系膜增生性肾炎T细胞免疫失衡及盐酸青藤碱单体的调节作用
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摘要
研究背景
在肾脏疾病如肾小球肾炎、小管间质性肾炎及同种异体移植物排斥反应的炎症反应中,T细胞在对肾脏免疫性损伤中占主导作用。T细胞的活化不仅需要MHC-肽-TCR方式提供第一信号,还需要共刺激分子提供第二信号。如果没有足够的共刺激信号刺激,T细胞表现为失能、耐受和凋亡。程序性死亡1共刺激分子(programmed death gene 1,PD-1)/PD-L(PD-1 ligand)途径是新近发现的共刺激信号,属CD28/B7超家族成员。已证实该信号途径的异常表达参与了自身免疫性疾病、同种异体移植物排斥反应及各种感染的发生及进展。目前针对共刺激信号途径的调节已经成为免疫治疗的新方法。
T细胞对疾病的影响不仅体现在活化水平,还体现在选择性分化水平。T-bet是新发现的Th1特异性转录因子,可促进Th0细胞向Th1细胞分化,GATA-3是Th2特异的转录因子,在Th2细胞分化过程中起关键作用。当机体免疫功能异常时,Th0细胞分化的Th1和Th2细胞亚群比例及功能失衡,出现相应的Th1和Th2漂移性疾病。研究显示T-bet/GATA-3比值能较准确地反映Th1/Th2细胞亚群的平衡状态,可用于评价慢性肾炎的免疫平衡状态。通过调节T-bet/GATA-3而恢复Th1/Th2的平衡,被认为是自身免疫性疾病的一个可能的治疗方向。
盐酸青藤碱(sinomenine, SN)是从中药青风藤(Sinomenium scutum Rehd et wils)中提取的生物碱单体,为新型中药免疫调节药物,疗效较好副作用小,用于慢性肾炎的治疗已有10多年的成功经验,但其具体机制依然不清。
本研究以常见的非IgA系膜增生性肾炎(mesangial proliferative nephritis,MsPGN)为观察对象(避免不同病理类型对结果的影响),观察这类患者体内PD-1/PD-L表达以及Th1/Th2平衡状态有何变化;并从临床的角度,前瞻性地观察SN对MsPGN患者蛋白尿等临床症状的影响,观察SN对患者外周血单个核细胞(peripheral blood mononuclear cell,PBMC) PD-1/PD-L、T-bet/GATA-3及Th1/Th2细胞因子表达的影响,以探讨SN治疗MsPGN的免疫机制,为应用SN治疗MsPGN提供理论依据。
研究方法
1.研究对象
实验观察MsPGN患者25例,另观察10例正常人作为对照。其中肾组织标本,MsPGN组标本来自25例MsPGN患者的肾活检标本,对照组标本10例来自亲属供肾的移植前肾活检标本和因肾肿瘤而切除的远离肿瘤端且经光镜证实正常的肾组织标本。
2.实验方法
(1)免疫组化检测治疗前MsPGN患者肾组织和对照组肾组织中PD-1、PD-L1、PD-L2蛋白的表达;定量RT-PCR和多色流式细胞技术检测治疗前MsPGN患者和对照组PBMC PD-1、PD-L1、PD-L2 mRNA和蛋白表达。以了解MsPGN患者是否存在共刺激信号PD-1/PD-L的异常表达。
(2)免疫组化检测治疗前MsPGN患者肾组织和对照组肾组织中T-bet、GATA-3蛋白的表达;定量RT-PCR检测治疗前MsPGN患者和对照组PBMC T-bet、GATA-3 mRNA表达;同时采用ELISA法测定治疗前MsPGN患者和对照组血清IFN-γ、IL-4、IL-10表达水平。以了解MsPGN患者在T细胞选择性分化水平是否存在Th1/Th2失衡。
(3)采用定量RT-PCR和多色流式细胞技术观察不同浓度SN(50μg/ml-200μg/ml)对体外培养的MsPGN患者外周血淋巴细胞PD-1、PD-L1、PD-L2 mRNA和蛋白表达的影响。
(4)采用定量RT-PCR和Western Blot方法观察不同浓度SN(50μg/ml-200μg/ml)对体外培养的MsPGN患者外周血淋巴细胞T-bet、GATA-3 mRNA和蛋白表达的影响;同时采用ELISA法检测培养上清中IFN-γ、IL-4、IL-10表达变化。
(5)留取对照组和治疗前后MsPGN患者血、尿标本,分别进行临床生化及尿蛋白检测,以观察SN治疗MsPGN的临床疗效。
(6)采用定量RT-PCR和多色流式细胞技术观察MsPGN患者接受SN治疗1月、3月后PBMC PD-1、PD-L1、PD-L2 mRNA和蛋白表达的变化。
(7)采用定量RT-PCR观察MsPGN患者接受SN治疗1月、3月后PBMC T-bet、GATA-3 mRNA表达的变化;同时采用ELISA法检测血清IFN-γ、IL-4、IL-10表达变化。
结果
1. MsPGN患者肾活检标本肾小管间质部位的淋巴细胞有PD-1阳性表达,而PD-L1表达在肾小管上皮细胞(renal tubular epithelial cells, TEC),肾小球无PD-1、PD-L1表达。对照组肾组织中无PD-1表达,PD-L1表达很少甚至无表达。两组肾组织标本中均未检测到PD-L2表达。MsPGN患者PBMC PD-1、PD-L1 mRNA和蛋白表达高于对照组,而PD-L2蛋白在MsPGN患者和对照组中表达很少,两组间无显著差异。
2.在TEC和肾间质渗出的淋巴细胞检测到T-bet、GATA-3的阳性表达,MsPGN肾组织T-bet表达强于对照组而GATA-3表达弱于对照组;MsPGN患者PBMC T-bet mRNA表达高于对照组,而GATA-3 mRNA表达低于对照组;MsPGN患者血清IFN-γ水平高于对照人群,而IL-10水平低于对照人群。
3.体外实验观察到SN可以抑制MsPGN患者外周血淋巴细胞PD-1、PD-L1 mRNA和蛋白表达,这种抑制作用随着SN浓度的增加而增强。
4.体外实验观察到SN可以抑制MsPGN患者外周血淋巴细胞T-bet mRNA和蛋白表达,下调T-bet/GATA-3比值,并抑制Th1细胞因子IFN-γ的表达,这种调节作用随着SN浓度的增加而增强。
5. SN治疗可有效控制MsPGN患者蛋白尿,并升高补体C3水平,副作用小。
6. SN治疗可以抑制MsPGN患者PBMC PD-1、PD-L1 mRNA和蛋白表达,这种抑制作用随着SN治疗时间的延长而增强。
7. SN治疗可以抑制MsPGN患者PBMC T-bet的mRNA表达,下调T-bet/GATA-3比值,并抑制Th1细胞因子IFN-γ的表达,这种调节作用随着SN治疗时间的延长而增强。
结论
SN治疗可有效控制轻、中度MsPGN患者蛋白尿,并升高补体C3水平,副作用小,但疗程至少要1月以上才有明显疗效。
MsPGN患者肾组织和PBMC上PD-1、PD-L1表达高于对照组,可能与MsPGN的免疫发病机制有关。SN可以抑制PD-1、PD-L1的病理性高表达,这种抑制作用随着SN浓度的增加、治疗时间的延长而增强,提示SN控制MsPGN患者蛋白尿的治疗机制可能与抑制PD-1、PD-L1表达有关。
MsPGN患者在T细胞选择性分化过程中,在转录调控水平和细胞因子水平存在Th1优势反应,这可能是MsPGN的重要免疫发病机制。而SN能抑制MsPGN患者T-bet表达,下调T-bet/GATA-3比值,并抑制Th1细胞因子IFN-γ的表达,从而在转录调控水平和细胞因子水平抑制Th1反应,恢复Th1/Th2的平衡。SN的这种调节作用随着SN浓度的增加、治疗时间的延长而增强。提示SN恢复MsPGN患者Th1/Th2平衡的免疫调节作用,可能是其能控制蛋白尿的重要的治疗机制。
Background
Experimental results from inflammatory renal diseases, such as allograft rejection, tubulointerstitial nephritis and glomerulonephritis, have suggested a prominent role of T cells in the injurious renal immune response. It is accepted that signals provide by TCR-peptide-MHC and co-stimulatory molecules are required for the optimal activation of T cells. Without sufficient co-stimulation, T cells are rendered anergy, tolerance or apoptosis. Recently, B7 superfamily molecules, PD-L1, PD-L2 and their receptor PD-1 (programmed death-1) have been identified. Previous studies indicated that the abnormality of PD-1/PD-L pathway might play a role in the development of autoimmune disease, allograft rejection, and various kinds of infection. Treatment aiming at regulating co-stimulatory pathway has now been recognized as a new way of immunotherapy.
Inappropriate T cell responses result from not only the abnormality of T cell activation but also the abnormality of T cell differentiation. Immune responses polarized by either Th1 or Th2 subset predominance result in the activation of different inflammatory effector pathways and disease outcomes. T-bet is a newly discovered Th1-specific transcription factor thought to initiate Th1 development, whereas GATA-3 plays a pivotal role in the development of the Th2 phenotype. It has been reported that the T-bet/GATA-3 ratio is representative of the balance between Th1 and Th2 cells. Maintaining the Th1/Th2 balance via the regulation of T-bet/GATA-3 ratio has been recognized as a promising therapy in the treatment of autoimmune diseases.
Sinomenine (SN), a pure alkaloid extracted from the Chinese medical plant Sinomenium acutum, has been effectively used for over ten years in the treatment of patients with chronic glomerulonephritis. Although sinomenine is effectively used in the clinic and is gaining international popularity, little is known about its exact mechanisms of action.
In this study, patients with mesangial proliferative nephritis (MsPGN), which is one of the most common pathological type of chronic GN, were examined. Both the PD-1/PD-L expression and the balanced state of Th1/Th2 were observed in patients with MsPGN. Then the effect of SN on proteinuria was detected. In order to elucidate the therapeutic mechanism of SN, the effect of SN on the expressions of PD-1/PD-L and T-bet/GATA-3 in peripheral blood mononuclear cells (PBMCs) and the serum levels of interferon-γ(IFN-γ), interleukin (IL)-4, and IL-10 were studied.
Patients and methods
1. Patients and renal specimens
A total of 25 patients with MsPGN and 10 healthy donors were enrolled in this study. Immunohistochemistry was enforced on the paraffin-embedded tissues from renal biopsies of 25 patients with MsPGN. Additionally, ten specimens obtained from pre-transplant renal biopsies and non-tumor kidney tissues from cancer patients served as controls.
2. methods
(1) In order to observe the expression pattern of PD-1/PD-L in patients with MsPGN, the intrarenal levels of PD-1, PD-L1, and PD-L2 were determined by immunohistochemistry and the expression levels of PD-1, PD-L1, and PD-L2 on PBMCs in both MsPGN patients and healthy donors were detected via real-time RT-PCR and flow cytometry.
(2) In order to observe if there is an imbalance between Th1 and Th2 cells in patients with MsPGN, the intrarenal levels of T-bet and GATA-3 were determined by immunohistochemistry, the expression levels of T-bet and GATA-3 in PBMCs in both MsPGN patients and healthy donors were detected via real-time RT-PCR, and the serum levels of IFN-γ, IL-4, and IL-10 were studied via ELISA.
(3) The effect of SN on the expression levels of PD-1, PD-L1, and PD-L2 on lymphocytes in vitro was studied via real-time RT-PCR and flow cytometry.
(4) The effect of SN on the expression levels of T-bet and GATA-3 on lymphocytes in vitro was studied via real-time RT-PCR and Western Blot. Additionally, ELISA was applied to detect the effect of SN on the supernatant levels of IFN-γ, IL-4, and IL-10.
