytokines,toll-like receptors and rheumatoid synovial fluid.BMC Immunol.2006;7:23.
[136]Shortman K,Naik SH.Steady-state and inflammatory dendritic-cell development.Nat Rev Immunol.2007;7:19-30.
[137]Allan RS,Smith CM,Belz GT,et al.Epidermal viral immunity induced by CD8alpha+ dendritic cells but not by Langerhans cells.Science.2003;301:1925-1928.
[138]Varol C,Landsman L,Fogg DK,et al.Monocytes give rise to mucosal,but not splenic,conventional dendritic cells.J Exp Med.2007;204:171-180.
[139]Qu C,Edwards EW,Tacke F,et al.Role of CCR8 and other chemokine pathways in the migration of monocytederived dendritic cells to lymph nodes.J Exp Med.2004;200:1231-1241.
[140]Vermaelen KY,Carro-Muino I,Lambrecht BN,et al.Specific migratory dendritic cells rapidly transport antigen from the airways to the thoracic lymph nodes.J Exp Med.2001;193:51-60.
[141]Kurts C,Kosaka H,Carbone FR,et al.Class I-restricted cross-presentation of exogenous self-antigens leads to deletion of autoreactive CD8(+) T cells.J Exp Med.1997;186:239-245.
[142]Serbina NV,Pamer EG.Monocyte emigration from bone marrow during bacterial infection requires signals mediated by chemokine receptor CCR2.Nat Immunol.2006;7:311-317.
[143]Tacke F,Alvarez D,Kaplan TJ,et al.Monocyte subsets differentially employ CCR2,CCR5,and CX3CR1 to accumulate within atherosclerotic plaques.J ClinInvest.2007;117:185-94.
[144]Bachmann MF,Kopf M,Marsland BJ.Chemokines:more than just road signs.Nat Rev Immunol.2006;6:159-164.
[145]Sallusto F,Schaerli P,Loetscher P,et al.Rapid and coordinated switch in chemokine receptor expression during dendritic cell maturation.Eur J Immunol.1998;28:2760-2769.
[146]Balashov KE,Rottman JB,Weiner HL,et al.CCR5(+) and CXCR3(+) T cells are increased in multiple sclerosis and their ligands MIP-1 alpha and IP-10 are expressed in demyelinating brain lesions.Proc.Natl.Acad.Sci.U.S.A.1999;96:6873-6878.
[147]Sallusto F,Mackay CR,Lanzavecchia A.Selective expression of the eotaxin receptor CCR3 by human T helper 2 cells.Science.1997;277:2005-2007.
[148]Gombert M,Dieu-Nosjean MC,Winterberg F,et al.CCL1-CCR8 interactions:an axis mediating the recruitment of T cells and Langerhans-type dendritic cells tosites of atopic skin inflammation.J Immunol.2005;174:5082-5091.
[149]Harrington PM,Newton DJ,Williams CM,et al.Eotaxin and eotaxin receptor (CCR3) expression in Sephadex particle-induced rat lung inflammation.Int J Exp Pathol.1999;80:177-185.
[150]Andrew DP,Ruffing N,Kim CH,et al.C-C chemokine receptor 4 expression defines a major subset of circulating nonintestinal memory T cells of both Thl and Th2 potential.J Immunol.2001;166:103-111.
[151]Sallusto F,Lenig D,Forster R,et al.Lanzavecchia:Two subsets of memory T lymphocytes with distinct homing potentials and effector functions.Nature.1999;401:708-712.
[152]Ginhoux F,Tacke F,Angeli V,et al.Langerhans cells arise from monocytes in vivo.Nat Immunol.2006;7:265-273.
[153]Nagasawa T,Kikutani H,Kishimoto T.Molecular cloning and structure of a pre-B-cell growthstimulating factor.Proc.Natl.Acad.Sci.U.S.A.1994;91:2305-2309.
[154]Peled A,Kollet O,Ponomaryov T,et al.The chemokine SDF-1 activates the integrins LFA-1,VLA-4,and VLA-5 on immature human CD34(+) cells:role in transendothelial/stromal migration and engraftment of NOD/SCID mice.Blood.2000;95:3289-3296.
[155]Sanz-Rodriguez F,Hidalgo A,Teixido J.Chemokine stromal cell-derived factor-1 alpha modulates VLA-4 integrin-mediated multiple myeloma cell adhesion to CS-1 /fibronectin and VCAM-1.Blood.2001;97:346-351.
[156]Zernecke A,Liehn EA,Gao JL,et al.Deficiency in CCR5 but not CCR1 protects against neointima formation in atherosclerosis-prone mice:involvement of IL-10.Blood.2006;107:4240-4243.
[157]Baltus T,Weber KS,Johnson Z,et al.Oligomerization of RANTES is required for CCR1 -mediated arrest but not CCR5-mediated transmigration of leukocytes on inflamed endothelium.Blood.2003;102:1985-1988.
[158]Oppermann M.Chemokine receptor CCR5:insights into structure,function,and regulation.Cell Signal.2004;16:1201-1210.
[159]Yurchenko E,Tritt M,Hay V,et al.CCR5-dependent homing of naturally occurring CD4+ regulatory T cells to sites of Leishmania major infection favors pathogen persistence.J Exp Med.2006;203:2451-2460.
[160]Floto RA,MacAry PA,Boname JM,et al.Dendritic cell stimulation by mycobacterial Hsp70 is mediated through CCR5.Science.2006;314:454-458.
[161]Smith CM,Wilson NS,Waithman J,et al.Cognate CD4(+) T cell licensing of dendritic cells in CD8(+) T cell immunity.Nat Immunol.2004;5:1143-1148.
[162]Castellino F,Huang AY,Altan-Bonnet G,et al.Chemokines enhance immunity by guiding naive CD8+ T cells to sites of CD4+ T cell-dendritic cell interaction.Nature.2006;440:890-895.
[163]Jin Y,Shen L,Chong EM,et al.The chemokine receptor CCR7 mediates corneal antigen-presenting cell trafficking.Mol Vis.2007;13:626-634.
[164]Lo JC,Chin RK,Lee Y,et al.Differential regulation of CCL21 in lymphoid/nonlymphoid tissues for effectively attracting T cells to peripheral tissues.J Clin Invest.2003;112:1495-1505.
[165]Luther SA,Tang HL,Hyman PL,et al.Coexpression of the chemokines ELC and SLC by T zone stromal cells and deletion of the ELC gene in the plt/plt mouse.Proc.Natl.Acad.Sci.U.S.A.2000;97;12694-1269.
[166]Czeloth N,Bernhardt G,Hofmann F,et al.Sphingosine-1-phosphate mediates migration of mature dendritic cells.J Immunol.2005;175;2960-2967.
[167]Sanchez-Sanchez N,Riol-Blanco L,de la Rosa G,et al.Chemokine receptor CCR7 induces intracellular signaling that inhibits apoptosis of mature dendritic cells.Blood.2004;104:619-625.
[168]Flanagan K,Moroziewicz D,Kwak H,et al.The lymphoid chemokine CCL21 costimulates naive T cell expansion and Th1 polarization of non-regulatory CD4+ T cells.Cell Immunol.2004;231:75-84.
