糖皮质激素对BXSB狼疮小鼠肾组织fractalkine/CX3CR1表达的影响

目的：研究BXSB狼疮小鼠肾组织趋化因子fractalkine及其受体CX3CR1的表达以及给予泼尼松治疗后的改变,探讨两者在狼疮肾炎发病机制中的可能作用。方法：12 周龄雄性BXSB狼疮小鼠随机分成泼尼松治疗组（n=6）和实验对照组（n=6）;另取同周龄雄性C57BL/6J小鼠6只作为正常对照组。正常对照组和实验对照组小鼠每天给予0.5 mL生理盐水灌胃;泼尼松治疗组小鼠每天给予0.18 mg/20 g BW的泼尼松溶于0.5 mL生理盐水灌胃。持续10周结束实验。应用逆转录-聚合酶链反应（RT-PCR）及Western印迹检测小鼠肾组织fractalkine和CX3CR1 mRNA和蛋白的表达,并检测小鼠实验室指标以及肾脏组织病理学的变化。结果： BXSB狼疮小鼠肾组织fractalkine以及CX3CR1 mRNA和蛋白表达均较C57BL/6J小鼠明显增高,而经过泼尼松治疗后的BXSB小鼠两者的表达均较未治疗组（实验对照组）明显下降,同时伴有血清免疫球蛋白G（IgG）、IgM、血清抗双链脱氧核糖核酸（dsDNA）抗体水平以及血尿素氮（BUN）、血肌酐（SCr）水平的明显改善,尿蛋白减少;肾小球内免疫复合物沉积和肾脏组织病理学改变亦显著减轻。结论： 实验结果提示fractalkine/CX3CR1可能参与了小鼠狼疮肾炎的发病机制,且糖皮质激素可能通过抑制肾脏fractalkine的表达而发挥其治疗效应。     

巨噬细胞衍生趋化因子在MRL/lpr小鼠肾组织中的异常表达

目的:研究巨噬细胞衍生趋化因子(MDC)在红斑狼疮小鼠狼疮肾炎中的作用。方法:比较MRL/lpr狼疮小鼠和BALB/c小鼠24h尿蛋白,并用逆转录-聚合酶链反应技术(RT-PCR)和免疫组织化学方法检测两组小鼠肾组织MDCmRNA及蛋白表达情况。结果:MRL/lpr小鼠24h尿蛋白高于BALB/c小鼠(P0.05);MRL/lpr小鼠肾组织MDCmRNA表达较BALB/c小鼠明显升高(P0.05);MRL/lpr小鼠肾小球、肾小管及间质均有明显MDC蛋白表达,而BALB/c小鼠肾脏未见MDC表达。结论:MDC在红斑狼疮小鼠肾脏组织中的表达增高,说明MDC可能参与介导了小鼠狼疮性肾炎的病变过程。     

中药狼疮方对狼疮样BXSB小鼠肺组织CD134/CD134L和RANTES表达的影响

目的探讨中药狼疮方对狼疮样BXSB小鼠肺组织CD134／CD134L和RANTES表达的影响。方法采用BXSB小鼠模型,随机分为3组：狼疮方治疗组、强的松治疗组、未治疗组,每组6只,疗程10周。另设与BXSB小鼠同基因的正常C57BL／6小鼠6只为正常对照组。分别取小鼠肺组织,应用逆转录-荧光定量-聚合酶链反应（RT-FQ-PCR）技术定量测定小鼠肺组织CD134、CD134L和趋化因子RANTES的mRNA表达水平。结果①未治疗组小鼠肺组织CD134、CD134L mRNA和RANTKS mRNA的表达水平都显著高于正常对照组（P〈0．01,P〈0．05）;经强的松或中药狼疮方治疗后,BXSB小鼠肺组织CD134、CD134L及RANTES的mRNA表达都受到明显抑制,显著低于未治疗组（P〈0．01,P〈0．05）;且接近正常水平,与正常对照组无显著性差异（P〉0．05）。②BXSB小鼠肺组织RANTES的mRNA表达水平与CD134L的mRNA表达水平呈显著的正相关关系（r=0．793,P〈0．05）,而与CD134的mRNA表达水平无显著的相关关系（r=0．412,P〉0．05）。结论中药狼疮方具有与强的松类似的免疫抑制作用,可显著抑制狼疮样小鼠肺组织CD134／CD134L共刺激信号表达;并下调肺组织RANTKS mRNA表达水平,具有一定的肺脏保护作用。     