(5) To evaluate the clinic effect of SN, blood biochemical parameters and proteinuria were detected at month 0, month 1, and month 3 in the course of treatment with SN.
(6) The effect of SN on the expression levels of PD-1, PD-L1, and PD-L2 on PBMCs in vivo was studied via real-time RT-PCR and flow cytometry at 1 month and 3 months after the start of treatment with SN.
(7) The effect of SN on the expression levels of T-bet and GATA-3 on PBMCs in vivo was studied via real-time RT-PCR and the serum levels of IFN-γ, IL-4, and IL-10 were studied by ELISA at 1 month and 3 months after the start of treatment with SN.
Results
1. Positive staining for PD-1 was detected on infiltrating lymphocytes in the interstitial space and PD-L1 was detected in tubular epithelial cells in renal tissues from MsPGN patients. However, the expression of PD-L2 was hardly observed in renal tissues in both MsPGN patients and control. Patients with MsPGN were found to have increased PD-1 and PD-L1 expression in both renal biopsy tissues and PBMCs compared with normal individuals.
2. Positive staining for both T-bet and GATA-3 was detected in tubular epithelial cells and infiltrating lymphocytes in the interstitial space. Patients with MsPGN were found to have increased T-bet and depressed GATA-3 expression in both renal biopsy tissues and PBMCs compared with normal individuals. Additionally, MsPGN patients were found to have elevated serum IFN-γvalues and decreased serum IL-10 values when compared with healthy donors.
3. The expression of PD-1 and PD-L1 on lymphocytes was suppressed by SN in vitro, and the inhibition effect was concentration dependent.
4. The expression of T-bet on lymphocytes was suppressed by SN in vitro, therefore, the T-bet/GATA-3 ratio was markedly decreased. SN was also found to markedly inhibit the secretion of IFN-γ. These effects above were concentration dependent.
5. SN could ameliorate proteinuria and increase the levels of Complement C3 in MsPGN patients with rare side effects.
6. SN could suppresse the expression of PD-1 and PD-L1 on PBMCs from MsPGN patients in vivo, and this effect was more significant at 3 months.
7. SN was found to cause a decrease in T-bet expression on PBMCs from MsPGN patients in vivo, resulting in a drop in the T-bet/GATA-3 ratio. SN could also inhibit the secretion of IFN-γ. These effects were more significant at 3 months.
Conclusions
SN could ameliorate proteinuria and increase the levels of Complement C3 in MsPGN patients with rare side effects, however, the course of treatment should last for more than 1 month.
Increased expression of PD-1 and PD-L1 in renal tissues and PBMCs from MsPGN patients may contribute to the development of MsPGN. Our results indicate that SN has the potential to inhibit the high expression of PD-1 and PD-L1 in PBMCs from MsPGN patients and thereby produce therapeutic effects.
A shift toward the Th1 pathway of Th cell activation at both transcriptional level and cytokine level occurs in MsPGN patients, and that SN has the potential to counter this shift in the Th1/Th2 balance and thereby produce therapeutic effects.
在肾脏疾病如肾小球肾炎、小管间质性肾炎及同种异体移植物排斥反应的炎症反应中,T细胞在对肾脏免疫性损伤中占主导作用。T细胞的活化不仅需要MHC-肽-TCR方式提供第一信号,还需要共刺激分子提供第二信号。如果没有足够的共刺激信号刺激,T细胞表现为失能、耐受和凋亡。程序性死亡1共刺激分子(programmed death gene 1,PD-1)/PD-L(PD-1 ligand)途径是新近发现的共刺激信号,属CD28/B7超家族成员。已证实该信号途径的异常表达参与了自身免疫性疾病、同种异体移植物排斥反应及各种感染的发生及进展。目前针对共刺激信号途径的调节已经成为免疫治疗的新方法。
T细胞对疾病的影响不仅体现在活化水平,还体现在选择性分化水平。T-bet是新发现的Th1特异性转录因子,可促进Th0细胞向Th1细胞分化,GATA-3是Th2特异的转录因子,在Th2细胞分化过程中起关键作用。当机体免疫功能异常时,Th0细胞分化的Th1和Th2细胞亚群比例及功能失衡,出现相应的Th1和Th2漂移性疾病。研究显示T-bet/GATA-3比值能较准确地反映Th1/Th2细胞亚群的平衡状态,可用于评价慢性肾炎的免疫平衡状态。通过调节T-bet/GATA-3而恢复Th1/Th2的平衡,被认为是自身免疫性疾病的一个可能的治疗方向。
盐酸青藤碱(sinomenine, SN)是从中药青风藤(Sinomenium scutum Rehd et wils)中提取的生物碱单体,为新型中药免疫调节药物,疗效较好副作用小,用于慢性肾炎的治疗已有10多年的成功经验,但其具体机制依然不清。
本研究以常见的非IgA系膜增生性肾炎(mesangial proliferative nephritis,MsPGN)为观察对象(避免不同病理类型对结果的影响),观察这类患者体内PD-1/PD-L表达以及Th1/Th2平衡状态有何变化;并从临床的角度,前瞻性地观察SN对MsPGN患者蛋白尿等临床症状的影响,观察SN对患者外周血单个核细胞(peripheral blood mononuclear cell,PBMC) PD-1/PD-L、T-bet/GATA-3及Th1/Th2细胞因子表达的影响,以探讨SN治疗MsPGN的免疫机制,为应用SN治疗MsPGN提供理论依据。
研究方法
1.研究对象
实验观察MsPGN患者25例,另观察10例正常人作为对照。其中肾组织标本,MsPGN组标本来自25例MsPGN患者的肾活检标本,对照组标本10例来自亲属供肾的移植前肾活检标本和因肾肿瘤而切除的远离肿瘤端且经光镜证实正常的肾组织标本。
2.实验方法
(1)免疫组化检测治疗前MsPGN患者肾组织和对照组肾组织中PD-1、PD-L1、PD-L2蛋白的表达;定量RT-PCR和多色流式细胞技术检测治疗前MsPGN患者和对照组PBMC PD-1、PD-L1、PD-L2 mRNA和蛋白表达。以了解MsPGN患者是否存在共刺激信号PD-1/PD-L的异常表达。
(2)免疫组化检测治疗前MsPGN患者肾组织和对照组肾组织中T-bet、GATA-3蛋白的表达;定量RT-PCR检测治疗前MsPGN患者和对照组PBMC T-bet、GATA-3 mRNA表达;同时采用ELISA法测定治疗前MsPGN患者和对照组血清IFN-γ、IL-4、IL-10表达水平。以了解MsPGN患者在T细胞选择性分化水平是否存在Th1/Th2失衡。
(3)采用定量RT-PCR和多色流式细胞技术观察不同浓度SN(50μg/ml-200μg/ml)对体外培养的MsPGN患者外周血淋巴细胞PD-1、PD-L1、PD-L2 mRNA和蛋白表达的影响。
(4)采用定量RT-PCR和Western Blot方法观察不同浓度SN(50μg/ml-200μg/ml)对体外培养的MsPGN患者外周血淋巴细胞T-bet、GATA-3 mRNA和蛋白表达的影响;同时采用ELISA法检测培养上清中IFN-γ、IL-4、IL-10表达变化。
(5)留取对照组和治疗前后MsPGN患者血、尿标本,分别进行临床生化及尿蛋白检测,以观察SN治疗MsPGN的临床疗效。
(6)采用定量RT-PCR和多色流式细胞技术观察MsPGN患者接受SN治疗1月、3月后PBMC PD-1、PD-L1、PD-L2 mRNA和蛋白表达的变化。
(7)采用定量RT-PCR观察MsPGN患者接受SN治疗1月、3月后PBMC T-bet、GATA-3 mRNA表达的变化;同时采用ELISA法检测血清IFN-γ、IL-4、IL-10表达变化。
结果
1. MsPGN患者肾活检标本肾小管间质部位的淋巴细胞有PD-1阳性表达,而PD-L1表达在肾小管上皮细胞(renal tubular epithelial cells, TEC),肾小球无PD-1、PD-L1表达。对照组肾组织中无PD-1表达,PD-L1表达很少甚至无表达。两组肾组织标本中均未检测到PD-L2表达。MsPGN患者PBMC PD-1、PD-L1 mRNA和蛋白表达高于对照组,而PD-L2蛋白在MsPGN患者和对照组中表达很少,两组间无显著差异。
2.在TEC和肾间质渗出的淋巴细胞检测到T-bet、GATA-3的阳性表达,MsPGN肾组织T-bet表达强于对照组而GATA-3表达弱于对照组;MsPGN患者PBMC T-bet mRNA表达高于对照组,而GATA-3 mRNA表达低于对照组;MsPGN患者血清IFN-γ水平高于对照人群,而IL-10水平低于对照人群。
3.体外实验观察到SN可以抑制MsPGN患者外周血淋巴细胞PD-1、PD-L1 mRNA和蛋白表达,这种抑制作用随着SN浓度的增加而增强。
4.体外实验观察到SN可以抑制MsPGN患者外周血淋巴细胞T-bet mRNA和蛋白表达,下调T-bet/GATA-3比值,并抑制Th1细胞因子IFN-γ的表达,这种调节作用随着SN浓度的增加而增强。
5. SN治疗可有效控制MsPGN患者蛋白尿,并升高补体C3水平,副作用小。
6. SN治疗可以抑制MsPGN患者PBMC PD-1、PD-L1 mRNA和蛋白表达,这种抑制作用随着SN治疗时间的延长而增强。
7. SN治疗可以抑制MsPGN患者PBMC T-bet的mRNA表达,下调T-bet/GATA-3比值,并抑制Th1细胞因子IFN-γ的表达,这种调节作用随着SN治疗时间的延长而增强。
结论
SN治疗可有效控制轻、中度MsPGN患者蛋白尿,并升高补体C3水平,副作用小,但疗程至少要1月以上才有明显疗效。
MsPGN患者肾组织和PBMC上PD-1、PD-L1表达高于对照组,可能与MsPGN的免疫发病机制有关。SN可以抑制PD-1、PD-L1的病理性高表达,这种抑制作用随着SN浓度的增加、治疗时间的延长而增强,提示SN控制MsPGN患者蛋白尿的治疗机制可能与抑制PD-1、PD-L1表达有关。
MsPGN患者在T细胞选择性分化过程中,在转录调控水平和细胞因子水平存在Th1优势反应,这可能是MsPGN的重要免疫发病机制。而SN能抑制MsPGN患者T-bet表达,下调T-bet/GATA-3比值,并抑制Th1细胞因子IFN-γ的表达,从而在转录调控水平和细胞因子水平抑制Th1反应,恢复Th1/Th2的平衡。SN的这种调节作用随着SN浓度的增加、治疗时间的延长而增强。提示SN恢复MsPGN患者Th1/Th2平衡的免疫调节作用,可能是其能控制蛋白尿的重要的治疗机制。
Background
Experimental results from inflammatory renal diseases, such as allograft rejection, tubulointerstitial nephritis and glomerulonephritis, have suggested a prominent role of T cells in the injurious renal immune response. It is accepted that signals provide by TCR-peptide-MHC and co-stimulatory molecules are required for the optimal activation of T cells. Without sufficient co-stimulation, T cells are rendered anergy, tolerance or apoptosis. Recently, B7 superfamily molecules, PD-L1, PD-L2 and their receptor PD-1 (programmed death-1) have been identified. Previous studies indicated that the abnormality of PD-1/PD-L pathway might play a role in the development of autoimmune disease, allograft rejection, and various kinds of infection. Treatment aiming at regulating co-stimulatory pathway has now been recognized as a new way of immunotherapy.