[169]Marsland BJ,Battig P,Bauer M,et al.CCL19 and CCL21 induce a potent proinflammatory differentiation program in licensed dendritic cells.Immunity.2005;22:493-505.
[170]Lasagni L,Francalanci M,Annunziato F,et al.An alternatively spliced variant of CXCR3 mediates the inhibition of endothelial cell growth induced by IP-10,Mig,and I-TAC,and acts as functional receptor for platelet factor 4.J Exp Med.2003;197:1537-1549.
[171]Qian C,An H,Yu Y,et al.TLR agonists induce regulatory dendritic cells to recruit Thl cells via preferential IP-10 secretion and inhibit Thl proliferation.Blood.2007;109:3308-3315.
[172]Molesworth-Kenyon S,Mates A,Yin R,et al.CXCR3,IP-10,and Mig are required for CD4+ T cell recruitment during the DTH response to HSV-1 yet are independent of the mechanism for viral clearance.Virology.2005;333:1-9.
[173]Guarda G,Hons M,Soriano SF,et al.L-selectin-negative CCR7(-) effector and memory CD8(+) T cells enter reactive lymph nodes and kill dendritic cells.Nat Immunol.2007;8:743-752.
[174]Loetscher P,Pellegrino A,Gong JH,et al.The ligands of CXC chemokine receptor 3,I-TAC,Mig,and IP 10,are natural antagonists for CCR3.J Biol Chem.2001;276:2986-2991.
[175]Gonzalez-Cuadrado S,Bustos C,Ruiz-Ortega M,et al.Expression of leucocyte chemoattractants by interstitial renal fibroblasts:up-regulation by drugs associated with interstitial fibrosis.Clin Exp Immunol.1996;106:518-522.
[176]Hyun JG,Lee G,Brown JB,et al.Anti-interferoninducible chemokine,CXCL10,reduces colitis by impairing T helper-1 induction and recruitment in mice.Inflamm Bowel Dis.2005;11:799-805.
[177]Bogyo M,Shin S,McMaster JS,et al.Substrate binding and sequence preference of the proteasome revealed by active-site-directed affinity probes.Chem Biol. 1998;5:307-320.
[178]Hershko A,Ciechanover A.The ubiquitin system.Annu Rev Biochem.1998;67:425-479.
[179]Goldberg AL.Protein degradation and protection against misfolded or damaged proteins.Nature.2003;426:895-899.
[180]Ciechanover A.The ubiquitin-proteasome proteolytic pathway.Cell.1994;79:13-21
[181]Adams J.The proteasome:structure,function,and role in the cell.Cancer Treat Rev.2003;29 Suppl 1:3-9.
[182]Rock KL,Goldberg AL.Degradation of cell proteins and the generation of MHC class Ⅰ-presented peptides.Annu Rev Immunol.1999;17:739-779.
[183]Rock KL,Gramm C,Rothstein L,et al.Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class Ⅰ molecules.Cell.1994;78:761-771.
[184]Cascio P,Call M,Petre BM,et al.Properties of the hybrid form of the 26S proteasome containing both 19S and PA28 complexes.EMBO J.2002;21:2636-2645.
[185]Cascio P,Hilton C,Kisselev AF,et al.26S proteasomes and immunoproteasomes produce mainly N-extended versions of an antigenic peptide.EMBO J.2001;20:2357-2366.
[186]Strehl B,Seifert U,Kruger E,et al.Interferon-γ,the functional plasticity of the ubiquitinproteasome system,and MHC class Ⅰ antigen processing.Immunol Rev.2005;207:19-30.
[187]Goldberg AL,Cascio P,Saric T,et al.The importance of the proteasome and subsequent proteolytic steps in the generation of antigenic peptides.Mol Immunol.2002;39:147-164.
[188]York IA,Goldberg AL,Mo XY,et al.Proteolysis and class Ⅰ major histocompatibility complex antigen presentation.Immunol Rev.1999;172:49-66.
[189]Zhang J,Bardos T,Li D,et al.Cutting edge:regulation of T cell activation threshold by CD28 costimulation through targeting Cbl-P for ubiquitination.J Immunol.2002;169:2236-2240.
[190]Qureshi N,Vogel SN,Van Way C,et al.a central regulator of inflammation and macrophage function.Immunol Res.2005;31:243-260.
[191]Matsushita M,Takasaki Y,Takeuchi K,et al.Autoimmune response to proteasome activator 28a in patients with connective tissue diseases.J Rheumatol.2004;31:252-259.
[192]Mountz JD.Significance of increased circulating proteasome in autoimmune disease.J Rheumatol.2002;29:2027-2030.
[193]Egerer K,Kuckelkorn U,Rudolph PE,et al.Circulating proteasomes are markers of cell damage and immunologic activity in autoimmune diseases.J Rheumatol.2002;29:2045-2052.
[194]Firestein GS.NF-kB:Holy Grail for rheumatoid arthritis? Arthritis Rheum.2004;50:2381-2386.
[195]Elliott PJ,Zollner TM,Boehncke WH.Proteasome inhibition:a new anti-inflammatory strategy.J Mol Med.2003;81:235-245.
[196]Niu H,Cattoretti G,Dalla-Favera R.BCL6 controls the expression of the B7-1/CD80 costimulatory receptor in germinal center B cells.J Exp Med.2003;198(2):211-221.
[197]Hofer S,Rescigno M,Granucci F,et al.Differential activation of NF-kappa B subunits in dendritic cells in response to Gram-negative bacteria and to lipopolysaccharide.Microbes Infect.2001;3(4):259-265.
[198]Pettit AR,Quinn C,MacDonald KP,et al.Nuclear localization of RelB is associated with effective antigen presenting cell function.J Immunol.1997;159(8):3681-3691.
[199]Ouaaz F,Arron J,Zheng Y,et al.Dendritic cell development and survival require distinct NF-kB subunits.Immunity.2002;16(2):257-270.
[200]Zheng Y,Ouaaz F,Bruzzo P,et al.NF-kappa B RelA(p65) is essential for TNF-alpha-induced fas expression but dispensable for both TCR-induced expression and activation-induced cell death.J Immunol.2001;166(8):4949-4957.
[201]Speirs K,Lieberman L,Caamano J,et al.Cutting edge:NF-kappa B2 is a negative regulator of dendritic cell function.J Immunol.2004;172(2):752-756.
[202]Ghosh S,May MJ,Kopp EB.NF-kappa B and Rel proteins:evolutionarily conserved mediators of immune respones.Annu Rev Immunol.1998;16:225-260.
[203]Tanaka K,Tsurumi C.The 26S proteasome:subunits and functions.Mol Biol Rep.1997;24(1-2):3-11.
[204]Yaron A,Gonen H,Alkalay I,et al.Inhibition of NF-kappa-B cellular function via specific targeting of the I-kappa-B-ubiquitin ligase.EMBO J.1997;16(21):6486-94.
[205]Marok R,Winyard PG,Coumbe A,et al.Activation of the transcription factor nuclear factor-kappaB in human inflamed synovial tissue.Arthritis Rheum.1996;39(4):583-91.
