Spontaneous B cell hyperactivity in autoimmune-prone MRL mice

The MRL-lpr/lpr mouse strain is a commonly used model of the human autoimmune disease systemic lupus erythematosus (SLE). Although much is known about the contribution of the lpr Fas mutation to B cell tolerance breakdown, the role of the genetic background of the MRL strain itself is less well explored. In this study, we use the MD4 anti-hen egg lysozyme Ig (IgHEL) transgenic system to explore B cell function in MRL+/+ and non-autoimmune mice. We demonstrate that MRL IgHEL B cells show spontaneous hyperactivity in the absence of self-antigen, which is associated with low total B cell numbers but an expansion of the marginal zone B cell population. However, B cell anergy is normal in the presence of soluble lysozyme [soluble hen egg lysozyme (sHEL)], and MRL IgHEL B cells undergo normal elimination in the presence of sHEL when competing with a polyclonal C57BL/6 B cell repertoire. We conclude that B cell hyperactivity may contribute to the autoimmune phenotype of MRL+/+ and MRL-lpr/lpr strains when it initiates antibody responses to rare or sequestered antigens that are below the threshold for tolerance induction, but that there is no B cell intrinsic defect in anergy in MRL mice.     

Interleukin-1 receptor associated kinase 1 is a potential therapeutic target of anti-inflammatory therapy for systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is a chronic autoimmune inflammatory disease and currently has no effective therapy. The genome-wide analyses indicate that interleukin-1 receptor associated kinase 1 (IRAK1) is associated with the susceptibility of SLE in humans. In the present study, we identified that IRAK1 was overexpressed and hyper-activated in splenic mononuclear cells from B6.MRL-Fas^lpr/Nju (B6.lpr) mice and peripheral blood mononuclear cells (PBMCs) from SLE patients. Intraperitoneal treatment with a small molecular inhibitor of IRAK1 (IRAK1/4 inhibitor or IRAK-Inh) significantly mitigated inflammatory responses and renal injury in B6.lpr mice. IRAK-Inh treatment or knockdown of IRAK1 by specific siRNA decreased the relative levels of NF-κBp65 phosphorylation in human PBMCs from SLE patients. Therefore, IRAK1 may be a potential target for anti-inflammatory therapy for SLE and other inflammatory diseases.     

白细胞介素10信号转导机制对C57BL/6和MRL/lpr~-小鼠腹腔巨噬细胞功能及活性影响的比较

目的：体外比较白细胞介素10（IL-10）信号转导机制对C57BL/6和MRL/lpr-小鼠腹腔巨噬细胞功能及活性的影响。方法：分别分离C57BL/6和MRL/lpr-小鼠腹腔巨噬细胞进行培养；用不同浓度IL-10（0.01、0.1、1和10ng/ml）进行刺激,用MTT法测定比较两种品系小鼠腹腔巨噬细胞的增殖反应能力；Real time PCR分别测定其产生前炎性细胞因子IL-1α、M-CSF、TNF-α的量；用100ng/mlIL-10分别刺激两种品系小鼠腹腔巨噬细胞10分钟,免疫印迹法比较两种细胞中JAK1、TYK2、STAT3及磷酸化水平。结果：C57BL/6小鼠巨噬细胞数及其产生的前炎性细胞因子量随IL-10刺激浓度的增加而递减；MRL/lpr-小鼠巨噬细胞数及其前炎性细胞因子IL-1α、M-CSF的量随IL-10刺激浓度的增加而递增,但巨噬细胞产生TNF-α的量随IL-10刺激浓度的增加而递减；MRL/lpr-小鼠细胞内信号转导分子的磷酸化水平低于C57BL/6。结论：MRL/lpr-小鼠的细胞免疫和炎症反应不能被IL-10抑制,可能是由于其信号转导过程中的缺陷所致。     