Inappropriate T cell responses result from not only the abnormality of T cell activation but also the abnormality of T cell differentiation. Immune responses polarized by either Th1 or Th2 subset predominance result in the activation of different inflammatory effector pathways and disease outcomes. T-bet is a newly discovered Th1-specific transcription factor thought to initiate Th1 development, whereas GATA-3 plays a pivotal role in the development of the Th2 phenotype. It has been reported that the T-bet/GATA-3 ratio is representative of the balance between Th1 and Th2 cells. Maintaining the Th1/Th2 balance via the regulation of T-bet/GATA-3 ratio has been recognized as a promising therapy in the treatment of autoimmune diseases.
Sinomenine (SN), a pure alkaloid extracted from the Chinese medical plant Sinomenium acutum, has been effectively used for over ten years in the treatment of patients with chronic glomerulonephritis. Although sinomenine is effectively used in the clinic and is gaining international popularity, little is known about its exact mechanisms of action.
In this study, patients with mesangial proliferative nephritis (MsPGN), which is one of the most common pathological type of chronic GN, were examined. Both the PD-1/PD-L expression and the balanced state of Th1/Th2 were observed in patients with MsPGN. Then the effect of SN on proteinuria was detected. In order to elucidate the therapeutic mechanism of SN, the effect of SN on the expressions of PD-1/PD-L and T-bet/GATA-3 in peripheral blood mononuclear cells (PBMCs) and the serum levels of interferon-γ(IFN-γ), interleukin (IL)-4, and IL-10 were studied.
Patients and methods
1. Patients and renal specimens
A total of 25 patients with MsPGN and 10 healthy donors were enrolled in this study. Immunohistochemistry was enforced on the paraffin-embedded tissues from renal biopsies of 25 patients with MsPGN. Additionally, ten specimens obtained from pre-transplant renal biopsies and non-tumor kidney tissues from cancer patients served as controls.
2. methods
(1) In order to observe the expression pattern of PD-1/PD-L in patients with MsPGN, the intrarenal levels of PD-1, PD-L1, and PD-L2 were determined by immunohistochemistry and the expression levels of PD-1, PD-L1, and PD-L2 on PBMCs in both MsPGN patients and healthy donors were detected via real-time RT-PCR and flow cytometry.
(2) In order to observe if there is an imbalance between Th1 and Th2 cells in patients with MsPGN, the intrarenal levels of T-bet and GATA-3 were determined by immunohistochemistry, the expression levels of T-bet and GATA-3 in PBMCs in both MsPGN patients and healthy donors were detected via real-time RT-PCR, and the serum levels of IFN-γ, IL-4, and IL-10 were studied via ELISA.
(3) The effect of SN on the expression levels of PD-1, PD-L1, and PD-L2 on lymphocytes in vitro was studied via real-time RT-PCR and flow cytometry.
(4) The effect of SN on the expression levels of T-bet and GATA-3 on lymphocytes in vitro was studied via real-time RT-PCR and Western Blot. Additionally, ELISA was applied to detect the effect of SN on the supernatant levels of IFN-γ, IL-4, and IL-10.
(5) To evaluate the clinic effect of SN, blood biochemical parameters and proteinuria were detected at month 0, month 1, and month 3 in the course of treatment with SN.
(6) The effect of SN on the expression levels of PD-1, PD-L1, and PD-L2 on PBMCs in vivo was studied via real-time RT-PCR and flow cytometry at 1 month and 3 months after the start of treatment with SN.
(7) The effect of SN on the expression levels of T-bet and GATA-3 on PBMCs in vivo was studied via real-time RT-PCR and the serum levels of IFN-γ, IL-4, and IL-10 were studied by ELISA at 1 month and 3 months after the start of treatment with SN.
Results
1. Positive staining for PD-1 was detected on infiltrating lymphocytes in the interstitial space and PD-L1 was detected in tubular epithelial cells in renal tissues from MsPGN patients. However, the expression of PD-L2 was hardly observed in renal tissues in both MsPGN patients and control. Patients with MsPGN were found to have increased PD-1 and PD-L1 expression in both renal biopsy tissues and PBMCs compared with normal individuals.
2. Positive staining for both T-bet and GATA-3 was detected in tubular epithelial cells and infiltrating lymphocytes in the interstitial space. Patients with MsPGN were found to have increased T-bet and depressed GATA-3 expression in both renal biopsy tissues and PBMCs compared with normal individuals. Additionally, MsPGN patients were found to have elevated serum IFN-γvalues and decreased serum IL-10 values when compared with healthy donors.
3. The expression of PD-1 and PD-L1 on lymphocytes was suppressed by SN in vitro, and the inhibition effect was concentration dependent.
4. The expression of T-bet on lymphocytes was suppressed by SN in vitro, therefore, the T-bet/GATA-3 ratio was markedly decreased. SN was also found to markedly inhibit the secretion of IFN-γ. These effects above were concentration dependent.
5. SN could ameliorate proteinuria and increase the levels of Complement C3 in MsPGN patients with rare side effects.
6. SN could suppresse the expression of PD-1 and PD-L1 on PBMCs from MsPGN patients in vivo, and this effect was more significant at 3 months.
7. SN was found to cause a decrease in T-bet expression on PBMCs from MsPGN patients in vivo, resulting in a drop in the T-bet/GATA-3 ratio. SN could also inhibit the secretion of IFN-γ. These effects were more significant at 3 months.
Conclusions
SN could ameliorate proteinuria and increase the levels of Complement C3 in MsPGN patients with rare side effects, however, the course of treatment should last for more than 1 month.
Increased expression of PD-1 and PD-L1 in renal tissues and PBMCs from MsPGN patients may contribute to the development of MsPGN. Our results indicate that SN has the potential to inhibit the high expression of PD-1 and PD-L1 in PBMCs from MsPGN patients and thereby produce therapeutic effects.
A shift toward the Th1 pathway of Th cell activation at both transcriptional level and cytokine level occurs in MsPGN patients, and that SN has the potential to counter this shift in the Th1/Th2 balance and thereby produce therapeutic effects.
引文
1.郑法雷等.肾脏病临床与进展.人民军医出版社. 2005;64.
2. Kuroiwa T, Lee EG. Cellular interactions in the pathogenesis of lupus nephritis: the role of T cells and macrophages in the amplification of the inflammatory process in the kidney. Lupus. 1998;7:597-603.
3. Burne MJ, Daniels F, El Ghandour A, Mauiyyedi S, Colvin RB, O’Donnell MP, et al. Identification of the CD4(+) T cell as a major pathogenic factor in ischemic acute renal failure. J Clin Invest. 2001;108:1283–1290.
4. Christian K, Felix H, Veronika LK, et al. Role of T cells and dendritic cells in glomerular immunopathology. Semin Immunopathol. 2007;29:317-335.
5. Chen L. Co-inhibitory molecules of the B7-CD28 family in the control of T-cell immunity. Nat Rev Immunol. 2004;4:336–47.
6. Baxter AG, Hodgkin PD. Activation rules: the two-signal theories of immune activation. Nat Rev Immunol. 2002;2:439–446.
7. Carreno BM, Collins M. The B7 family of ligands and its receptors: new pathways for costimulation and inhibition of immune responses. Annu Rev Immunol. 2002;20:29–53.
8. Schwartz RH. A cell culture model for T lymphocyte clonal anergy. Science. 1990;248:1349–1356.
9. Seo SK, Seo HM, Jeong HY, et al. Co-inhibitory role of T-cell-associated B7-H1 and B7-DC in the T-cell immune response. Immunol Lett. 2006;102:222-228.
10. Chen Y, Zhang J, Li J, et al. Expression of B7-H1 in Inflammatory Renal Tubular Epithelial Cells. Nephron Exp Nephrol. 2005;102:81-92.
11. Zhang J, Chen Y, Li J, et al. Renal tubular epithelial expression of the coinhibitory molecule B7-DC (programmed death-1 ligand). J Nephrol. 2006;19:429-438.
12. Mataki N, Kikuchi K, Kawai T, et al. Expression of PD-1, PD-L1, and PD-L2 in the liver in autoimmune liver diseases. Am J Gastroenterol. 2007;102:302-312.
13. Piconi S, Trabattoni D, Saresella M, et al. Effects of specific immunotherapy on the B7 family of costimulatory molecules in allergic inflammation. J Immunol.2007;178:1931-1937.
14. Tipping PG, Kitching AR. Glomerulonephritis, Th1 and Th2: what's new? Clin Exp Immunol. 2005;142:207-215.
15. Jia L, Wang C, Kong H, et al. Effect of PA-MSHA vaccine on plasma phospholipids metabolic profiling and the ratio of Th2/Th1 cells within immune organ of mouse IgA nephropathy.J Pharm Biomed Anal.2007;43:646-654.
16. Szabo SJ, Kim ST, Costa GL, et al. A novel transcription factor, T-bet, directs Th1 lineage commit-ment. Cell. 2000;100:655-669.
17. Zheng W, Flavell RA. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell. 1997;89:587-596.
18. Yu HR, Chang JC, Chen RF, et al. Different antigens trigger different Th1/Th2 reactions in neonatal mononuclear cells (MNCs) relating to T-bet/GATA-3 expression. J Leukoc Biol. 2003;74:952-958.
19. Chakir H, Wang H, Lefebvre DE, et al. T-bet/GATA-3 ratio as a measure of the Th1/Th2 cytokine profile in mixed cell population:Predominant role of GATA-3. J Immunol Methods. 2003;278:157-169.
20. Yamasaki H. Pharmacology of sinomenine, an anti-rheumatic alkaloid from Sinomenium acutum. Acta Med Okayama 1976;30:1–20.
21. 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;7:637-645.
22. Shu L, Yin W, Zhang J, et al. Sinomenine inhibits primary CD4 (+) T-cell proliferation via apoptosis. Cell Biol Int. 2007;31:784-789.
23. Liu L, Riese J, Resch K, Kaever V. Impairment of macrophage eicosanoid and nitric oxide production by an alkaloid from Sinomenium acutum. Arzneimittelforschung. 1994;44:1223–1226.
24. Dai YB, Liu HX, Zao LI. Immunosuppressive effect of Sinomenine on ICAM-1 expression in rat renal allograf t rejection. Mod J Integr Tradit Chin West Med. 2003;12:1358–1361.
25. Feng H, Yamaki K, Takano H, Suppression of Th1 and Th2 immune responses in mice by Sinomenine, an alkaloid extracted from the chinese medicinal plant Sinomeniumacutum. Planta Med. 2006;72:1383-1388.
26. Wang Y, Fang Y, Huang W, Effect of sinomenine on cytokine expression of macrophages and synoviocytes in adjuvant arthritis rats. J Ethnopharmacol. 2005;98:37-43.
27. Mark W, Schneeberger S, Seiler R, Sinomenine blocks tissue remodeling in a rat model of chronic cardiac allograft rejection. Transplantation. 2003;75:940-945.
28.杨汝春,王永钧,林京莲,等.青藤碱对白介素1β诱导的肾小管上皮细胞骨架变化和明胶酶活性的影响.中华肾脏病杂志. 2006;22:499-502.
29. Liu L, Buchner E, Beitze D, Schmidt-Weber CB, Kaever V, Emmrich F, et al. Amelioration of rat experimental arthritides by treatment with the alkaloid sinomenine. Int J Immunopharmacol. 1996;18:529–543.
30. Kondo Y, Takano F, Yoshida K, Hojo H. Protection by sinomenine against endotoxin-induced fulminant hepatitis in galactosamine-sensitized mice. Biochem Pharmacol. 1994;48:1050–1052.