[206]Rodriguez MS,Michalopoulos I,Arenzana-Seisdedos F,et al.Inducible degradation of I kappa B alpha in vitro and in vivo requires the acidic C-terminal domain of the protein.Mol Cell Biol.1995;15(5):2413-9.
[207]Kroll M,Conconi M,Desterro MJ.The carboxy-terminus of I kappaB alpha determines susceptibility to degradation by the catalytic core of the proteasome.Oncogene.1997;15(15):1841-50.
[208]Antonelli A,Crinelli R,Bianchi M,et al.Efficient inhibition of macrophage TNF-alpha production upon targeted delivery of K48R ubiquitin.Br J Haematol.1999;104(3):475-81.
[209]Palombella VJ,Conner EM,Fuseler JW,et al.Role of the proteasome and NF-kappaB in streptococcal cell wall-induced polyarthritis.Proc Natl Acad Sci U SA.1998;95(26):15671-15676.
[2]Drosos AA.Epidemiology of rheumatoid arthritis.Autoimmun Rev.2004;3 Suppl 1:S20-22.
[3]Terlman H,Pagliari LJ,Liu H,et al.Rheumatoid arthritis synovial macrophages express the Fas-associated death domain-like interleukin-1β-converting enzyme inhibitory protein and are refractory to Fas-mediated apoptosis.Arthritis rheum.2001 ,44:21-30.
[4]Yocum DE.T cells:pathogenic cells and therapeutic targets in rheumatoid arthritis.Semin Arthritis Rheum.1999;29(1):27-35.
[5]Hang HG,Wang YM,Xie JF,et al.Regulation of tumor necrosis factora-mediated apoptosis of rheumatoid arthritis synovial fibroblasts by the protein kinase Akt.Arthritis rheum.2001;44:1555-1567.
[6]Black RA,Rauch CT,Kozlosky CJ,et al.A metalloproteinase disintegrin that releases tumour-necrosis factor-alpha from cells.Nature.1997;385(6618):729-733.
[7]Nawroth PP,Bank I,Handley D,et al.Tumor necrosis factor/cachectin interacts with endothelial cell receptors to induce release of interleukin 1.J Exp Med.1986;163(6):1363-1375.
[8]Chin JE,Winterrowd GE,Krzesicki RF,et al.Role of cytokines in inflammatory synovitis:the coordinate regulation of intercellular adhesion molecule 1 and HLA class Ⅰ and class Ⅱ antigens in rheumatoid synovial fibroblasts.Arthritis Rheum.1990;33(12):1776-1786.
[9]Butler DM,Maini RN,Feldmann M,et al.Modulation of proinflammatory cytokine release in rheumatoid synovial membrane cell cultures.Comparison of monoclonal anti TNF-alpha antibody with the interleukin-1 receptor antagonist.Eur Cytokine Netw.1995;6(4):225-230.
[10]Keffer J,Probert L,Cazlaris H,et al.Transgenic mice expressing human turnout necrosis factor:a predictive genetic model of arthritis.EMBO J.1991;10(13):4025-4031.
[11]Wooley PH,Dutcher J,Widmer MB,et al.Influence of a recombinant human soluble tumor necrosis factor receptor Fc fusion protein on type Ⅱ collagen-induced arthritis in mice.J Immunol.1993;151(11):6602-6607.
[12]Williams RO,Feldmann M,Maini RN.Anti-tumor necrosis factor ameliorates joint disease in marine collagen-induced arthritis.Proc Natl Acad Sci USA.1992;89(20):9784-9788.
[13]Koch AE,Kunkel SL,Strieter RaM.Cytokines in rheumatoid arthritis.J Invest Med.1995;43(1):28-38.
[14]Arend WP,Dayer JM.Inhibition of the production and effects of interleukin-1 and tumor necrosis factor alpha in rheumatoid arthritis.Arthritis Rheum.1995;38(2):151-160.
[15]MacNaul KL,Chartrain N,Lark M,et al.Discoordinate expression of stromelysin,collagenase,and tissue inhibitor of metalloproteinases-1 in rheumatoid human synovial fibroblasts:synergistic effects of interleukin-1 and tumor necrosis factor-alpha on stromelysin expression.J Biol Chem.1990;265(28):17238-17245.
[16]Strand V,Kavanaugh AF.The role of interleukin- 1 in bone resorption in rheumatoid arthritis.Rheumatology.2004;43(Suppl-3):Ⅲ10-Ⅲ16.
[17]Dayer J M.The pivotal role of interleukin- 1 in the clinical manifestations of rheumatoid arthritis.Rheumatology(Oxford).2003;42(Suppl 2):3-10.
[18]Mosmann TR,Sad S.The expanding universe of T-cell subsets:Th1,Th2 and more.Immunol Today.1996;17(3):138-146.
[19]Aarvak T,Chabaud M,Miossec P,et al.IL-17 is produced by some proinflammatory Thl/ThO cells but not by Th2 cells.J Immunol.1999;162(3):1246-1251.
[20]Romagnani S.Regulation of the development of type 2 T-helper cells in allergy.Curr Opin Immunol.1994;6(6):838-846.
[21]Yudoh K,Matsuno H,Osada R,et al.Decreased cellular activity and replicative capacity of osteoblastic cells isolated from the periarticular bone of rheumatoid arthritis patients compared with osteoarthritis patients. Arthritis Rheum. 2000;43(10):2178-2188.
[22] Shimoyama Y, Nagafuchi H, Suzuki N, et al. Synovium infiltrating T cells induce excessive synovial cell function through CD28/B7 pathway in patients with rheumatoid arthritis. J Rheumatol. 1999;26(10):2094-2101.
[23] 张利宁,曹英林,赵文璞,等.CD137在SLE和RA病人外周血T细胞上的表达特点及意义.中华微生物学和免疫学杂志.2000;20(2):126-127.
[24] El Ewau TD, De Keyser F, De Wever N, et al. A comparative phenotypical analysis of rheumatoid nodules and rheumatoid synovium with special reference to adhesion molecules and activation markers. Ann Rheum Dis. 1998; 57(8): 480-486.
[25] Miltenburg AM, van Laar JM, de Kuiper R, et al. T cells cloned from human rheumatoid synovial membrance functionally represent the Th1 subset. Scand J Immunol. 1992;35(5):603-610.
[26] Quayle AJ, Chomarat P, Miossec P, et al. Rheumatoid inflammatory T-cell clones express mostly Thl but also Th2 and mixed (Th0-like) cytokine patterns. Scand J Immunol. 1993;38(1):75-82.
[27] Davis LS, Cush JJ, Schulze-Koops H, et al. Rheumatoid synovial CD4+T cells exhibit a reduced capacity to differentiate into IL-4-producing T-helper-2 effector cells. Arthritis Res. 2001;3(1):54-64.
[28] Park W, Weyand CM, Schmidt D, et al. Co-stimulatory pathways controlling activation and perpheral tolerance of human CD4+CD28-T cell. Eur J Immunol. 1997;27(5):1082-1090.
[29] Josien R, Wong BR, Li HL, et al. TRANCE, a TNF family member, is differentially expressed on T cell subsets and induces cytokine production in dendritic cells. J Immunol. 1999;162(5):2562-2568.
[30] Ross FP. Interleukin 7 and estrogen-induced bone loss. Trends Endocrinol Metab. 2003;14(4):147-149.