IL-18对肾小管上皮细胞表型转化的影响

目的：研究IL-18对体外培养肾小管上皮细胞表型转化的影响，以明确IL-18在慢性肾脏疾病中的可能作用机制。方法：应用体外细胞培养技术培养人肾小管上皮细胞侏（HK-2）。应用RT-PCR技术检测α-平滑肌肌动蛋白（α-SMA）、转化生长因子-β1（TGF-β1）mRNA水平，用免疫细胞化学方法（ICC）及Western blot技术分别检测IL-18对HK-2细胞表达仪-SMA蛋白的影响。结果：（1）IL-18可促进HK-2细胞表达α-SMA、TGF-β1 mRNA，且两者之间呈正相关（P〈0．05）。（2）IL-18增加α-SMA阳性HK-2细胞百分数（P〈0．05）。（3）IL-18使HK-2细胞α-SMA蛋白表达水平增加。结论：IL-18可剂量和时间依赖性地促进肾小管上皮细胞转分化为肌成纤维细胞，促进肾间质纤维化。     

系统性红斑狼疮小鼠脾细胞中IL-12信号传递途径的研究

目的探讨在狼疮小鼠脾细胞中是否存在IL-12通过Lck/p38/c-jun传递信号及其对脾细胞的影响。方法以慢性移植物抗宿主病(graft versus host disease,GVHD)小鼠为狼疮小鼠模型,分别采用放射自显影、Western blot和Northern blot,检测培养的脾细胞中Lck的活性、p38磷酸化活化及c-jun基因表达的变化。结果狼疮小鼠脾细胞经IL-12刺激后,与正常对照组相比较,Lck的活性增高、p38异常活化,c-junmRNA的水平增高。加入Lck抑制剂PP1组Lck的活性及p38磷酸化消失,无c-jun基因的表达;加入p38抑制剂SB203580组亦无p38磷酸化及c-jun基因的表达。结论狼疮小鼠的脾细胞异常,IL-12可通过Lck/p38/c-jun在细胞内传递信号,直接参与免疫损伤。     

系统性斑狼疮小鼠脾细胞中IL—12信号传递途径的研究

目的 探讨在狼疮小鼠脾细胞中是否存在IL-12通过Lck/p38/c-jun传递信号及其对脾细胞的影响。方法 以慢性移植物抗宿主病（graft versus host disease,CVHD)小鼠为狼疮小鼠模型。分别采用放射自显影、Western blot和Northern blot，检测培养的脾细胞中Lck的活性、p38磷酸化活化及c-jun基因表达的变化。结果 狼疮小鼠脾细胞经IL-12刺激后，与正常对照组相比较，Lck抑制剂PP1组Lck的活性及p38磷酸化消失，无c-jun基因的表达；加入p38抑制剂SB203580组亦无p38磷酸化及c-jun基因的表达。结果 狼疮小鼠的脾细胞异常，IL-12可通过Lck/p38/c-jun在细胞内传递信号，直接参考免疫损伤。     

维生素D3对肺炎支原体感染小鼠肺组织ICAM-1及IL-4表达的影响

目的探讨维生素D3(VD3)对肺炎支原体感染(MP)小鼠肺组织ICAM-1及IL-4表达的影响。方法 45只BALB/c小鼠随机分成对照组、MP感染组与维生素D3组。模型组与维生素D3组小鼠采用滴鼻法进行MP感染。取肺组织制作病理切片,HE染色观察组织病理学变化,Western blot方法与real time PCR方法分别检测各组小鼠肺组织ICAM-1及IL-4蛋白与mRNA的表达水平。结果 MP感染BALB/c小鼠7天后,肺组织有大量炎性细胞浸润,给予维生素D3处理后,炎性明显减轻。MP感染组BALB/c小鼠肺组织ICAM-1及IL-4蛋白与mRNA的表达水平显著高于对照组,给予维生素D3处理后,感染MP的BALB/c小鼠肺组织ICAM-1及IL-4蛋白与mRNA的表达水平显著降低,P0.01。结论维生素D3能够降低感染肺炎支原体感染小鼠的肺内炎性反应,可能与降低ICAM-1及IL-4表达相关。     