Murine lupus in MRL/lpr mice lacking CD4 or CD8 T cells

MRL/lpr mice develop a systemic autoimmune disease similar to systemic lupus erythematosus in humans. The mice show progressive lymphadenopathy due to the accumulation of an unusual population of CD4^?8^?(DN) B220⁺ αβ⁺ T cells. We bred MRL/lpr mice with mice lacking CD4⁺ or CD8⁺ T cells by gene targeting via homologous recombination in embryonal stem cells to determine the roles of these cells in the autoimmune disease. No difference in survival or autoantibody levels was noted between CD8-/- lpr and littermate controls. Interestingly, these CD8-/- lpr mice have a reduced level of B220⁺ DN T cells despite the fact that the degree of lymphadenopathy was unaltered. CD4-/- lpr mice had a diminished autoimmune disease with a reduction in autoantibody production and skin vasculitits, and increased survival compared to littermate controls. However, CD4-/- lpr mice had an enhanced splenomegaly that developed massively by 16–20 weeks of age (5 to 8 greater than lpr control mice) due to the accumulation of DN B220⁺ T cells. In addition, there were no differences in peripheral lymph node enlargement, although the proportion of DN B220⁺ T cells was about twofold higher in the CD4-/- lpr mice. These cells were phenotypically identical to the DN population in control lpr mice, indicating that the accumulating DN T cells can be dissociated from the autoimmune disease in these mice. Collectively, our results reveal that the autoimmune disease is dependent on CD4⁺, but not CD8⁺ T cells, and that many of the B220⁺ DN T cells traverse a CD8 developmental pathway.     

Ets-1在系统性红斑狼疮中的研究进展

系统性红斑狼疮(SCE)是一种典型的系统性自身免疫性疾病,其发病机制尚未明确,其中T、B细胞功能异常起着重要作用.转录因子Ets-1作为SLE的易感基因之一在淋巴细胞分化与细胞因子调节上起重要作用.Ets-1除影响B细胞分化和功能外,对T细胞的生存、增殖、发育和功能起重要作用.虽然Ets-1在SLE发病中的确切机制仍尚未明确,但越来越多的研究表明Ets-1在SLE的发生发展中起着重要作用.     

P-糖蛋白单克隆抗体改善MRL/lpr狼疮鼠肾脏病变的研究

目的 探讨P-糖蛋白(P-glyeoprotein,P-gp)单克隆抗体(UIC2)在改善MRL/lpr狼疮鼠肾脏病变中的作用.方法 24只14周龄雌性MRl/lpr狼疮鼠分为3组:P-gp单克隆抗体1次治疗组(G1)、P-gp单克隆抗体3次治疗组(G2)、对照组(G0).观察体重;采用考马斯亮蓝法检测24 h尿蛋白定量;采用酶联免疫吸附法试验(enzyme linked immunosorbent assay,ELISA)检测抗双链DNA(dsDNA)抗体水平;采用酶法检测血清肌酐水平;采用苏木素-伊红染色、免疫荧光和电镜观察肾脏病理改变.结果 ①22周时G1组和G2组体重高于G0组(P<0.05).② 24 h尿蛋白定量22周时Gl组[(1.9±1.1)mg]和G2组[(1.4±0.9)mg]低于G0组[(3.1±1.9)mg](P<0.05);26周时G1组[(2.4±1.4)mg]和G2组[(1.8±1.1)mg]低于G0组[(5.3±2.2)mg](P<0.05).③26周时血清肌酐G1组[(7.0±2.9)μmol/L]和G2组[(6.1±2.5)μmol/L]低于G0组[(12.7±1.3)umol/L](P<0.05).④19周时,抗dsDNA抗体滴度G1组[(43±19)×102 U/mL]和G2组[(45±32)×102 U/mL]低于G0组[(87±39)×102 U/mL](P<0.05);26周时G2组[(35±11)×102 U/mL]低于G0组[(59±35)×102 U/mL].⑤肾小球新月体形成率G1组(0.11±0.05)和G2组(0.09土0.01)低于G0组(0.23±0.07),G2组较Gl组减低(P均<0.05).结论 P-gp单克隆抗体可改善MRl/lpr狼疮鼠肾脏病变的进展,减少尿蛋白、降低血清肌酐,对狼疮肾炎有显著疗效.     

系统性红斑狼疮病人外周血中TNF和IL—1的检测

肿瘤坏死因子（ＴＮＦ）和白细胞介素１（ＩＬ－１）是一对介导炎性反应的主要细胞因子，用细胞生物法，ＥＬＩ０Ａ法和３Ｈ－ＴｄＲ渗入法对２０例系统性红斑狼疮（ＳＬＥ）患者的外周血单个核细胞（ＰＢＭＣ）培养上清和血清进行了ＴＮＦ和ＩＬ－１水平的检测。结果表明：ＳＬＥ病人的ＰＢＭＣ在体外对有丝分裂原的刺激不敏感。其上清液中的ＴＮＰ的活性和上清液及血清中ＴＮＦ－α蛋白含量的水平与疾病的活动性及是否合并感染均无相关性。ＳＬＥ病人ＩＬ－１的生物活性和健康人无明显差异。活动期病人ＩＬ－１的分泌水平低于恢复期患者，ＩＬ－１分泌能力的降低和病情活动有关。     