[31] Banchereau J, Briere F, Caux C, et al. Immunobiology of dendritic cells. Annu Rev Immunol.2000;18:767-811.
[32]Steinman RM,Cohn ZA.Identification of a novel cell type in peripheral lymphoid organs of mice.I.Morphology,quantitation,tissue distribution.J Exp Med.1973;137(5):1142-1162.
[33]Banchereau J,Steinman RM.Dendritic cells and the control of immunity.Nature.1998;392:245-252.
[34]Kitamura H,Iwakabe K,Yahata T,et al.The natural killer T (NKT) cell ligand alpha-galactosylceramide demonstrates its immunopotentiating effect by inducing interleukin (IL)-12 production by dendritic cells and IL-12 receptor expression on NKT cells.J Exp Med.1999;189:1121-1128.
[35]Brocker T.Survival of mature CD4 T lymphocytes is dependent on major histocompatibility complex class Ⅱ-expressing dendritic cells.J Exp Med.1997;186:1223-1232.
[36]Lutzky V,Thomas R:Dendritic cells.Contemporary Targeted Therapies in Rheumatology.Edited by Smolen JS,Lipsky PE.London:Informa Healthcare;2007:63-78.
[37]Marquez C,Trigueros C,Fernandez E,et al.The development of T and non-T-cell lineages from CD34+human thymic precursors can be traced by the differential expression of CD44.J Exp Med.1995;181(2):475-483.
[38]O'Keeffe M,Hochrein H,Vremec D,et al.Dendritic cell precursor populations of mouse blood:identification of the murine homologues of human blood plasmacytoid pre-DC2 and CD11c+ DC1 precursors.Blood.2003;101(4):1453-1459.
[39]Mohty M,Isnardon D,Blaise D,et al.Identification of precursors of leukemic dendritic cells differentiated from patients with acute myeloid leukemia.Leukemia.2002;16(11):2267-2274.
[40]Itano AA,McSorley SJ,Reinhardt RL,et al.Distinct dendritic cell populations sequentially present antigen to CD4 T cells and stimulate different aspects of cell-mediated immunity.Immunity.2003;19( 1 ):47-57.
[41]Ardav(?)n C.Thymic dendritic cells.Immunol Today.1997;18(7):350-361.
[42]Dauer M,Obermaier B,Herten J,et al.Mature dendritic cells derived from human monocytes within 48 hours:a novel strategy for dendritic cell differentiation from blood precursors.J Immunol.2003;170(8):4069-4076.
[43]Merrick A,Errington F,Milward K,et al.Immunosuppressive effects of radiation on human dendritic cells:reduced IL-12 production on activation and impairment of naive T-cell priming.Br J Cancer.2005;92(8):1450-1458.
[44]Dustin ML,Cooper JA.The immunological synapse and the actin cytoskeleton:molecular hardware for T cell signaling.Nat.Immunol.2000;1(1):23-29.
[45]Pettit AR,MacDonald KPA,O'Sullivan B,et al.Differentiated dendritic cells expressing nuclear RelB are predominantly located in rheumatoid synovial tissue perivascular mononuclear cell aggregates.Arthritis Rheum.2000;43:791-800.
[46]Steinman RM,Swanson J.The endocytic activity of dendritic cells.J Exp Med.1995;182:283-288.
[47]Sallusto F,Palermo B,Lenig D,et al.Distinct patterns and kinetics of chemokine production regulate dendritic cell function.Eur J Immunol.1999.29:1617-1625.
[48]Sallusto F,Lanzavecchia A.Understanding dendritic cell and Tlymphocyte traffic through the analysis of chemokine receptor expression.Immunol Rev.2000.177:134-140.
[49]Hermans IF,Ritchie DS,Yang J,et al.CD8+ T cell-dependent elimination of dendritic cells in vivo limits the induction of antitumor immunity.J Immunol.2000.164:3095-3101.
[50]Kalinski P,Vieira P,Schuitemaker JH,et al.Generation of human type 1- and type 2-polarized dendritic cells from peripheral blood.Methods Mol Biol.2003.215:427-436.
[51]Steptoe RJ,Ritchie JM,Jones LK,et al.Autoimmune diabetes is suppressed by transfer of proinsulin-encoding Gr-1+ myeloid progenitor cells that differentiate in vivo into resting dendritic cells.Diabetes.2005;54:434-442.
[52]Sallusto F,Lanzavecchia A.Mobilizing dendritic cells for tolerance,priming and chronic inflammation.J Exp Med.1999;189:611-614.
[53]Yan M,Peng J,Jabbar IA,et al.Activation of dendritic cells by human papillomavirus-like particlesthrough TLR4 and NF-kappaB-mediated signalling,moderated by TGF-beta.Immunol Cell Biol.2005;83:83-91.
[54]Reise Sousa C.Dendritic cells in a mature age.Nat Rev Immunol.2006;6:476-483.
[55]Siebenlist U,Franzoso G,Brown K.Structure,regulation and function of NF-kappa B.Annu Rev Cell Biol.1994;10:405-455.
[56]O'Sullivan B,Thompson AG,Thomas R:NF-kappa B as a therapeutic target in autoimmune disease.Curr Opin Ther Targets 2007,11:111-122.
[57]Bondeson J,Foxwell B,Brennan F,et al.Defining therapeutic targets by using adenovirus:blocking NF-kappaB inhibits both inflammatory and destructive mechanisms in rheumatoid synovium but spares anti-inflammatory mediators.Proc Natl Acad Sci USA.1999;96:5668-5673.
[58]Koch AE,Burrows JC,Haines GK,et al.Immunolocalization of endothelial and leukocyte adhesion molecules in human rheumatoid and osteoarthritic synovial tissues.Lab Invest.1991;64:313-320.
[59]Lo JC,Basak S,James ES,et al.Coordination between NF-kappaB family members p50 and p52 is essential for mediating LTbetaR signals in the development and organization of secondary lymphoid tissues.Blood.2006;107:1048-1055.
[60]Banchereau J,Paczesny S,Blanco P,et al.Dendritic cells:controllers of the immune system and a new promise for immunotherapy.Ann N Y Acad Sci.2003.987:180-187.
[61]Thomas R,Davis LS,Lipsky PE.Rheumatoid synovium is enriched in mature antigen-presenting dendritic cells.J Immunol.1994;152(5):2613-2623.
[62]Waalen K,Thoen J,Forre O,et al.Rheumatoid synovial dendritic cells as stimulators in allogeneic and autologous mixed leukocyte reactions—Comparison with autologous monocytes as stimulator cells.Scand J Immunol.1986;23(2):233-241.
[63]Devereux D,RE OH,McGuire J,et al.HLA-DR4Dw4-restricted T cell recognition of self antigen(s) in the rheumatoid synovial compartment.Int Immunol.1991;3(7):635-640.
[64]Palmer DG.The anatomy of the rheumatoid lesion.Br Med Bull.1995;51(2):286-295.
[65]Chen E,Keystone EC,Fish EN.Restricted cytokine expression in rheumatoid arthritis.Arthr Rheum.1993;36(7):901-910.