白细胞介素—12信号转导参与小鼠肾小管上皮细胞炎性损伤

为观察白细胞介素-12（IL-12）在小鼠肾小管上皮细胞（TEC）炎症损伤的信号传递,以培养的正常小鼠TEC作为空白对照组,狼疮肾炎（LN）TEC作为实验组,以IL-12（10μg/L)刺激5min,利用放射自显影检测发现正常对照及LN组lck活性增强,后者更明显,应用lck抑制剂PP1后其活性消失。再以等浓度IL-12刺激TEC15min,采用免疫印迹观察到LN组P38磷酸化强于正常对照组,应用PP1或P38抑制剂SB203580则不发生P38磷酸化。给予IL-12刺激时发现LN组c-Jun基因表达水平强于正常对照组,应用PP1或SB203580后则未有c-Jun基因表达,提示IL-12可通过lck/P38/c-Jun信号途径参与对狼疮小鼠TEC的炎症损伤。     

HMGB1/TLR/NF-κB在狼疮性肾炎小鼠肾组织中的表达及意义

目的:探讨HMGB1/TLR/NF-κB在狼疮性肾炎小鼠肾组织中的表达及作用机制.方法:选取16周龄的雄性BXSB小鼠(狼疮性肾炎模型)和同周龄C57BL/6 小鼠(正常对照)作为研究对象,透射电镜观察肾组织的超微结构改变;RT-PCR检测肾皮质中HMGB1mRNA的表达变化;免疫组织化学和流式细胞术检测肾组织中HMGB1、NF-κB、TLR2和PCNA蛋白的表达变化.结果:(1) 16周时,BXSB小鼠血清中BUN水平明显升高;(2) 16周时,与正常的C57BL/6 小鼠相比,BXSB基底膜明显增厚,部分足突融合,内皮细胞下可见团块状电子致密物沉积;(3) 狼疮性肾炎模型组小鼠肾组织的肾小球中可见较多的PCNA阳性表达,肾小管上皮细胞核内也可见少量的表达;(4) 与对照组相比,BXSB小鼠肾组织中HMGB1mRNA及蛋白表达升高,HMGB1蛋白尤其在细胞增生明显而肥大的肾小球呈高表达,主要位于细胞浆和细胞外;而在C57BL/6小鼠肾脏组织中以小管细胞核表达为主;(5) 狼疮性肾炎模型组小鼠肾组织p-NF-κB和TLR2蛋白表达明显升高;(6) HMGB1蛋白与p-NF-κB蛋白表达呈显著正相关(r=0.833,P=0.000);p-NF-κB蛋白与TLR2蛋白表达呈显著正相关(r=0.765,P=0.001).结论:(1) HMGB1能促进肾小球固有细胞的增生,导致增生性肾小球肾炎形成;(2) HMGB1在小鼠狼疮性肾炎中的致炎作用可能部分通过结合其受体TLR2,激活NF-κB信号途径而实现的.     