31. Yang P, Yang L, Luo Z. Effects of Sinomenine on T lymphocyte subsets in rat renal allograft transplantation model. J Clin Urol. 2003;10:620–622.
32. Yongwen Chen, Jingyi Li, Jingbo Zhang, et al. Sinomenine inhibits B7-H1 and B7-DC expression on human renal tubular epithelial cells. International Immunopharmacology. 2005;5:1446– 1457.
33. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real- time quantitative PCR and the 2(- Delta Delta C (T)) Method. Methods. 2001;25: 402-408.
34. Zhang X, Schwartz JC, Guo XL, et al. Structural and functional analysis of the costimulatory receptor programmed death-1. Immunity. 2004;20:337-347.
35. Shinohara T, Taniwaki M, Ishida Y, et al. Structure and chromosomal localization of the human PD-1 gene (PDCD1). Genomics. 1994;23:704-706.
36. Dong HD, Zhu GF, Tamada K, et al. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat Med. 1999;5:1365-1369.
37. Freeman G, Long AJ, Iwai Y, et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med. 2000;192:1027-1034.
38. Latchman Y, Wood CR, Chernova T, et al. PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nat Immunol. 2001;2:261-268.
39. Anthony JC, Jose C, Gutierrez R. The expanding B7 superfamily:increasing complexity in costimulatory signals regulating T cell function. Nature Immunol. 2001;3:203-209.
40. Okazaki T, Maeda A, Nishimura H, et al. PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting Srchomology 2-domain-containing tyrosine phosphatase 2 to phosphotyrodine. Proc Natl Acad Sci USA. 2001;98:13866-13871.
41. Wang SH, Lin CH, Li RN, et al. Polymorphisms of Genes for Programmed Cell Death 1 Ligands in Patients with Rheumatoid Arthritis. J Clin Immunol. 2007;27:563-567.
42. Wang SH, Chen YJ, Ou TT, et al. Programmed Death-1 Gene Polymorphisms in Patients With Systemic Lupus Erythematosus in Taiwan. J Clin Immunol. 2006; 26:506-511.
43. Nielsen C, Hansen D, Husby S, et al. Association of a putative regulatory polymorphism in the PD-1 gene with susceptibility to type 1 diabetes. Tissue Antigens. 2003;62:492-497.
44. Nishimura H, Nose M, Hiai H, et al. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity. 1999;11:141-151.
45. Nishimura H, Okazaki T, Tanaka Y, et al. Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science. 2001;291:319-322.
46. Tamura H, Dong H, Zhu G, et al. B7–H1 costimulation preferentially enhances CD28-independent T-helper cell function. Blood. 2001;97:1809-1816.
47. Subudhi SK, Zhou P, Yerian LM, et al. Local expression of B7-H1 promotes organ-specific autoimmunity and transplant rejection. J Clin Invest. 2004;113:694-700.
48. Norikazu M, Kentaro K, Toshihiro K, et al. Expression of PD-1, PD-L1, and PD-L2 in the liver in autoimmune liver diseases. Am J Gastroenterol. 2007;102:302-312.
49. Takanori K, Teruji T, Koji U, et al. Blockade of B7-H1 suppresses the development of chronic intestinal inflammation. J Immunol. 2003;171:4156-4163.
50. Menke J, Lucas JA, Zeller GC. Programmed death 1 ligand (PD-L) 1 and PD-L2 limit autoimmune kidney disease: distinct roles. J Immunol. 2007;179:7466-7477.
51. Mackay CR. Homing of native, memory and effector lymphocytes. Curr Opin Immunol.1993;5:423-427.
52. Masopust D, Vezys V, Marzo A.L, et al. Preperential localization of effector memory cells in nonlymphoid tissue. Science. 2001;291:2413-2417.
53. Yamazaki T, Akiba H, Iwai H, et al. Expression of programmed death 1 ligands by murine T cells and APC. J Immunol. 2002;169:5538-5545.
54. Zhu B, Guleria I, Khosroshahi A, et al. Differential role of programmed death-ligand 1 and programmed death-ligand 2 in regulating the susceptibility and chronic progression of experimental autoimmune encephalomyelitis. J Immunol. 2006;176:3480-3489.
55. Matsumoto K, Inoue H, Nakano T, et al. B7-DC regulates asthmatic response by an IFN- -dependent mechanism. J Immunol. 2004;172:2530-2541.
56. Agata Y, Kawasaki A, Nishimura H, et al. Expression of the PD-1 antigen on the surface of stimulated mouse T and B lymphocytes. Int Immunol. 1996;8:765-772.
57. Evans A, Cooksley H, Phillips S, et al. Programmed death 1 expression during antiviral treatment of chronic hepatitis B: Impact of hepatitis B e-antigen seroconversion. Hepatology. 2008;48:759-769.
58.彭国平,孙雯,吴炜,等.慢性乙型肝炎患者外周血树突状细胞PD-L1表达的相关研究.浙江大学学报. 2008; 37:364-372.
59. Dong H, Chen X. Immunoregulatory role of B7-H1 in chronicity of inflammatory responses. Cell Mol Immunol. 2006;3:179-187.
60. Tim M, Bettina S, Thomas K, et al. Microglial expression of the B7 family member B7 Homolog 1 confers strong immune inhibition:implications for immune responses and autoimmunity in the CNS. J Neuroscience. 2005;25:2537-2546.
61. Ding H, Wu X, Gao W. PD-L1 is expressed by human renal tubular epithelial cells and suppresses T cell cytokine synthesis. Clinical Immunology. 2005;115:184-191.
62. Waeckerle MY, Starke A, Wuthrich RP. PD-L1 partially protects renal tubular epithelial cells from the attack of CD8+ cytotoxic T cells. Nephrol Dial Transplant. 2007;22:1527-1536.
63. Beswick EJ, Pinchuk IV, Das S, et al. B7-H1 expression on gastric epithelial cells after Helicobacter pylori exposure promotes the development of CD4+CD25+FoxP3+ regulatory T cells. Infect Immun. 2007;75:4334-4341.
64. Seo SK, Seo HM, Jeong HY, et al. Co-inhibitory role of T-cell-associated B7-H1 and
B7-DC in the T-cell immune response. Immunol Lett. 2006;102:222-228.
65. Chen Y, Yang C, Jin N, et al. Sinomenine promotes differentiation but impedes maturation and co-stimulatory molecule expression of human monocyte-derived dendritic cells. International Immunopharmacology. 2007;7:1102-1110.
66. Dong H, Strome SE, Matteson EL, et al. Costimulating aberrant T cell responses by B7-H1 autoantibodies in rheumatoid arthritis. J Clin Invest. 2003;111:363-370.
67. Yamazaki T, Akiba H, Koyanagi A, et al. Blockade of B7-H1 on macrophages suppresses CD4+ T cell proliferation by augmenting IFN-gamma-induced nitric oxide production. J Immunol. 2005;175:1586-1592.
68. Nishimura H, Nose M, Hiai H, et al. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity. 1999;11:141-151.
69. Sharpe AH, Freeman GJ. The B7-CD28 superfamily. Nat Rev Immunol. 2002;2:116-126.
70. Wang S, Bajorath J, Flies DB, et al. Molecular modeling and functional mapping of B7–H1 and B7-DC uncouple costimulatory function from PD-1 interaction. J Exp Med. 2003;197:1083-1091.
71. Holdsworth SR, Kitching AR, Tipping PG. Th1 and Th2 T helper cell subsets affect patterns of injury and outcomes in glomerulonephritis. Kidney Int. 1999;55:1198-1216.
72. Mosmann TR, Cherwinski H, Bond MW, et al. Two type of murine helper T cell clone.Ⅰ.Definition according to profies of activities and secreted proteins. J Imunol. 1986;136:2348-2357.
73. Arakawa S, Hatano Y, Katagiri K, et al. Differential expression of mRNA for Th1 and Th2 cytokine-associated transcription factors and suppressors of cytokine signaling in peripheral blood mononuclear cells of patients with atopic dermatitis. Clin Exp Immunol. 2004;135:505-510.
74. Nakamura Y, Hoshino M. TH2 cytokines and associated transcription factors as therapeutic targets in asthma.. Curr Drug Targets Inflamm Allergy.2005;4:267-270.
75. Horiuchi Y, Hanazawa A, Nakajima Y, et al. T-helper (Th)1/Th2 imbalance in the peripheral blood of dogs with malignant tumor. Microbiol Immunol. 2007; 51:1135-1138.
76. Li B, Yang P, Zhou H, et al. T-bet expression is upregulated in active Behcet’s disease. Br J Ophthalmol. 2003;87:1264-1267.
77. Obata F, Yoshida K, Ohkubo M, et al. Contribution of CD4+ and CD8+ T cells and interferon-gamma to the progress of chronic rejection of kidney allografts: the Th1 response mediates both acute and chronic rejection.Transpl Immunol. 2005;14:21-25.
78. Cousins DJ, Lee TH, Staynov DZ. Cytokine coexpression during human Th1/Th2 cell differentiation:direct evidence for coordinated expression of Th2 cytokines. J Immunol. 2002;169:2498-2506.
79. Yu HR, Chang JC, Chen RF, et al. Different antigens trigger different Th1/Th2 reactions in neonatal mononuclear cells (MNCs) relating to T-bet/GATA-3 expression. J Leukoc Biol. 2003;74:952-958.
80. Chakir H, Wang H, Lefebvre DE, et al. T-bet/GATA-3 ratio as a measure of the Th1/Th2 cytokine profile in mixed cell population:Predominant role of GATA-3. J Immunol Methods. 2003;278:157-169.
81. Chan RW, Lai FM, Li EK, et al. Imbalance of Th1/Th2 transcription factors in patients with lupus nephritis. Rheumatology. 2006;45:951-957.
82. Segelmark M, Butkowski R, Wieslander J. Antigen restriction and IgG subclasses among anti-GBM autoantibodies. Nephrol Dial Transplant. 1990;5:991-996.
83. Noel LH, Aucouturier P, Monteiro RC, Preud’Homme JL, et al. Glomerular and serum immunoglobulin G subclasses in membranous nephropathy and anti-glomerular basement membrane nephritis. Clin Immunol Immunopathol. 1988;46:186-194.
84. Graninger WB, Hassfeld W, Pesau BB, et al. Induction of systemic lupus erythematosus by interferon-gamma in a patient with rheumatoid arthritis. J Rheumatol. 1991;18:1621-1622.
85. Machold KP, Smolen JS. Interferon-gamma induced exacerbation of systemic lupus erythematosus. J Rheumatol. 1990;17:831-832.
86. Parker MG, Atkins MB, Ucci AA, et al. Rapidly progressive glomerulonephritis after immunotherapy for cancer. J Am Soc Nephrol. 1995;5:1740-1744.
87. Feuerer M, Eulenburg K, Loddenkemper C, et al. Self-limitation of Th1-mediated inflammation by IFN-gamma. J Immunol. 2006;176:2857-2863.
88.向莉,高小夏,潘家荣.慢性肾小球肾炎患者血清IFN-γ、IL-10的变化及意义.中国临床医学. 2006;13:269-272.
89. Couper K N, Blount D G, Riley E M. IL-10:the master regulator of immunity to infection. J Immunol. 2008;180:5771-5777.
90.周燕斌,李幼姬,吴玉红.狼疮肾炎患者血清及外周血单个核细胞中IL-10和γ干扰素的表达及意义.中国现代医学杂志. 2006;16:529-532.
91. Tipping P G, Holdsworth S R. Cytokines in glomerulonephritis. Semin Nephrol. 2007;27:275-285.
92. Tipping P G, Kitching A R. Glomerulonephritis, Th1 and Th2: what’s new. Clin Exp Immunol. 2005;142:207-215.
93. Graninger W B, Hassfeld W, Pesau B B, et al. Induction of systemic lupus erythematosus by interferon-gamma in a patient with rheumatoid arthritis. J Rheumatol. 1991;18:1621-1622.