[66]Piemonti L,Bernasconi S,Luini W,et al.IL-13 supports differentiation of dendritic cells from circulating precursors in concert with GM-CSF.Eur Cytokine Netw.1995;6(4):245-252.
[67]MacPherson GG,Jenkins CD,Stein MJ,et al.Endotoxin-mediated dendritic cell release from the intestine.Characterization of released dendritic cells and TNF dependence.J Immunol.1995;154(3):1317-1322.
[68]Santiago Schwarz F,Sullivan C,Rappa D,et al.Distinct alterations in lineage committed progenitor cells exist in the peripheral blood of patients with rheumatoid arthritis and primary Sjogren's syndrome.J Rheumatol.1996;23(3):439-446.
[69]Thomas R,Lipsky PE.Could endogenous self-peptides presented by dendritic cells initiate rheumatoid arthritis? Immunol Today.1996;17(12):559-564.
[70]MacDonald KP,Nishioka Y,Lipsky PE,et al.Functional CD40-ligand is expressed by T cells in rheumatoid arthritis.J Clin Invest.1997;100(9):2404-2414.
[71]Dittel BN,Visintin I,Merchant RM,et al.Presentation of the self antigen myelin basic protein by dendritic cells leads to experimental autoimmune encephalomyelitis.J Immunol.1999;163:32-39.
[72]Thomas R,Davis LS,Lipsky PE.Rheumatoid synovium is enriched in mature antigen-presenting dendritic cells.J Immunol.1994;152:2613-2623.
[73]Page G,Lebecque S,Miossec P.Anatomic localization of immature and mature dendritic cells in an ectopic lymphoid organ:correlation with selective chemokine expression in rheumatoid synovium.J Immunol.2002;168:5333-5341.
[74]Cavanagh LL,Boyce A,Smith L,et al.Rheumatoid arthritis synovium contains plasmacytoid dendritic cells.Arthritis Res Ther.2005;7:R230-R240.
[75]Steinman RM,Pack M,Inaba K.Dendritic cells in the T-cell areas of lymphoid organs.Immunol Rev.1997;156:25-37.
[76]Guermonprez P,Valladeau J,Zitvogel L,et al.Antigen presentation and T cell stimulation by dendritic cells.Annu Rev Immunol.2002;20:621-667.
[77]Liu YJ.Dendritic cell subsets and lineages,and their functions in innate and adaptive immunity.2001;106(3):259-62.
[78]Mc William AS,Napoli S,Marsh AM,et al.Dendritic cells are recruited into the airway epithelium during the inflammatory response to a broad spectrum of stimuli.J Exp Med.1996;184(6):2429-32.
[79]Cook DN,Prosser DM,Forster R? et al.CCR6 mediates dendritic cell localization,lymphocyte homeostasis,and immune responses in mucosal tissue.Immunity.2000;12(5):495-503.
[80]Steinman RM,Turley S,Mellman I,et al.The induction of tolerance by dendritic cells that have captured apoptotic cells.J Exp Med.2000 Feb 7;191(3):411-416.
[81]Shortman K,Liu YJ.Mouse and human dendritic cell subtypes.Nat Rev Immunol.2002 Mar;2(3):151-61.
[82]Kadowaki N,Ho S,Antonenko S,et al.Subsets of human dendritic cell precursors express different toll-like receptors and respond to different microbial antigens.J Exp Med.2001 Sep 17;194(6):863-9.
[83]Jahnsen FL,Lund-Johansen F,Dunne JF,et al.Experimentally induced recruitment of plasmacytoid (CD123high) dendritic cells in human nasal allergy.J Immunol.2000 Oct l;165(7):4062-8.
[84]Rossi D,Zlotnik A.The biology of chemokines and their receptors.Annu Rev Immunol.2000;18:217-42.
[85]Vanbervliet B,Homey B,Durand I,et al.Sequential involvement of CCR2 and CCR6 ligands for immature dendritic cell recruitment:possible role at inflamed epithelial surfaces.Eur J Immunol.2002;32(1):231-42.
[86]Dieu MC,Vanbervliet B,Vicari A,et al.Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites. J Exp Med. 1998;188(2):373-86.
[87] Gunn MD, Tangemann K, Tarn C, et al. A chemokine expressed in lymphoid high endothelial venules promotes the adhesion and chemotaxis of naive T lymphocytes. Proc Natl Acad Sci USA. 1998;95(1):258-63.
[88] Nakano H, Tamura T, Yoshimoto T, et al. Genetic defect in T lymphocyte-specific homing into peripheral lymph nodes. Eur J Immunol. 1997;27(1):215-21.
[89] Gunn MD, Kyuwa S, Tam C, et al. Mice lacking expression of secondary lymphoid organ chemokine have defects in lymphocyte homing and dendritic cell localization. J Exp Med. 1999;189(3):451-60.
[90] Mori S, Nakano H, Aritomi K, et al. Mice lacking expression of the chemokines CCL21-ser and CCL19 (pit mice) demonstrate delayed but enhanced T cell immune responses. J Exp Med. 2001;193(2):207-18.
[91] Forster R, Schubel A, Breitfeld D, et al. CCR7 coordinates the primary immune response by establishing functional microenvironments in secondary lymphoid organs. Cell. 1999;99(1):23-33.
[92] 李时珍.本草纲目.第1版,第18卷,草目部.北京:北京人民出版社,1977;31340.
[93] 刘东平.青藤碱的研究进展.安徽医药.2005;9(11):859-860.
[94] 王文君,王培训,李晓娟,等.青藤碱抗炎机制-青藤碱对人外周血单个核细胞环氧化酶活性及其基因表达的影响.中国中药杂志.2003;28(4):352-356.
[95] 涂胜豪,胡永红,陆付耳.青藤碱对人淋巴细胞产生IL-2,IL-2R和IL-6的影响.中国实验临床免疫学杂志.1998;10(5):268-269.
[96] 王勇,方勇飞,周新,等.青藤碱对佐剂性关节炎大鼠腹腔巨噬细胞表达细胞因子的影响.中华风湿病杂志.2003;7(7):415-418.
[97] 刘良,李晓娟,王培训,等.青藤碱对人外周血单个核细胞IL-1β、IL-8细胞因子基因表达的影响.中国免疫学杂志.2002;18(4):241-244.
[98] 李晓娟,王培训,刘良,等.青藤碱对T淋巴细胞活化及TH1类细胞内细胞因子表达的影响.中国免疫学杂志.2004;20:249-252.
[99] 徐琳本,邱赛红.正清风痛宁对免疫作用影响的实验研究.湖南中医杂志.1996;12(2):47-48.
[100] 戴英波,黄循,罗志刚,等.青藤碱对大鼠肾移植排斥反应时细胞间黏附分子-1表达的抑制作用.现代中西医结合杂志.2003;12(13):1358-1361.
[101] 王毅,陈正,熊烈,等.青藤碱对肾移植大鼠急性排斥反应及T细胞增殖的影响.中华实验外科杂志.2004;21(5):573-574.
[102] 孔庆峰,李军.青藤碱研究新进展.中国中医杂志.2005;30(20):1573-1576.
[103] 罗烈,杨剑,宋金春,等.苯甲酰青藤碱的镇痛抗炎作用.中国医院药学杂志.2005;25(3):196-198.