狼疮肾炎患者IL-18/IL-18BP的表达及免疫抑制治疗对其的影响

目的：观察狼疮肾炎（Lupus nephritis，LN）病人血清白细胞介素18（Interleukin 18，IL-18）和白细胞介素18结合蛋白（Interleukin 18 binding protein, IL-18BP）的水平及外周血单个核细胞（PBMC）中IL-18和IL-18BP mRNA的表达水平与疾病的关系，及免疫抑制治疗对其的影响。方法：酶联免疫吸附法（ELISA）法检测25例活动性LN病人采用环磷酰胺和糖皮质激素治疗前后及25例正常对照者的血清IL-18和1L-18BP水平，同时逆转录多聚酶链反应（RT-PCR）法测定外周血PBMC中IL-18和IL-18BP mRNA的表达水平。结果：活动性患者治疗前血清IL-18、IL-18BP及PBMC中IL-18和IL-18BP mRNA的表达水平均较正常对照组明显升高（P〈0．01）。游离IL-18水平明显增高，血清游离IL-18水平与患者肾脏病理活动指数（AI）及24小时尿蛋白定量呈正相关，与补体C3水平呈负相关，与患者GFR水平无明显相关。采用免疫抑制剂治疗后IL-18和IL-18BP在蛋白及mRNA水平均下降，血清游离IL-18水平明显下降（P〈0．01）。结论：狼疮肾炎患者血清游离IL-18水平升高并与疾病活动性相关，IL-18／IL-18BP失衡参与了狼疮肾脏病理过程，增加内外源性IL-18BP可能为IN的治疗提供新的途径。     

IL-4Ralpha polymorphism in regulation of IL-4 synthesis by T cells: implication in susceptibility to a subset of murine lupus

To thoroughly understand the role of IL-4 in the pathogenesis of systemic lupus erythematosus (SLE), a prototypic antibody-mediated systemic autoimmune disease, we examined the potential of in vitro IL-4 production by anti-CD3 mAb-stimulated splenic T cells in SLE model of NZB, BXSB and related mouse strains. Unexpectedly, both SLE-prone NZB and BXSB mice had a limited potential to produce IL-4, while disease-free NZW mice had a high potential. Levels in (NZB x NZW) F1 and (NZW x BXSB) F1 were in between. Genome-wide search for quantitative trait loci (QTL) controlling this variation identified a single significant QTL in the vicinity of IL-4Ralpha gene on chromosome 7. Sequence analysis of IL-4Ralpha cDNA revealed that there are 17 nucleotide substitutions resulting in eight amino acid changes between NZB and NZW strains. BXSB showed the identical sequence, as did NZB. Thus, it was suggested that the NZW-type polymorphism controls a high potential and the NZB/BXSB-type polymorphism controls a low potential for IL-4 production by T cells. Linkage studies using NZW x (NZW x BXSB) F1 male and (NZB x NZW) F1 x NZW female back-cross mice revealed that the BXSB/NZB-type IL-4Ralpha polymorphism significantly linked to BXSB, but not to (NZB x NZW) F1 lupus. Thus, the low IL-4-producing phenotype appears to predispose to SLE in BXSB, but not NZB-related strains, suggesting that the role of IL-4 in the pathogenesis may differ between certain subsets of SLE, even if they show similar disease phenotypes.     

NF-κB信号途径在小鼠狼疮性肾炎发病中的可能作用

目的:探讨NF-κB信号途径在小鼠狼疮性肾炎发病中的可能作用。方法:选取16周龄的雄性BXSB小鼠(狼疮性肾炎模型组)和同周龄C57BL/6小鼠(正常对照组)作为研究对象,透射电镜和PAS染色观察肾组织的超微结构形态改变;RT-PCR技术检测小鼠全血中HMGB1mRNA的表达变化。采用ELISA方法检测血清中HMGB1蛋白浓度;免疫组织化学检测肾组织中HMGB1和PCNA蛋白的表达变化;Western blot和流式细胞术检测肾组织中RAGE、p-NF-κB和IκB蛋白的表达。结果:16周时,与正常的C57BL/6小鼠相比,BXSB小鼠血清中BUN水平及尿中微球白蛋白水平明显升高;与正常的C57BL/6小鼠相比,BXSB小鼠全血中HMGB1mRNA水平和血清中HMGB1蛋白浓度明显升高;16周时,与正常的C57BL/6小鼠相比,BXSB基底膜明显增厚,部分足突融合,内皮细胞下可见团块状电子致密物沉积;与正常的C57BL/6小鼠相比,BXSB小鼠肾组织的肾小球中可见较多的PCNA阳性表达,肾小管上皮细胞核内也可见少量的表达;BXSB小鼠肾组织中HMGB1蛋白表达升高,HMGB1蛋白尤其在细胞增生明显而肥大的肾小球呈高表达,主要位于细胞浆和细胞外;而在C57BL/6小鼠肾脏组织中以小管细胞核表达为主;与对照组相比,BXSB小鼠肾组织p-NF-κB和RAGE蛋白表达明显升高;而IκB蛋白表达明显降低;HMGB1蛋白与p-NF-κB蛋白表达呈显著正相关(r=0.833,P=0.000);p-NF-κB蛋白与RAGE蛋白表达呈显著正相关(r=0.621,P=0.018);HMGB1蛋白与RAGE蛋白表达呈显著正相关(r=0.848,P=0.000);p-NF-κB蛋白与IκB蛋白表达呈显著负相关(r=-0.759,P=0.002)。结论:HMGB1在小鼠狼疮性肾炎中的致炎作用可能部分通过结合其受体RAGE,激活NF-κB信号途径,促进肾小球固有细胞的增生,从而导致增生性肾小球肾炎形成而实现的。     