94. Machold K P, Smolen J S. Interferon-gamma induced exacerbation of systemic lupus erythematosus. J Rheumatol. 1990;17:831-832.
95. Parker M G, Atkins M B, Ucci A A, et al. Rapidly progressive glomerulonephritis after immunotherapy for cancer. J Am Soc Nephrol. 1995;5:1740-1744.
96. Kawasaki Y, Suzuki J, Sakai N, et al. Evaluation of T helper-1/-2 balance on the basis of IgG subclasses and serum cytokines in children with glomerulonephritis. Am J Kidney Dis. 2004;44:42-49.
97.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;20:249-258.
98. Shimohata H, Yamada A, Yoh K, et al. Overexpression of T-bet in T cells accelerates autoimmune glomerulonephritis in mice with a dominant Th1 background. J Nephrol. 2009;22:123-129.
99. Zeng Y, Gu B, Ji X, et al. Sinomenine, an antirheumatic alkaloid, ameliorates clinical signs of disease in the Lewis rat model of acute experimental autoimmune encephalolmyelitis. Biol Pharm Bull. 2007;30:1438-1444.
100.戴英波,黄循,罗志刚,等.青藤碱对大鼠肾移植急性排斥反应的抑制作用.中国现代医学杂志. 2004;14:49-54.
101.王毅,熊烈,陈正,等.青藤碱对肾移植大鼠Th1细胞生物学特性的影响.美国中华临床医学杂志. 2004;6:4-6.
1. Christian K, Felix H, Veronika LK, et al. Role of T cells and dendritic cells in glomerular immunopathology. Semin Immunopathol. 2007;29:317-335.
2. Liang SC, Latchman YE, Buhimann JE, et al. Regulation of PD-1, PD-L1, and PD-L2 expression during normal and autoimmune responses. Eur J Immunol. 2003;33:2706-2716.
3. Nishimura H, Nose M, Hiai H, et al. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity. 1999;11:141-151.
4. Chen Y, Zhang J, Li J, et al. Expression of B7-H1 in Inflammatory Renal Tubular Epithelial Cells. Nephron Exp Nephrol. 2005;11;102:81-92.
5. Didier AM, Mohamed HS. Role of novel T-cell costimulatory pathways in transplantation. Curr Opin Organ Transplant. 2003;8:25-33.
6. Lindsten T, Lee KP, Harris ES, et al. Characterization of CTLA-4 structure and expression on human T cells. J Immunol. 1993;151:3498-3499.
7. Complexities of CD28/B72:CTLA-4 costimulatory pathways in antoimmunity and transplantation. Annu Rev Immunol. 2001;19:225-252.
8. Niemann M, Mueller A, Yard BA, et al. B7-1(CD80) and B7-2(CD86) expression in human tubular epithelial cells in vivo and in vitro. Nephron. 2002;92:542-556.
9. Wu Q, Jinde K, Endoh M, et al. Clinical significance of costimulatory molecules CD80/CD86 expression in IgA nephropathy. Kidney Int. 2004;65:888-896.
10. Wu Q, Jinde K, Endoh M, et al. Costimulatory molecules CD80 and CD86 in human crescentic glomerulonephritis. Am J Kidney Dis. 2003;41:950-961.
11. Jochen R, Gero VG, Martin L, et al. Induction of B7-1 in podocytes is associated with nephrotic syndrome. J Clin Invest. 2004;113:1390-1397.
12. Singbartl K, Bockhorn SG, Zarbock A, et al. T cells modulate neutrophil-dependent acute renal failure during endotoxemia:critical role for CD28. J Am Soc Nephrol. 2005;16:720-728.
13. Laskowski IA, Pratschke J, Wilhelm MJ, et al. Anti-CD28 monoclonal antibody therapyprevents chronic rejection of renal allografts in rats. J Am Soc Nephrol. 2002;13:519-527.
14. Vincenti F, Luggen M. T cell costimulation:a rational target in the therapeutic armamentarium for autoimmune diseases and tranaplantation. Annu Rev Med. 2007;58:347-358.
15. Cunnane G, Chan OT, Cassafer G, et al. Prevention of renal damage in murine lupus nephritis by CTLA-4Ig and cyclophosphamide. Arthritis Rheum. 2004;50:1539-1548.
16. Anthony JC, Jose C, Gutierrez R. The expanding B7 superfamily:increasing complexity in costimulatory signals regulating T cell function. Nature Immunol. 2001;3:203-209.
17. Zhang X, Schwartz JC, Guo XL, et al. Structural and functional analysis of the costimulatory receptor programmed death-1. Immunity. 2004;20:337-347.
18. Seo SK, Seo HM, Jeong HY, et al. Co-inhibitory role of T-cell-associated B7-H1 and B7-DC in the T-cell immune response. Immunol Lett. 2006;102:222-228.
19. Okazaki T, Maeda A, Nishimura H, et al. PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting Srchomology 2-domain-containing tyrosine phosphatase 2 to phosphotyrodine. Proc Natl Acad Sci USA. 2001;98:13866-13871.
20. Freeman GJ, Long AJ, Iwai Y, et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med. 2000;192:1027-1037.
21. Nishimura H, Okazaki T, Tanaka Y, et al. Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science. 2001;291:319-322.
22. Subudhi SK, Zhou P, Yerian LM, et al. Local expression of B7-H1 promotes organ-specific autoimmunity and transplant rejection. J Clin Invest. 2004;113:694-700.
23. Zhang J, Chen Y, Li J, et al. Renal tubular epithelial expression of the coinhibitory molecule B7-DC (programmed death-1 ligand). J Nephrol. 2006;19:429-438.
24. Yongwen Chen, Jingyi Li, Jingbo Zhang, et al. Sinomenine inhibits B7-H1 and B7-DC expression on human renal tubular epithelial cells. International Immunopharmacology. 2005;5:1446-1457.
25. Waeckerle MY, Starke A, Wuthrich RP. PD-L1 partially protects renal tubular epithelial cells from the attack of CD8+ cytotoxic T cells. Nephrol Dial Transplant. 2007;22:1527-1536.
26. Hanlu D, Xiongfei W, Jun W. Delivering PD-1 inhibitory signal concomitant with blocking ICOS co-stimulation suppresses lupus-like syndrome in autoimmune BXSB mice. Clinical Immunology. 2006;118:258-267.
27. Coyle AJ, Lehar S, Loyd C, et al. The CD28-related molecule ICOS is required for effective T cell-dependent immune responses. Immunity. 2000;13:95-105.
28. Wahl P, Schoop R, Bilic G, et al. Renal tubular epithelial expression of the costimulatory molecule B7RP-1(inducible costimulatory ligand). I Am Soc Nephrol. 2002;13:1517-1526.
29. De HS, Woltman AM, Trouw LA, et al. Renal tubular epithelial cells modulate T-cell responses via ICOS-L and B7-H1. Kidney Int. 2005;68:2091-2102.
30. Wahl P, Wuthrich RP. Role of the B7RP-1/ICOS pathway in renal tubular epithelial antigen presentation to CD4+ Th1 and Th2 cells. Nephron Exp Nephrol. 2004;98:31-38.
31. Toubi E, Shoenfeld Y. The role of CD40-CD154 interactions in autoimmunity and the benefit of disrupting this pathway. Autoimmunity. 2004;37:457-464.
32. Revnolds J, Khan SB, Allen AR, et al. Blockade of the CD154-CD40 costimulatory pathway prevents the development of experimental autoimmune glomerulonephritis. Kidney Int. 2004;66:1444-1452.
33. Boumpas DT, Furie R, Manzi S, et al. A short course of BG9588(anti-CD40 ligand antibody) improves serologic activity and decreases hematuria in patients with proliferative lupus glomerulonephritis. Arthritis Rheum. 2003;48:719-727.
34. Kairaitis L, Wang Y, Zheng L, et al. Blockade of CD40-CD40 ligand protects against renal injury in chronic proteinuric renal disease. Kidney Int. 2003,64:1265-1272.
35. Pearson TC, Trambley J, Odom K, et al. Anti-CD40 therapy extends renal allograft survival in rhesus macaques. Transplantation. 2002;74:933-940.
1.中国医药大词典.香港,建文书局出版社. 1963:1171.
2. Wang Y, Fang Y, Huang W, Effect of sinomenine on cytokine expression of macrophages and synoviocytes in adjuvant arthritis rats. J Ethnopharmacol. 2005;98:37-43.
3. Mark W, Schneeberger S, Seiler R, Sinomenine blocks tissue remodeling in a rat model of chronic cardiac allograft rejection. Transplantation. 2003;75:940-945.
4.扬汝春,王永钧,林京莲,等.青藤碱对白介素1β诱导的肾小管上皮细胞骨架变化和明胶酶活性的影响.中国肾脏病杂志杂志. 2006;22:499-502.
5. 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: 637-645.
6. Shu L, Yin W, Zhang J, et al. Sinomenine inhibits primary CD4 (+) T-cell proliferation via apoptosis. Cell Biol Int. 2007;31: 784-789.
7. Feng H, Yamaki K, Takano H, Suppression of Th1 and Th2 immune responses in mice by Sinomenine, an alkaloid extracted from the chinese medicinal plant Sinomenium acutum. Planta Med. 2006;72: 1383-1388.
8.中华人民共和国药典(一部).广东科技出版社. 1995:165.
9.叶木荣,刘良,曾元儿,等.青藤碱在大鼠体内分布与脏器毒理的关系研究.中国药理学通报. 2001;17:65-69.
10.莫志贤,周吉银,王彩云.青藤碱的身体依赖性和精神依赖性实验研究.中国药物滥用防治杂志. 2004;10:190-193.
11.邱赛红,黄宇明,仇萍,等.青藤碱致突变性的实验研究.中药药理与临床. 1999;15:23-24.
12.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;20:249-258.
13.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;21:573-574.
14. Shu L, Yin W, Zhang J, et al. Sinomenine inhibits primary CD4+ T-cell proliferation viaapoptosis. Cell Biol Int. 2007;31:784-789.
15. He X, Wang J, Guo Z, et al. Requirement for ERK activation in sinomenine-induced apoptosis of macrophages. Immunol Lett. 2005;98:91-96.
16.徐道华,周晨慧.树突状细胞CD80和CD86表达及胞外白细胞介素12分泌与不同剂量青藤碱干预的影响.中国组织工程研究与临床康复. 2007;11:5654-5656.
17.杨承英,陈永文,傅晓岚,等.青藤碱促进树突状细胞分化抑制其成熟.免疫学杂志. 2007;23:265-269.
18.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-κB活性的影响.广东医学. 2006;27:55-57.
19.高永翔,龚立,田艳勋,等.青藤碱对小鼠骨髓树突状细胞免疫功能的影响.成都中医药大学学报. 2005;28:13-14.
20. Wang Y, Fang Y, Huang W, et al. Effect of sinomenine on cytokine expression of macrophages and synoviocytes in adjuvant arthritis rats. J Ethnopharmacol. 2005;98:37-43.
21. Zeng Y, Gu B, Ji X, et al. Sinomenine, an antirheumatic alkaloid, ameliorates clinical signs of disease in the Lewis rat model of acute experimental autoimmune encephalolmyelitis. Biol Pharm Bull. 2007;30:1438-1444.
22.戴英波,黄循,罗志刚,等.青藤碱对大鼠肾移植急性排斥反应的抑制作用.中国现代医学杂志. 2004;14:49-54.
23.王毅,熊烈,陈正,等.青藤碱对肾移植大鼠Th1细胞生物学特性的影响.中华临床医学杂志. 2004;6:4-6.