[104] 王晓洪,邱赛红,董绍象.青藤碱片的药效学研究.中药药理与临床.1997;13(4):23.
[105] 刘继红,李卫东,藤慧玲,等.青藤碱治疗类风湿关节炎免疫作用和机制.药学学报.2005;40(2):127-131.
[106] 杨德森,刘芳,曾繁典,等.青藤碱对大鼠佐剂性关节炎治疗作用及机制的研究.中国中药杂志.2005;30(17):1361-1363.
[107] Pap T, Aupperle KR, Gay S, et al. Invasiveness of synovial fibroblasts is regulated by P53 in the SCID morse in vivo model of cartilage invasion. Arthritis Rheum. 2001; 44:676-681.
[108] 陈光星,刘良,赵诗哲,等.青藤碱对胶原诱导型关节大鼠滑膜细胞增殖及凋亡影响的研究.中华风湿病学杂志.2005;9(5):284-287.
[109] 易丹丹,蔡鸿生,罗顺德,等.藤碱凝胶对大鼠佐剂性关节炎的治疗作用.中国医院药学杂志.2002;22(8):465-468.
[110] 刘晓玲,陈光星,李晓娟,等.青藤碱对Ⅱ型胶原诱导关节炎大鼠滑膜炎症的抑制作用及其机理探讨.广州中医药大学学报.2002;19(3):214-217.
[111] Zhao Y, Li J, Yu K, et al. Sinomenine inhibits maturation of monocyte-derived dendritic cells through blocking activation of NF-kappa B. Int Immunopharmacol. 2007;7(5):637-645.
[112] 赵毅,余克强,李娟.青藤碱对类风湿关节炎病人树突状细胞免疫活性的影响.中国药学杂志.2006;41(20):1543-1545.
[113] 赵毅,余克强,李娟.青藤碱对类风湿关节炎树突状细胞核转录因子-кB活性的影响.广东医学.2006;27(1):55-57.
[114] 余克强,赵毅,李娟.青藤碱对类风湿关节炎树突状细胞CD80、CD86mRNA表达的影响.中国药房.2006;17(5):331-333.
[115] Robbiani DF, Finch RA, Jager D, Muller WA, Sartorelli AC, Randolph GJ. The leukotriene C (4) transporter MRP1 regulates CCL19 (MIP-3beta, ELC) -dependent mobilization of dendritic cells to lymph nodes. Cell. 2000; 103: 757-768.
[116] Baggiolini M. Chemokines and leukocyte traffic. Nature. 1998; 392: 565-568.
[117] Rossi D, Zlotnik A. The Biology of Chemokines and Their Receptors. Annual Reviews. 2000; 18: 217-243.
[118] Ardavin C. Thymic dendritic cells. Immunol Today. 1997; 18: 350-361.
[119] Kappler JW, Roehm N, Marrack P. T cell tolerance by clonal elimination in the thymus. Cell. 1987; 49:273-280.
[120] Sakaguchi N, Takahashi T, Hata H, et al. Altered thymic T-cell selection due to a mutation of the ZAP-70 gene causes autoimmune arthritis in mice. Nature. 2003; 426:454-460.
[121] Drakesmith H, O'Neil D, Schneider SC, et al. In vivo priming of T cells against cryptic determinants by dendritic cells exposed to interleukin 6 and native antigen. Proc Natl Acad Sci USA. 1998; 95:14903-14908.
[122] Kuhn KA, Kulik L, Tomooka B, et al. Antibodies against citrullinated proteins enhance tissue injury in experimental autoimmune arthritis. J Clin Invest 2006;l16:961-973.
[123] van Gaalen FA, van Aken J, Huizinga TW, et al. Association between HLA class Ⅱ genes and autoantibodies to cyclic citrullinated peptides (CCPs) influences the severity of rheumatoid arthritis. Arthritis Rheum. 2004; 50: 2113-2121.
[124] Hill JA, Southwood S, Sette A, et al. Cutting edge: the conversion of arginine to citrulline allows for a high-affinity peptide interaction with the rheumatoid arthritis-associated HLA-DRB1*0401 MHC class Ⅱ molecule.J Immunol.2003;171:538-541.
[125]Dessen A,Lawrence CM,Cupo S,et al.X-ray crystal structure of HLA-DR4 (DRA*0101,DRB1*0401) complexed with a peptide from human collagen Ⅱ.Immunity.1997;7:473-481.
[126]Michaelsson E,Holmdahl M,Engstrom A,et al.Macrophages,but not dendritic cells,present collagen to T cells.Eur J Immunol.1995;25:2234-2241.
[127]Yoshida M,Tsuji M,Kurosaka D,et al.Autoimmunity to citrullinated type Ⅱ collagen in rheumatoid arthritis.Mod Rheumatol.2006;16:276-281.
[128]Binstadt BA,Patel PR,Alencar H,et al.Particularities of the vasculature can promote the organ specificity of autoimmune attack.Nat Immunol.2006;7:284-292.
[129]Baeten D,Steenbakkers PG,Rijnders AM,et al.Detection of major histocompatibility complex/ human cartilage gp-39 complexes in rheumatoid arthritis synovitis as a specific and independent histologic marker.ArthritisRheum.2004;50:444-451.
[130]Thomas R,Quinn C.Functional differentiation of dendritic cells in rheumatoid arthritis:role of CD86 in the synovium.J Immunol.1996;156:3074-3086.
[131]Highton J,Kean A,Hessian PA,et al.Cells expressing dendritic cell markers are present in the rheumatoid nodule.J Rheumatol.2000;27:339-346.
[132]Summers KL,O'Donnell JL,Heiser A,et al.Synovial fluid transforming growth factor beta inhibits dendritic cell-T lymphocyte interactions in patients with chronic arthritis.Arthritis Rheum.1999;42:507-518.
[133]Fan L,Reilly CR,Luo Y,et al.Cutting edge:ectopic expression of the chemokine TCA4/SLC is sufficient to trigger lymphoid neogenesis.J Immunol.2000;164:3955-3959.
[134]Nanki T,Urasaki Y,Imai T,et al.Inhibition of fractalkine ameliorates murine collagen-induced arthritis.J Immunol.2004;173:7010-7016.
[135]van Lieshout AW,van der Voort R,le Blanc LM,et al.Novel insights in the regulation of CCL18 secretion by monocytes and dendritic cells via cytokines,toll-like receptors and rheumatoid synovial fluid.BMC Immunol.2006;7:23.
[136]Shortman K,Naik SH.Steady-state and inflammatory dendritic-cell development.Nat Rev Immunol.2007;7:19-30.
[137]Allan RS,Smith CM,Belz GT,et al.Epidermal viral immunity induced by CD8alpha+ dendritic cells but not by Langerhans cells.Science.2003;301:1925-1928.
[138]Varol C,Landsman L,Fogg DK,et al.Monocytes give rise to mucosal,but not splenic,conventional dendritic cells.J Exp Med.2007;204:171-180.
[139]Qu C,Edwards EW,Tacke F,et al.Role of CCR8 and other chemokine pathways in the migration of monocytederived dendritic cells to lymph nodes.J Exp Med.2004;200:1231-1241.