PD-L1 is expressed by human renal tubular epithelial cells and suppresses T cell cytokine synthesis

T cell activation is affected by both stimulatory and inhibitory co-signaling. MHC class II-expressing renal tubular epithelial cells (TEC) can function as APC for T cells. To study the influence of inhibitory ligands on TEC-mediated T cell activation, we examined the expression of programmed death ligand-1 (PD-L1) on human TEC line HK-2 cells, as well as in normal and diseased kidney samples. RT-PCR, FACS, and immunocytochemistry showed that PD-L1 is constitutively expressed on HK-2 cells, and is dramatically upregulated by IFN-gamma. In situ hybridization and immunohistochemical staining revealed constitutive low expression of PD-L1 on proximal tubules at both mRNA and protein levels in normal kidneys, but much higher expression in kidneys with type IV lupus nephritis. In vitro, pretreatment of IFN-gamma-stimulated HK-2 cells with anti-PD-L1 significantly enhanced IL-2 secretion from cocultured, mitogen-activated Jurkat or human peripheral blood T cells. These results suggest that the PD-L1:PD-1 pathway negatively regulates T cell activation by TEC, and may play an inhibitory role in TEC-mediated immune activation and immunopathology in the kidney.     

Association of elevated serum glycoprotein gp70 with increased gp70 immune complex formation and accelerated lupus nephritis in autoimmune male BXSB mice

BXSB male mice, which spontaneously develop a systemic lupus erythematosus (SLE) like disease, were the only strain to have a significant incidence of abnormally elevated levels of gp70 in sera. Concentrations of gp70 in some mice were more than 10 times (greater than 500 micrograms/ml) those of young BXSB and any other murine strain. The presence of high serum levels of gp70 was significantly associated with hepatic sinusoidal lymphocytosis, a high incidence of which was only observed in male BXSB mice. Serum levels of gp70-anti-gp70 immune complexes were greatly increased in mice with high levels of gp70, presumably associated with increased anti-gp70 antibody production. Such mice developed fatal glomerulonephritis significantly earlier than those with lower levels of gp70. These results suggest that (1) hepatic inflammation of unknown aetiology occurring uniquely in male BXSB mice during the course of their SLE may be responsible for the enhanced expression of serum gp70 antigen, because of its nature as an acute phase reactant and (2) enhanced expression of gp70 antigen is associated with increased formation of anti-gp70 antibodies and exacerbation of lupus nephritis in male BXSB mice.     