24. Kuroiwa T, Lee EG. Cellular interactions in the pathogenesis of lupus nephritis: the role of T cells and macrophages in the amplification of the inflammatory process in the kidney. Lupus. 1998;7:597-603.
25. Chen L. Co-inhibitory molecules of the B7-CD28 family in the control of T-cell immunity. Nat Rev Immunol. 2004;4:336-347.
26. Seo SK, Seo HM, Jeong HY, et al. Co-inhibitory role of T-cell-associated B7-H1 and B7-DC in the T-cell immune response. Immunol Lett. 2006;102:222-228.
27. Su KS, Hyoun MS, Hye YJ, et al. Co-inhibitory role of T-cell-associated B7-H1 and B7-DC in the T-cell immune response.Imlet.2005;9:007-013.
28. Mataki N, Kikuchi K, Kawai T, et al. Expression of PD-1, PD-L1, and PD-L2 in theliver in autoimmune liver diseases. Am J Gastroenterol. 2007;102:302-312.
29. Piconi S, Trabattoni D, Saresella M, et al. Effects of specific immunotherapy on the B7 family of costimulatory molecules in allergic inflammation. J Immunol. 2007;178:1931-1937.
30. Chen Y, Zhang J, Li J, et al. Expression of B7-H1 in Inflammatory Renal Tubular Epithelial Cells. Nephron Exp Nephrol. 2005;102:81-92.
31. Zhang J, Chen Y, Li J, et al. Renal tubular epithelial expression of the coinhibitory molecule B7-DC (programmed death-1 ligand). J Nephrol. 2006;19:429-38.
32.余克强,赵毅,李娟,等.青藤碱对类风湿关节炎树突状细胞CD80mRNA、CD86mRNA表达的影响.中国药房. 2006;17:331-333.
33.梁瑞燕,曹柳英,王文君,等.青藤碱抗炎作用机理研究.广州中医药大学学报. 2007;24:141-143.
34.王文君,王培训.青藤碱对环氧化酶2活性的选择性抑制作用.广州中医药大学学报. 2002;19:46-47.
35.陈炜,沈悦娣,赵光树,等.青藤碱对脂多糖诱导的神经细胞环氧化酶-2表达的影响.中国中药杂志. 2004;29:900-903.
36.徐广全,王斌,金相元,等.青藤碱对心脏移植大鼠急性排斥反应及环氧化酶2活性的影响.中华医学杂志. 2006;86:911-914.
37. Mark W, Schneeberger S, Seiler R, et al. Sinomenine blocks tissue remodeling in a rat model of chronic cardiac allograft rejection. Transplantation. 2003;75:940-945.
38. Kok TW, Yue PY, Mak NK, et al. The anti-angiogenic effect of sinomenine. Angiogenesis. 2005;8:3-12.
2. Kuroiwa T, Lee EG. Cellular interactions in the pathogenesis of lupus nephritis: the role of T cells and macrophages in the amplification of the inflammatory process in the kidney. Lupus. 1998;7:597-603.
3. Burne MJ, Daniels F, El Ghandour A, Mauiyyedi S, Colvin RB, O’Donnell MP, et al. Identification of the CD4(+) T cell as a major pathogenic factor in ischemic acute renal failure. J Clin Invest. 2001;108:1283–1290.
4. Christian K, Felix H, Veronika LK, et al. Role of T cells and dendritic cells in glomerular immunopathology. Semin Immunopathol. 2007;29:317-335.
5. Chen L. Co-inhibitory molecules of the B7-CD28 family in the control of T-cell immunity. Nat Rev Immunol. 2004;4:336–47.
6. Baxter AG, Hodgkin PD. Activation rules: the two-signal theories of immune activation. Nat Rev Immunol. 2002;2:439–446.
7. Carreno BM, Collins M. The B7 family of ligands and its receptors: new pathways for costimulation and inhibition of immune responses. Annu Rev Immunol. 2002;20:29–53.
8. Schwartz RH. A cell culture model for T lymphocyte clonal anergy. Science. 1990;248:1349–1356.
9. Seo SK, Seo HM, Jeong HY, et al. Co-inhibitory role of T-cell-associated B7-H1 and B7-DC in the T-cell immune response. Immunol Lett. 2006;102:222-228.
10. Chen Y, Zhang J, Li J, et al. Expression of B7-H1 in Inflammatory Renal Tubular Epithelial Cells. Nephron Exp Nephrol. 2005;102:81-92.
11. Zhang J, Chen Y, Li J, et al. Renal tubular epithelial expression of the coinhibitory molecule B7-DC (programmed death-1 ligand). J Nephrol. 2006;19:429-438.
12. Mataki N, Kikuchi K, Kawai T, et al. Expression of PD-1, PD-L1, and PD-L2 in the liver in autoimmune liver diseases. Am J Gastroenterol. 2007;102:302-312.
13. Piconi S, Trabattoni D, Saresella M, et al. Effects of specific immunotherapy on the B7 family of costimulatory molecules in allergic inflammation. J Immunol.2007;178:1931-1937.
14. Tipping PG, Kitching AR. Glomerulonephritis, Th1 and Th2: what's new? Clin Exp Immunol. 2005;142:207-215.
15. Jia L, Wang C, Kong H, et al. Effect of PA-MSHA vaccine on plasma phospholipids metabolic profiling and the ratio of Th2/Th1 cells within immune organ of mouse IgA nephropathy.J Pharm Biomed Anal.2007;43:646-654.
16. Szabo SJ, Kim ST, Costa GL, et al. A novel transcription factor, T-bet, directs Th1 lineage commit-ment. Cell. 2000;100:655-669.
17. Zheng W, Flavell RA. The transcription factor GATA-3 is necessary and sufficient for Th2 cytokine gene expression in CD4 T cells. Cell. 1997;89:587-596.
18. Yu HR, Chang JC, Chen RF, et al. Different antigens trigger different Th1/Th2 reactions in neonatal mononuclear cells (MNCs) relating to T-bet/GATA-3 expression. J Leukoc Biol. 2003;74:952-958.
19. Chakir H, Wang H, Lefebvre DE, et al. T-bet/GATA-3 ratio as a measure of the Th1/Th2 cytokine profile in mixed cell population:Predominant role of GATA-3. J Immunol Methods. 2003;278:157-169.
20. Yamasaki H. Pharmacology of sinomenine, an anti-rheumatic alkaloid from Sinomenium acutum. Acta Med Okayama 1976;30:1–20.
21. 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;7:637-645.
22. Shu L, Yin W, Zhang J, et al. Sinomenine inhibits primary CD4 (+) T-cell proliferation via apoptosis. Cell Biol Int. 2007;31:784-789.
23. Liu L, Riese J, Resch K, Kaever V. Impairment of macrophage eicosanoid and nitric oxide production by an alkaloid from Sinomenium acutum. Arzneimittelforschung. 1994;44:1223–1226.
24. Dai YB, Liu HX, Zao LI. Immunosuppressive effect of Sinomenine on ICAM-1 expression in rat renal allograf t rejection. Mod J Integr Tradit Chin West Med. 2003;12:1358–1361.
25. Feng H, Yamaki K, Takano H, Suppression of Th1 and Th2 immune responses in mice by Sinomenine, an alkaloid extracted from the chinese medicinal plant Sinomeniumacutum. Planta Med. 2006;72:1383-1388.
26. Wang Y, Fang Y, Huang W, Effect of sinomenine on cytokine expression of macrophages and synoviocytes in adjuvant arthritis rats. J Ethnopharmacol. 2005;98:37-43.
27. Mark W, Schneeberger S, Seiler R, Sinomenine blocks tissue remodeling in a rat model of chronic cardiac allograft rejection. Transplantation. 2003;75:940-945.
28.杨汝春,王永钧,林京莲,等.青藤碱对白介素1β诱导的肾小管上皮细胞骨架变化和明胶酶活性的影响.中华肾脏病杂志. 2006;22:499-502.
29. Liu L, Buchner E, Beitze D, Schmidt-Weber CB, Kaever V, Emmrich F, et al. Amelioration of rat experimental arthritides by treatment with the alkaloid sinomenine. Int J Immunopharmacol. 1996;18:529–543.
30. Kondo Y, Takano F, Yoshida K, Hojo H. Protection by sinomenine against endotoxin-induced fulminant hepatitis in galactosamine-sensitized mice. Biochem Pharmacol. 1994;48:1050–1052.
31. Yang P, Yang L, Luo Z. Effects of Sinomenine on T lymphocyte subsets in rat renal allograft transplantation model. J Clin Urol. 2003;10:620–622.
32. Yongwen Chen, Jingyi Li, Jingbo Zhang, et al. Sinomenine inhibits B7-H1 and B7-DC expression on human renal tubular epithelial cells. International Immunopharmacology. 2005;5:1446– 1457.
33. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real- time quantitative PCR and the 2(- Delta Delta C (T)) Method. Methods. 2001;25: 402-408.
34. Zhang X, Schwartz JC, Guo XL, et al. Structural and functional analysis of the costimulatory receptor programmed death-1. Immunity. 2004;20:337-347.
35. Shinohara T, Taniwaki M, Ishida Y, et al. Structure and chromosomal localization of the human PD-1 gene (PDCD1). Genomics. 1994;23:704-706.
36. Dong HD, Zhu GF, Tamada K, et al. B7-H1, a third member of the B7 family, co-stimulates T-cell proliferation and interleukin-10 secretion. Nat Med. 1999;5:1365-1369.
37. Freeman G, Long AJ, Iwai Y, et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med. 2000;192:1027-1034.
38. Latchman Y, Wood CR, Chernova T, et al. PD-L2 is a second ligand for PD-1 and inhibits T cell activation. Nat Immunol. 2001;2:261-268.
39. Anthony JC, Jose C, Gutierrez R. The expanding B7 superfamily:increasing complexity in costimulatory signals regulating T cell function. Nature Immunol. 2001;3:203-209.
40. Okazaki T, Maeda A, Nishimura H, et al. PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting Srchomology 2-domain-containing tyrosine phosphatase 2 to phosphotyrodine. Proc Natl Acad Sci USA. 2001;98:13866-13871.
41. Wang SH, Lin CH, Li RN, et al. Polymorphisms of Genes for Programmed Cell Death 1 Ligands in Patients with Rheumatoid Arthritis. J Clin Immunol. 2007;27:563-567.
42. Wang SH, Chen YJ, Ou TT, et al. Programmed Death-1 Gene Polymorphisms in Patients With Systemic Lupus Erythematosus in Taiwan. J Clin Immunol. 2006; 26:506-511.
43. Nielsen C, Hansen D, Husby S, et al. Association of a putative regulatory polymorphism in the PD-1 gene with susceptibility to type 1 diabetes. Tissue Antigens. 2003;62:492-497.
44. Nishimura H, Nose M, Hiai H, et al. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity. 1999;11:141-151.
45. Nishimura H, Okazaki T, Tanaka Y, et al. Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science. 2001;291:319-322.
46. Tamura H, Dong H, Zhu G, et al. B7–H1 costimulation preferentially enhances CD28-independent T-helper cell function. Blood. 2001;97:1809-1816.
47. Subudhi SK, Zhou P, Yerian LM, et al. Local expression of B7-H1 promotes organ-specific autoimmunity and transplant rejection. J Clin Invest. 2004;113:694-700.
48. Norikazu M, Kentaro K, Toshihiro K, et al. Expression of PD-1, PD-L1, and PD-L2 in the liver in autoimmune liver diseases. Am J Gastroenterol. 2007;102:302-312.
49. Takanori K, Teruji T, Koji U, et al. Blockade of B7-H1 suppresses the development of chronic intestinal inflammation. J Immunol. 2003;171:4156-4163.