[140]Vermaelen KY,Carro-Muino I,Lambrecht BN,et al.Specific migratory dendritic cells rapidly transport antigen from the airways to the thoracic lymph nodes.J Exp Med.2001;193:51-60.
[141]Kurts C,Kosaka H,Carbone FR,et al.Class I-restricted cross-presentation of exogenous self-antigens leads to deletion of autoreactive CD8(+) T cells.J Exp Med.1997;186:239-245.
[142]Serbina NV,Pamer EG.Monocyte emigration from bone marrow during bacterial infection requires signals mediated by chemokine receptor CCR2.Nat Immunol.2006;7:311-317.
[143]Tacke F,Alvarez D,Kaplan TJ,et al.Monocyte subsets differentially employ CCR2,CCR5,and CX3CR1 to accumulate within atherosclerotic plaques.J ClinInvest.2007;117:185-94.
[144]Bachmann MF,Kopf M,Marsland BJ.Chemokines:more than just road signs.Nat Rev Immunol.2006;6:159-164.
[145]Sallusto F,Schaerli P,Loetscher P,et al.Rapid and coordinated switch in chemokine receptor expression during dendritic cell maturation.Eur J Immunol.1998;28:2760-2769.
[146]Balashov KE,Rottman JB,Weiner HL,et al.CCR5(+) and CXCR3(+) T cells are increased in multiple sclerosis and their ligands MIP-1 alpha and IP-10 are expressed in demyelinating brain lesions.Proc.Natl.Acad.Sci.U.S.A.1999;96:6873-6878.
[147]Sallusto F,Mackay CR,Lanzavecchia A.Selective expression of the eotaxin receptor CCR3 by human T helper 2 cells.Science.1997;277:2005-2007.
[148]Gombert M,Dieu-Nosjean MC,Winterberg F,et al.CCL1-CCR8 interactions:an axis mediating the recruitment of T cells and Langerhans-type dendritic cells tosites of atopic skin inflammation.J Immunol.2005;174:5082-5091.
[149]Harrington PM,Newton DJ,Williams CM,et al.Eotaxin and eotaxin receptor (CCR3) expression in Sephadex particle-induced rat lung inflammation.Int J Exp Pathol.1999;80:177-185.
[150]Andrew DP,Ruffing N,Kim CH,et al.C-C chemokine receptor 4 expression defines a major subset of circulating nonintestinal memory T cells of both Thl and Th2 potential.J Immunol.2001;166:103-111.
[151]Sallusto F,Lenig D,Forster R,et al.Lanzavecchia:Two subsets of memory T lymphocytes with distinct homing potentials and effector functions.Nature.1999;401:708-712.
[152]Ginhoux F,Tacke F,Angeli V,et al.Langerhans cells arise from monocytes in vivo.Nat Immunol.2006;7:265-273.
[153]Nagasawa T,Kikutani H,Kishimoto T.Molecular cloning and structure of a pre-B-cell growthstimulating factor.Proc.Natl.Acad.Sci.U.S.A.1994;91:2305-2309.
[154]Peled A,Kollet O,Ponomaryov T,et al.The chemokine SDF-1 activates the integrins LFA-1,VLA-4,and VLA-5 on immature human CD34(+) cells:role in transendothelial/stromal migration and engraftment of NOD/SCID mice.Blood.2000;95:3289-3296.
[155]Sanz-Rodriguez F,Hidalgo A,Teixido J.Chemokine stromal cell-derived factor-1 alpha modulates VLA-4 integrin-mediated multiple myeloma cell adhesion to CS-1 /fibronectin and VCAM-1.Blood.2001;97:346-351.
[156]Zernecke A,Liehn EA,Gao JL,et al.Deficiency in CCR5 but not CCR1 protects against neointima formation in atherosclerosis-prone mice:involvement of IL-10.Blood.2006;107:4240-4243.
[157]Baltus T,Weber KS,Johnson Z,et al.Oligomerization of RANTES is required for CCR1 -mediated arrest but not CCR5-mediated transmigration of leukocytes on inflamed endothelium.Blood.2003;102:1985-1988.
[158]Oppermann M.Chemokine receptor CCR5:insights into structure,function,and regulation.Cell Signal.2004;16:1201-1210.
[159]Yurchenko E,Tritt M,Hay V,et al.CCR5-dependent homing of naturally occurring CD4+ regulatory T cells to sites of Leishmania major infection favors pathogen persistence.J Exp Med.2006;203:2451-2460.
[160]Floto RA,MacAry PA,Boname JM,et al.Dendritic cell stimulation by mycobacterial Hsp70 is mediated through CCR5.Science.2006;314:454-458.
[161]Smith CM,Wilson NS,Waithman J,et al.Cognate CD4(+) T cell licensing of dendritic cells in CD8(+) T cell immunity.Nat Immunol.2004;5:1143-1148.
[162]Castellino F,Huang AY,Altan-Bonnet G,et al.Chemokines enhance immunity by guiding naive CD8+ T cells to sites of CD4+ T cell-dendritic cell interaction.Nature.2006;440:890-895.
[163]Jin Y,Shen L,Chong EM,et al.The chemokine receptor CCR7 mediates corneal antigen-presenting cell trafficking.Mol Vis.2007;13:626-634.
[164]Lo JC,Chin RK,Lee Y,et al.Differential regulation of CCL21 in lymphoid/nonlymphoid tissues for effectively attracting T cells to peripheral tissues.J Clin Invest.2003;112:1495-1505.
[165]Luther SA,Tang HL,Hyman PL,et al.Coexpression of the chemokines ELC and SLC by T zone stromal cells and deletion of the ELC gene in the plt/plt mouse.Proc.Natl.Acad.Sci.U.S.A.2000;97;12694-1269.
[166]Czeloth N,Bernhardt G,Hofmann F,et al.Sphingosine-1-phosphate mediates migration of mature dendritic cells.J Immunol.2005;175;2960-2967.
[167]Sanchez-Sanchez N,Riol-Blanco L,de la Rosa G,et al.Chemokine receptor CCR7 induces intracellular signaling that inhibits apoptosis of mature dendritic cells.Blood.2004;104:619-625.
[168]Flanagan K,Moroziewicz D,Kwak H,et al.The lymphoid chemokine CCL21 costimulates naive T cell expansion and Th1 polarization of non-regulatory CD4+ T cells.Cell Immunol.2004;231:75-84.
[169]Marsland BJ,Battig P,Bauer M,et al.CCL19 and CCL21 induce a potent proinflammatory differentiation program in licensed dendritic cells.Immunity.2005;22:493-505.
[170]Lasagni L,Francalanci M,Annunziato F,et al.An alternatively spliced variant of CXCR3 mediates the inhibition of endothelial cell growth induced by IP-10,Mig,and I-TAC,and acts as functional receptor for platelet factor 4.J Exp Med.2003;197:1537-1549.
[171]Qian C,An H,Yu Y,et al.TLR agonists induce regulatory dendritic cells to recruit Thl cells via preferential IP-10 secretion and inhibit Thl proliferation.Blood.2007;109:3308-3315.
[172]Molesworth-Kenyon S,Mates A,Yin R,et al.CXCR3,IP-10,and Mig are required for CD4+ T cell recruitment during the DTH response to HSV-1 yet are independent of the mechanism for viral clearance.Virology.2005;333:1-9.