Elevated Expression of Transmembrane IL-15 in Immune Cells Correlates with the Development of Murine Lupus: a Potential Target for Immunotherapy Against SLE

Presentation in trans by the Interleukin-15 receptor α chain (IL-15Rα) has been suggested as the main mechanism for IL-15 anchoring to the cell surface, but it is also evident that IL-15 can exist as a transmembrane protein. We herein demonstrate that replacement of the first 41 residues of human IL-15 (hIL-15) with Ig κ chain leader sequence resulted in secretion of most of the recombinant hIL-15 expressed in transfectant cells, thus identifying the transmembrane region of IL-15. A fusion protein (hIL-15Rα-Fc) between the extracellular domain of hIL-15Rα and the Fc fragment of IgG1 was prepared and shown to be able to bind with transmembrane IL-15 (tmIL-15). The level of tmIL-15 expression in macrophages, activated T cells and B cells from 6-month-old BXSB male mice, an animal model for systemic lupus erythematosus (SLE), was significantly increased compared with that from BXSB females or young males. In addition, hIL-15Rα-Fc was able to block the T cell stimulating and anti-apoptotic effect of the tmIL-15-positive BXSB macrophages in vitro . Intravenous administration of hIL-15Rα-Fc reduced the titre of autoantibodies against dsDNA and also proteinuria in aged BXSB males, implying that neutralization of IL-15 activity in vivo may be an effective way of treating SLE.     

Isolation and functional analysis of autoreactive T cells from BXSB mice with murine lupus

CD4(+) T helper cells play a pivotal role in the pathogenesis of SLE, although the mechanism is still unclear. The present study was designed to isolate and characterize autoreactive T lymphocytes from BXSB mice, a mouse model for human SLE. Splenocytes from 6-month-old male BXSB mice with murine lupus were repeatedly stimulated in vitro with irradiated syngeneic B cells in the presence of recombinant IL-2, resulting in six autoreactive T-cell lines and two T-cell clones. TCR analysis showed that, one of the T-cell lines, ATL1, was almost clonal, as a Vbeta2.1-Jbeta2, a Valpha5.1-Jalpha15 and a Valpha10.1-Jalpha15 chains were predominantly expressed in this line. The two clones derived from ATL1 turned out to be sister clones, using the TCR Vbeta2.1-Jbeta2 and Valpha10.1-Jalpha15 chains. ATL1 cells proliferated in response to stimulation of syngeneic and H-2-matched allogeneic B cells and secreted IFN-gamma. Monoclonal Ab against CD4 and CD28 inhibited the proliferative response of ATL1 for syngeneic B cells. Interestingly, ATL1 did not respond to BXSB spleen or peritoneal macrophages, suggesting that B cells were able to either express accessory molecules necessary for T-cell triggering or present cryptic epitopes recognized by the autoreactive T cells. Moreover, ATL1 was able to help BXSB, but not C57BL/6, B cells producing IgG and IgM Abs against dsDNA and histone in vitro. Passive transfer of viable ATL1 cells into young female BXSB mice significantly accelerated the production of autoantibodies. Possible mechanisms of interaction between ATL1 and lupus B cells are further discussed.     

Heterozygosity of the major histocompatibility complex controls the autoimmune disease in (NZW x BXSB) F1 mice.

In the F1 hybrid of phenotypically normal NZW (H-2z) and systemic lupus erythematosus (SLE)-prone BXSB mice (H-2b), features of the disease became more severe than those seen in the BXSB mice, regardless of the presence or absence of the Yaa (Y-chromosome-linked autoimmune acceleration) mutant gene. To determine whether the gene(s) linked to the major histocompatibility complex (MHC) of NZW mice is involved in this event, we developed the H-2-congenic NZW.H-2d strain and compared the severity of autoimmune disease between (NZW x BXSB) F1 (H-2z/b) and (NZW.H-2d x BXSB) F1 mice (H-2d/b). The H-2z/b, but not H-2d/b, heterozygous F1 mice of both sexes showed an accelerated, higher incidence of proteinuria and a more severe thrombocytopenia than did the BXSB mice. In NZW x (NZW x BXSB) F1 backcross mice, the H-2z/b heterozygous progeny showed more severe disease than did the H-2z/z homozygotes. Thus, disease-accelerating events in (NZW x BXSB) F1 mice are linked to the H-2z/b heterozygosity. Because H-2d/z heterozygosity plays a crucial role for SLE in (NZB x NZW) F1 mice, in which SLE features differ from those in (NZW x BXSB) F1 mice, the present observations may imply that the different but related MHC heterozygosity acts as a predisposing genetic element in these different SLE syndromes.