50. Menke J, Lucas JA, Zeller GC. Programmed death 1 ligand (PD-L) 1 and PD-L2 limit autoimmune kidney disease: distinct roles. J Immunol. 2007;179:7466-7477.
51. Mackay CR. Homing of native, memory and effector lymphocytes. Curr Opin Immunol.1993;5:423-427.
52. Masopust D, Vezys V, Marzo A.L, et al. Preperential localization of effector memory cells in nonlymphoid tissue. Science. 2001;291:2413-2417.
53. Yamazaki T, Akiba H, Iwai H, et al. Expression of programmed death 1 ligands by murine T cells and APC. J Immunol. 2002;169:5538-5545.
54. Zhu B, Guleria I, Khosroshahi A, et al. Differential role of programmed death-ligand 1 and programmed death-ligand 2 in regulating the susceptibility and chronic progression of experimental autoimmune encephalomyelitis. J Immunol. 2006;176:3480-3489.
55. Matsumoto K, Inoue H, Nakano T, et al. B7-DC regulates asthmatic response by an IFN- -dependent mechanism. J Immunol. 2004;172:2530-2541.
56. Agata Y, Kawasaki A, Nishimura H, et al. Expression of the PD-1 antigen on the surface of stimulated mouse T and B lymphocytes. Int Immunol. 1996;8:765-772.
57. Evans A, Cooksley H, Phillips S, et al. Programmed death 1 expression during antiviral treatment of chronic hepatitis B: Impact of hepatitis B e-antigen seroconversion. Hepatology. 2008;48:759-769.
58.彭国平,孙雯,吴炜,等.慢性乙型肝炎患者外周血树突状细胞PD-L1表达的相关研究.浙江大学学报. 2008; 37:364-372.
59. Dong H, Chen X. Immunoregulatory role of B7-H1 in chronicity of inflammatory responses. Cell Mol Immunol. 2006;3:179-187.
60. Tim M, Bettina S, Thomas K, et al. Microglial expression of the B7 family member B7 Homolog 1 confers strong immune inhibition:implications for immune responses and autoimmunity in the CNS. J Neuroscience. 2005;25:2537-2546.
61. Ding H, Wu X, Gao W. PD-L1 is expressed by human renal tubular epithelial cells and suppresses T cell cytokine synthesis. Clinical Immunology. 2005;115:184-191.
62. Waeckerle MY, Starke A, Wuthrich RP. PD-L1 partially protects renal tubular epithelial cells from the attack of CD8+ cytotoxic T cells. Nephrol Dial Transplant. 2007;22:1527-1536.
63. Beswick EJ, Pinchuk IV, Das S, et al. B7-H1 expression on gastric epithelial cells after Helicobacter pylori exposure promotes the development of CD4+CD25+FoxP3+ regulatory T cells. Infect Immun. 2007;75:4334-4341.
64. Seo SK, Seo HM, Jeong HY, et al. Co-inhibitory role of T-cell-associated B7-H1 and
B7-DC in the T-cell immune response. Immunol Lett. 2006;102:222-228.
65. Chen Y, Yang C, Jin N, et al. Sinomenine promotes differentiation but impedes maturation and co-stimulatory molecule expression of human monocyte-derived dendritic cells. International Immunopharmacology. 2007;7:1102-1110.
66. Dong H, Strome SE, Matteson EL, et al. Costimulating aberrant T cell responses by B7-H1 autoantibodies in rheumatoid arthritis. J Clin Invest. 2003;111:363-370.
67. Yamazaki T, Akiba H, Koyanagi A, et al. Blockade of B7-H1 on macrophages suppresses CD4+ T cell proliferation by augmenting IFN-gamma-induced nitric oxide production. J Immunol. 2005;175:1586-1592.
68. Nishimura H, Nose M, Hiai H, et al. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity. 1999;11:141-151.
69. Sharpe AH, Freeman GJ. The B7-CD28 superfamily. Nat Rev Immunol. 2002;2:116-126.
70. Wang S, Bajorath J, Flies DB, et al. Molecular modeling and functional mapping of B7–H1 and B7-DC uncouple costimulatory function from PD-1 interaction. J Exp Med. 2003;197:1083-1091.
71. Holdsworth SR, Kitching AR, Tipping PG. Th1 and Th2 T helper cell subsets affect patterns of injury and outcomes in glomerulonephritis. Kidney Int. 1999;55:1198-1216.
72. Mosmann TR, Cherwinski H, Bond MW, et al. Two type of murine helper T cell clone.Ⅰ.Definition according to profies of activities and secreted proteins. J Imunol. 1986;136:2348-2357.
73. Arakawa S, Hatano Y, Katagiri K, et al. Differential expression of mRNA for Th1 and Th2 cytokine-associated transcription factors and suppressors of cytokine signaling in peripheral blood mononuclear cells of patients with atopic dermatitis. Clin Exp Immunol. 2004;135:505-510.
74. Nakamura Y, Hoshino M. TH2 cytokines and associated transcription factors as therapeutic targets in asthma.. Curr Drug Targets Inflamm Allergy.2005;4:267-270.
75. Horiuchi Y, Hanazawa A, Nakajima Y, et al. T-helper (Th)1/Th2 imbalance in the peripheral blood of dogs with malignant tumor. Microbiol Immunol. 2007; 51:1135-1138.
76. Li B, Yang P, Zhou H, et al. T-bet expression is upregulated in active Behcet’s disease. Br J Ophthalmol. 2003;87:1264-1267.
77. Obata F, Yoshida K, Ohkubo M, et al. Contribution of CD4+ and CD8+ T cells and interferon-gamma to the progress of chronic rejection of kidney allografts: the Th1 response mediates both acute and chronic rejection.Transpl Immunol. 2005;14:21-25.
78. Cousins DJ, Lee TH, Staynov DZ. Cytokine coexpression during human Th1/Th2 cell differentiation:direct evidence for coordinated expression of Th2 cytokines. J Immunol. 2002;169:2498-2506.
79. Yu HR, Chang JC, Chen RF, et al. Different antigens trigger different Th1/Th2 reactions in neonatal mononuclear cells (MNCs) relating to T-bet/GATA-3 expression. J Leukoc Biol. 2003;74:952-958.
80. Chakir H, Wang H, Lefebvre DE, et al. T-bet/GATA-3 ratio as a measure of the Th1/Th2 cytokine profile in mixed cell population:Predominant role of GATA-3. J Immunol Methods. 2003;278:157-169.
81. Chan RW, Lai FM, Li EK, et al. Imbalance of Th1/Th2 transcription factors in patients with lupus nephritis. Rheumatology. 2006;45:951-957.
82. Segelmark M, Butkowski R, Wieslander J. Antigen restriction and IgG subclasses among anti-GBM autoantibodies. Nephrol Dial Transplant. 1990;5:991-996.
83. Noel LH, Aucouturier P, Monteiro RC, Preud’Homme JL, et al. Glomerular and serum immunoglobulin G subclasses in membranous nephropathy and anti-glomerular basement membrane nephritis. Clin Immunol Immunopathol. 1988;46:186-194.
84. Graninger WB, Hassfeld W, Pesau BB, et al. Induction of systemic lupus erythematosus by interferon-gamma in a patient with rheumatoid arthritis. J Rheumatol. 1991;18:1621-1622.
85. Machold KP, Smolen JS. Interferon-gamma induced exacerbation of systemic lupus erythematosus. J Rheumatol. 1990;17:831-832.
86. Parker MG, Atkins MB, Ucci AA, et al. Rapidly progressive glomerulonephritis after immunotherapy for cancer. J Am Soc Nephrol. 1995;5:1740-1744.
87. Feuerer M, Eulenburg K, Loddenkemper C, et al. Self-limitation of Th1-mediated inflammation by IFN-gamma. J Immunol. 2006;176:2857-2863.
88.向莉,高小夏,潘家荣.慢性肾小球肾炎患者血清IFN-γ、IL-10的变化及意义.中国临床医学. 2006;13:269-272.
89. Couper K N, Blount D G, Riley E M. IL-10:the master regulator of immunity to infection. J Immunol. 2008;180:5771-5777.
90.周燕斌,李幼姬,吴玉红.狼疮肾炎患者血清及外周血单个核细胞中IL-10和γ干扰素的表达及意义.中国现代医学杂志. 2006;16:529-532.
91. Tipping P G, Holdsworth S R. Cytokines in glomerulonephritis. Semin Nephrol. 2007;27:275-285.
92. Tipping P G, Kitching A R. Glomerulonephritis, Th1 and Th2: what’s new. Clin Exp Immunol. 2005;142:207-215.
93. Graninger W B, Hassfeld W, Pesau B B, et al. Induction of systemic lupus erythematosus by interferon-gamma in a patient with rheumatoid arthritis. J Rheumatol. 1991;18:1621-1622.
94. Machold K P, Smolen J S. Interferon-gamma induced exacerbation of systemic lupus erythematosus. J Rheumatol. 1990;17:831-832.
95. Parker M G, Atkins M B, Ucci A A, et al. Rapidly progressive glomerulonephritis after immunotherapy for cancer. J Am Soc Nephrol. 1995;5:1740-1744.
96. Kawasaki Y, Suzuki J, Sakai N, et al. Evaluation of T helper-1/-2 balance on the basis of IgG subclasses and serum cytokines in children with glomerulonephritis. Am J Kidney Dis. 2004;44:42-49.
97.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;20:249-258.
98. Shimohata H, Yamada A, Yoh K, et al. Overexpression of T-bet in T cells accelerates autoimmune glomerulonephritis in mice with a dominant Th1 background. J Nephrol. 2009;22:123-129.
99. Zeng Y, Gu B, Ji X, et al. Sinomenine, an antirheumatic alkaloid, ameliorates clinical signs of disease in the Lewis rat model of acute experimental autoimmune encephalolmyelitis. Biol Pharm Bull. 2007;30:1438-1444.
100.戴英波,黄循,罗志刚,等.青藤碱对大鼠肾移植急性排斥反应的抑制作用.中国现代医学杂志. 2004;14:49-54.
101.王毅,熊烈,陈正,等.青藤碱对肾移植大鼠Th1细胞生物学特性的影响.美国中华临床医学杂志. 2004;6:4-6.
1. Christian K, Felix H, Veronika LK, et al. Role of T cells and dendritic cells in glomerular immunopathology. Semin Immunopathol. 2007;29:317-335.
2. Liang SC, Latchman YE, Buhimann JE, et al. Regulation of PD-1, PD-L1, and PD-L2 expression during normal and autoimmune responses. Eur J Immunol. 2003;33:2706-2716.
3. Nishimura H, Nose M, Hiai H, et al. Development of lupus-like autoimmune diseases by disruption of the PD-1 gene encoding an ITIM motif-carrying immunoreceptor. Immunity. 1999;11:141-151.
4. Chen Y, Zhang J, Li J, et al. Expression of B7-H1 in Inflammatory Renal Tubular Epithelial Cells. Nephron Exp Nephrol. 2005;11;102:81-92.
5. Didier AM, Mohamed HS. Role of novel T-cell costimulatory pathways in transplantation. Curr Opin Organ Transplant. 2003;8:25-33.
6. Lindsten T, Lee KP, Harris ES, et al. Characterization of CTLA-4 structure and expression on human T cells. J Immunol. 1993;151:3498-3499.
7. Complexities of CD28/B72:CTLA-4 costimulatory pathways in antoimmunity and transplantation. Annu Rev Immunol. 2001;19:225-252.
8. Niemann M, Mueller A, Yard BA, et al. B7-1(CD80) and B7-2(CD86) expression in human tubular epithelial cells in vivo and in vitro. Nephron. 2002;92:542-556.