[173]Guarda G,Hons M,Soriano SF,et al.L-selectin-negative CCR7(-) effector and memory CD8(+) T cells enter reactive lymph nodes and kill dendritic cells.Nat Immunol.2007;8:743-752.
[174]Loetscher P,Pellegrino A,Gong JH,et al.The ligands of CXC chemokine receptor 3,I-TAC,Mig,and IP 10,are natural antagonists for CCR3.J Biol Chem.2001;276:2986-2991.
[175]Gonzalez-Cuadrado S,Bustos C,Ruiz-Ortega M,et al.Expression of leucocyte chemoattractants by interstitial renal fibroblasts:up-regulation by drugs associated with interstitial fibrosis.Clin Exp Immunol.1996;106:518-522.
[176]Hyun JG,Lee G,Brown JB,et al.Anti-interferoninducible chemokine,CXCL10,reduces colitis by impairing T helper-1 induction and recruitment in mice.Inflamm Bowel Dis.2005;11:799-805.
[177]Bogyo M,Shin S,McMaster JS,et al.Substrate binding and sequence preference of the proteasome revealed by active-site-directed affinity probes.Chem Biol. 1998;5:307-320.
[178]Hershko A,Ciechanover A.The ubiquitin system.Annu Rev Biochem.1998;67:425-479.
[179]Goldberg AL.Protein degradation and protection against misfolded or damaged proteins.Nature.2003;426:895-899.
[180]Ciechanover A.The ubiquitin-proteasome proteolytic pathway.Cell.1994;79:13-21
[181]Adams J.The proteasome:structure,function,and role in the cell.Cancer Treat Rev.2003;29 Suppl 1:3-9.
[182]Rock KL,Goldberg AL.Degradation of cell proteins and the generation of MHC class Ⅰ-presented peptides.Annu Rev Immunol.1999;17:739-779.
[183]Rock KL,Gramm C,Rothstein L,et al.Inhibitors of the proteasome block the degradation of most cell proteins and the generation of peptides presented on MHC class Ⅰ molecules.Cell.1994;78:761-771.
[184]Cascio P,Call M,Petre BM,et al.Properties of the hybrid form of the 26S proteasome containing both 19S and PA28 complexes.EMBO J.2002;21:2636-2645.
[185]Cascio P,Hilton C,Kisselev AF,et al.26S proteasomes and immunoproteasomes produce mainly N-extended versions of an antigenic peptide.EMBO J.2001;20:2357-2366.
[186]Strehl B,Seifert U,Kruger E,et al.Interferon-γ,the functional plasticity of the ubiquitinproteasome system,and MHC class Ⅰ antigen processing.Immunol Rev.2005;207:19-30.
[187]Goldberg AL,Cascio P,Saric T,et al.The importance of the proteasome and subsequent proteolytic steps in the generation of antigenic peptides.Mol Immunol.2002;39:147-164.
[188]York IA,Goldberg AL,Mo XY,et al.Proteolysis and class Ⅰ major histocompatibility complex antigen presentation.Immunol Rev.1999;172:49-66.
[189]Zhang J,Bardos T,Li D,et al.Cutting edge:regulation of T cell activation threshold by CD28 costimulation through targeting Cbl-P for ubiquitination.J Immunol.2002;169:2236-2240.
[190]Qureshi N,Vogel SN,Van Way C,et al.a central regulator of inflammation and macrophage function.Immunol Res.2005;31:243-260.
[191]Matsushita M,Takasaki Y,Takeuchi K,et al.Autoimmune response to proteasome activator 28a in patients with connective tissue diseases.J Rheumatol.2004;31:252-259.
[192]Mountz JD.Significance of increased circulating proteasome in autoimmune disease.J Rheumatol.2002;29:2027-2030.
[193]Egerer K,Kuckelkorn U,Rudolph PE,et al.Circulating proteasomes are markers of cell damage and immunologic activity in autoimmune diseases.J Rheumatol.2002;29:2045-2052.
[194]Firestein GS.NF-kB:Holy Grail for rheumatoid arthritis? Arthritis Rheum.2004;50:2381-2386.
[195]Elliott PJ,Zollner TM,Boehncke WH.Proteasome inhibition:a new anti-inflammatory strategy.J Mol Med.2003;81:235-245.
[196]Niu H,Cattoretti G,Dalla-Favera R.BCL6 controls the expression of the B7-1/CD80 costimulatory receptor in germinal center B cells.J Exp Med.2003;198(2):211-221.
[197]Hofer S,Rescigno M,Granucci F,et al.Differential activation of NF-kappa B subunits in dendritic cells in response to Gram-negative bacteria and to lipopolysaccharide.Microbes Infect.2001;3(4):259-265.
[198]Pettit AR,Quinn C,MacDonald KP,et al.Nuclear localization of RelB is associated with effective antigen presenting cell function.J Immunol.1997;159(8):3681-3691.
[199]Ouaaz F,Arron J,Zheng Y,et al.Dendritic cell development and survival require distinct NF-kB subunits.Immunity.2002;16(2):257-270.
[200]Zheng Y,Ouaaz F,Bruzzo P,et al.NF-kappa B RelA(p65) is essential for TNF-alpha-induced fas expression but dispensable for both TCR-induced expression and activation-induced cell death.J Immunol.2001;166(8):4949-4957.
[201]Speirs K,Lieberman L,Caamano J,et al.Cutting edge:NF-kappa B2 is a negative regulator of dendritic cell function.J Immunol.2004;172(2):752-756.
[202]Ghosh S,May MJ,Kopp EB.NF-kappa B and Rel proteins:evolutionarily conserved mediators of immune respones.Annu Rev Immunol.1998;16:225-260.
[203]Tanaka K,Tsurumi C.The 26S proteasome:subunits and functions.Mol Biol Rep.1997;24(1-2):3-11.
[204]Yaron A,Gonen H,Alkalay I,et al.Inhibition of NF-kappa-B cellular function via specific targeting of the I-kappa-B-ubiquitin ligase.EMBO J.1997;16(21):6486-94.
[205]Marok R,Winyard PG,Coumbe A,et al.Activation of the transcription factor nuclear factor-kappaB in human inflamed synovial tissue.Arthritis Rheum.1996;39(4):583-91.
[206]Rodriguez MS,Michalopoulos I,Arenzana-Seisdedos F,et al.Inducible degradation of I kappa B alpha in vitro and in vivo requires the acidic C-terminal domain of the protein.Mol Cell Biol.1995;15(5):2413-9.
[207]Kroll M,Conconi M,Desterro MJ.The carboxy-terminus of I kappaB alpha determines susceptibility to degradation by the catalytic core of the proteasome.Oncogene.1997;15(15):1841-50.
[208]Antonelli A,Crinelli R,Bianchi M,et al.Efficient inhibition of macrophage TNF-alpha production upon targeted delivery of K48R ubiquitin.Br J Haematol.1999;104(3):475-81.
[209]Palombella VJ,Conner EM,Fuseler JW,et al.Role of the proteasome and NF-kappaB in streptococcal cell wall-induced polyarthritis.Proc Natl Acad Sci U SA.1998;95(26):15671-15676.