9. Wu Q, Jinde K, Endoh M, et al. Clinical significance of costimulatory molecules CD80/CD86 expression in IgA nephropathy. Kidney Int. 2004;65:888-896.
10. Wu Q, Jinde K, Endoh M, et al. Costimulatory molecules CD80 and CD86 in human crescentic glomerulonephritis. Am J Kidney Dis. 2003;41:950-961.
11. Jochen R, Gero VG, Martin L, et al. Induction of B7-1 in podocytes is associated with nephrotic syndrome. J Clin Invest. 2004;113:1390-1397.
12. Singbartl K, Bockhorn SG, Zarbock A, et al. T cells modulate neutrophil-dependent acute renal failure during endotoxemia:critical role for CD28. J Am Soc Nephrol. 2005;16:720-728.
13. Laskowski IA, Pratschke J, Wilhelm MJ, et al. Anti-CD28 monoclonal antibody therapyprevents chronic rejection of renal allografts in rats. J Am Soc Nephrol. 2002;13:519-527.
14. Vincenti F, Luggen M. T cell costimulation:a rational target in the therapeutic armamentarium for autoimmune diseases and tranaplantation. Annu Rev Med. 2007;58:347-358.
15. Cunnane G, Chan OT, Cassafer G, et al. Prevention of renal damage in murine lupus nephritis by CTLA-4Ig and cyclophosphamide. Arthritis Rheum. 2004;50:1539-1548.
16. Anthony JC, Jose C, Gutierrez R. The expanding B7 superfamily:increasing complexity in costimulatory signals regulating T cell function. Nature Immunol. 2001;3:203-209.
17. Zhang X, Schwartz JC, Guo XL, et al. Structural and functional analysis of the costimulatory receptor programmed death-1. Immunity. 2004;20:337-347.
18. Seo SK, Seo HM, Jeong HY, et al. Co-inhibitory role of T-cell-associated B7-H1 and B7-DC in the T-cell immune response. Immunol Lett. 2006;102:222-228.
19. Okazaki T, Maeda A, Nishimura H, et al. PD-1 immunoreceptor inhibits B cell receptor-mediated signaling by recruiting Srchomology 2-domain-containing tyrosine phosphatase 2 to phosphotyrodine. Proc Natl Acad Sci USA. 2001;98:13866-13871.
20. Freeman GJ, Long AJ, Iwai Y, et al. Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J Exp Med. 2000;192:1027-1037.
21. Nishimura H, Okazaki T, Tanaka Y, et al. Autoimmune dilated cardiomyopathy in PD-1 receptor-deficient mice. Science. 2001;291:319-322.
22. Subudhi SK, Zhou P, Yerian LM, et al. Local expression of B7-H1 promotes organ-specific autoimmunity and transplant rejection. J Clin Invest. 2004;113:694-700.
23. Zhang J, Chen Y, Li J, et al. Renal tubular epithelial expression of the coinhibitory molecule B7-DC (programmed death-1 ligand). J Nephrol. 2006;19:429-438.
24. Yongwen Chen, Jingyi Li, Jingbo Zhang, et al. Sinomenine inhibits B7-H1 and B7-DC expression on human renal tubular epithelial cells. International Immunopharmacology. 2005;5:1446-1457.
25. Waeckerle MY, Starke A, Wuthrich RP. PD-L1 partially protects renal tubular epithelial cells from the attack of CD8+ cytotoxic T cells. Nephrol Dial Transplant. 2007;22:1527-1536.
26. Hanlu D, Xiongfei W, Jun W. Delivering PD-1 inhibitory signal concomitant with blocking ICOS co-stimulation suppresses lupus-like syndrome in autoimmune BXSB mice. Clinical Immunology. 2006;118:258-267.
27. Coyle AJ, Lehar S, Loyd C, et al. The CD28-related molecule ICOS is required for effective T cell-dependent immune responses. Immunity. 2000;13:95-105.
28. Wahl P, Schoop R, Bilic G, et al. Renal tubular epithelial expression of the costimulatory molecule B7RP-1(inducible costimulatory ligand). I Am Soc Nephrol. 2002;13:1517-1526.
29. De HS, Woltman AM, Trouw LA, et al. Renal tubular epithelial cells modulate T-cell responses via ICOS-L and B7-H1. Kidney Int. 2005;68:2091-2102.
30. Wahl P, Wuthrich RP. Role of the B7RP-1/ICOS pathway in renal tubular epithelial antigen presentation to CD4+ Th1 and Th2 cells. Nephron Exp Nephrol. 2004;98:31-38.
31. Toubi E, Shoenfeld Y. The role of CD40-CD154 interactions in autoimmunity and the benefit of disrupting this pathway. Autoimmunity. 2004;37:457-464.
32. Revnolds J, Khan SB, Allen AR, et al. Blockade of the CD154-CD40 costimulatory pathway prevents the development of experimental autoimmune glomerulonephritis. Kidney Int. 2004;66:1444-1452.
33. Boumpas DT, Furie R, Manzi S, et al. A short course of BG9588(anti-CD40 ligand antibody) improves serologic activity and decreases hematuria in patients with proliferative lupus glomerulonephritis. Arthritis Rheum. 2003;48:719-727.
34. Kairaitis L, Wang Y, Zheng L, et al. Blockade of CD40-CD40 ligand protects against renal injury in chronic proteinuric renal disease. Kidney Int. 2003,64:1265-1272.
35. Pearson TC, Trambley J, Odom K, et al. Anti-CD40 therapy extends renal allograft survival in rhesus macaques. Transplantation. 2002;74:933-940.
1.中国医药大词典.香港,建文书局出版社. 1963:1171.
2. Wang Y, Fang Y, Huang W, Effect of sinomenine on cytokine expression of macrophages and synoviocytes in adjuvant arthritis rats. J Ethnopharmacol. 2005;98:37-43.
3. Mark W, Schneeberger S, Seiler R, Sinomenine blocks tissue remodeling in a rat model of chronic cardiac allograft rejection. Transplantation. 2003;75:940-945.
4.扬汝春,王永钧,林京莲,等.青藤碱对白介素1β诱导的肾小管上皮细胞骨架变化和明胶酶活性的影响.中国肾脏病杂志杂志. 2006;22:499-502.
5. 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: 637-645.
6. Shu L, Yin W, Zhang J, et al. Sinomenine inhibits primary CD4 (+) T-cell proliferation via apoptosis. Cell Biol Int. 2007;31: 784-789.
7. Feng H, Yamaki K, Takano H, Suppression of Th1 and Th2 immune responses in mice by Sinomenine, an alkaloid extracted from the chinese medicinal plant Sinomenium acutum. Planta Med. 2006;72: 1383-1388.
8.中华人民共和国药典(一部).广东科技出版社. 1995:165.
9.叶木荣,刘良,曾元儿,等.青藤碱在大鼠体内分布与脏器毒理的关系研究.中国药理学通报. 2001;17:65-69.
10.莫志贤,周吉银,王彩云.青藤碱的身体依赖性和精神依赖性实验研究.中国药物滥用防治杂志. 2004;10:190-193.
11.邱赛红,黄宇明,仇萍,等.青藤碱致突变性的实验研究.中药药理与临床. 1999;15:23-24.
12.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;20:249-258.
13.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;21:573-574.
14. Shu L, Yin W, Zhang J, et al. Sinomenine inhibits primary CD4+ T-cell proliferation viaapoptosis. Cell Biol Int. 2007;31:784-789.
15. He X, Wang J, Guo Z, et al. Requirement for ERK activation in sinomenine-induced apoptosis of macrophages. Immunol Lett. 2005;98:91-96.
16.徐道华,周晨慧.树突状细胞CD80和CD86表达及胞外白细胞介素12分泌与不同剂量青藤碱干预的影响.中国组织工程研究与临床康复. 2007;11:5654-5656.
17.杨承英,陈永文,傅晓岚,等.青藤碱促进树突状细胞分化抑制其成熟.免疫学杂志. 2007;23:265-269.
18.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-κB活性的影响.广东医学. 2006;27:55-57.
19.高永翔,龚立,田艳勋,等.青藤碱对小鼠骨髓树突状细胞免疫功能的影响.成都中医药大学学报. 2005;28:13-14.
20. Wang Y, Fang Y, Huang W, et al. Effect of sinomenine on cytokine expression of macrophages and synoviocytes in adjuvant arthritis rats. J Ethnopharmacol. 2005;98:37-43.
21. Zeng Y, Gu B, Ji X, et al. Sinomenine, an antirheumatic alkaloid, ameliorates clinical signs of disease in the Lewis rat model of acute experimental autoimmune encephalolmyelitis. Biol Pharm Bull. 2007;30:1438-1444.
22.戴英波,黄循,罗志刚,等.青藤碱对大鼠肾移植急性排斥反应的抑制作用.中国现代医学杂志. 2004;14:49-54.
23.王毅,熊烈,陈正,等.青藤碱对肾移植大鼠Th1细胞生物学特性的影响.中华临床医学杂志. 2004;6:4-6.
24. Kuroiwa T, Lee EG. Cellular interactions in the pathogenesis of lupus nephritis: the role of T cells and macrophages in the amplification of the inflammatory process in the kidney. Lupus. 1998;7:597-603.
25. Chen L. Co-inhibitory molecules of the B7-CD28 family in the control of T-cell immunity. Nat Rev Immunol. 2004;4:336-347.
26. Seo SK, Seo HM, Jeong HY, et al. Co-inhibitory role of T-cell-associated B7-H1 and B7-DC in the T-cell immune response. Immunol Lett. 2006;102:222-228.
27. Su KS, Hyoun MS, Hye YJ, et al. Co-inhibitory role of T-cell-associated B7-H1 and B7-DC in the T-cell immune response.Imlet.2005;9:007-013.
28. Mataki N, Kikuchi K, Kawai T, et al. Expression of PD-1, PD-L1, and PD-L2 in theliver in autoimmune liver diseases. Am J Gastroenterol. 2007;102:302-312.
29. Piconi S, Trabattoni D, Saresella M, et al. Effects of specific immunotherapy on the B7 family of costimulatory molecules in allergic inflammation. J Immunol. 2007;178:1931-1937.
30. Chen Y, Zhang J, Li J, et al. Expression of B7-H1 in Inflammatory Renal Tubular Epithelial Cells. Nephron Exp Nephrol. 2005;102:81-92.
31. Zhang J, Chen Y, Li J, et al. Renal tubular epithelial expression of the coinhibitory molecule B7-DC (programmed death-1 ligand). J Nephrol. 2006;19:429-38.
32.余克强,赵毅,李娟,等.青藤碱对类风湿关节炎树突状细胞CD80mRNA、CD86mRNA表达的影响.中国药房. 2006;17:331-333.
33.梁瑞燕,曹柳英,王文君,等.青藤碱抗炎作用机理研究.广州中医药大学学报. 2007;24:141-143.
34.王文君,王培训.青藤碱对环氧化酶2活性的选择性抑制作用.广州中医药大学学报. 2002;19:46-47.
35.陈炜,沈悦娣,赵光树,等.青藤碱对脂多糖诱导的神经细胞环氧化酶-2表达的影响.中国中药杂志. 2004;29:900-903.
36.徐广全,王斌,金相元,等.青藤碱对心脏移植大鼠急性排斥反应及环氧化酶2活性的影响.中华医学杂志. 2006;86:911-914.
37. Mark W, Schneeberger S, Seiler R, et al. Sinomenine blocks tissue remodeling in a rat model of chronic cardiac allograft rejection. Transplantation. 2003;75:940-945.
38. Kok TW, Yue PY, Mak NK, et al. The anti-angiogenic effect of sinomenine. Angiogenesis. 2005;8:3-12.