Methyl- rich diet ameliorates lupus-like disease in MRL/lpr mice

Genetic susceptibility is necessary but not sufficient for systemic lupus erythematosus (SLE) to appear indicating that environmental factors are also key components in the disease onset. Aberrant DNA methylation profile positively correlates with the development of lupus-like disease in MRL/lpr mice. In the present study, we evaluate the effect of long term administration of methyl-rich diet in MRL mice. The results showed that supplemented diet decreased the levels of proteinuria and of anti-dsDNA antibodies and modulated cytokine profiles. Limited kidney failure and prevented development of skin lesions in MRL/lpr mice were another positive effects of the high-dose methyl diet. These data suggest that it is possible to modulate the disease course by altering the amount of particular dietary micronutrients and that nutrition-mediated changes in DNA methylation may have potential clinical relevance.     

Selective silencing of DNA-specific B lymphocytes delays lupus activity in MRL/lpr mice

The pathological DNA-specific B lymphocytes in lupus are logical targets for a selected therapeutic intervention. We have hypothesized that it should be possible to suppress selectively the activity of these B cells in lupus mice by administering to them an artificial molecule that cross-links their surface immunoglobulins with the inhibitory FcgammaIIb surface receptors. A hybrid molecule was constructed by coupling the DNA-mimicking DWEYSVWLSN peptide to a monoclonal anti-mouse FcgammaRIIb antibody. This chimeric antibody was added to cultured spleen cells from sick MRL/lpr mice, immunized with diphtheria toxoid, resulting in reduction of the numbers of anti-DNA but not of anti-diphtheria IgG antibody-producing cells. Intravenous infusions with the DNA-peptide antibody chimera to 7-wk-old animals prevented the appearance of IgG anti-DNA antibodies and of albuminuria in the next 2 months. The administration of the DNA-peptide chimeric antibody to 18 wk-old mice with full-blown disease resulted in the maintenance of a flat level of IgG anti-DNA antibodies and in delay of the aggravation of the lupus glomerulonephritis. The use of chimeric antibodies targeting inhibitory B lymphocyte receptors represents a novel approach for the selective suppression of autoreactive disease-associated B cells in autoimmune diseases.     

肾怡对狼疮小鼠免疫器官内Th1/Th2细胞比例的调节作用

目的：探讨复方中药肾怡对MRL/lpr小鼠免疫器官内Th1/Th2细胞比例的调节作用。方法：将MRL/lpr狼疮小鼠随机分成对照组和中药治疗组，从12周观察到28周后于实验终点处死小鼠。通过流式细胞术分别检测胸腺和脾脏中CD4^ CD30^-(Th1)、CD4^ CD30^ (Th2)淋巴细胞所占百分比，进一步对脾细胞进行CD^ T淋巴细胞分选，并于体外利用PHA—P进行刺激后，通过RT—PCR方法检测IL-4(Th2细胞因子)和IFN—γ(Th1细胞因子)的表达丰度，比较二者的比值在对照组和中药治疗组之间的差别。结果：胸腺中几乎检测不到CD4^ CD30^ T淋巴细胞；脾脏中虽可检测到CD4^ CD30^ T淋巴细胞，但在未加入PHA-P的条件下，CD4^ CD30^ T淋巴细胞在对照组和中药治疗组中所占的比例没有显著差异；经PHAP刺激后对照组和治疗组脾细胞中CD4^ CD30^ T淋巴细胞所占百分比均明显增加，但治疗组中CD4^ CD30^ T淋巴细胞所占百分比的增加程度明显低于对照组；将脾细胞中CD4^ T淋巴细胞分选出来后进行RT-PCR检测，结果显示中药治疗组CD^ T淋巴细胞所表达IL-4／IFN—γ(Th2／Th1)比值明显低于对照组。结论：复方中药肾怡能够纠正MRL/lpr狼疮小鼠脾脏中CD^ T淋巴细胞在PHA-P刺激的条件下朝向Th2过度分化的倾向性。     

外周血CD19+B细胞PD-L1的表达与系统性红斑狼疮发病的相关性分析

目的:探讨程序性死亡配体1(Programmed death ligand-1,PD-L1)在系统性红斑狼疮(Systemic lupus erythema-tosus,SLE)患者外周血B细胞上的表达及临床意义。方法:应用流式细胞仪检测51例SLE患者和38例健康对照者外周血CD19+B细胞表面PD-L1的表达水平,比较SLE稳定组、活动组和健康对照组以及狼疮肾炎组和无狼疮肾炎组之间CD19+B细胞表面PD-L1表达阳性细胞的百分比,并分析其与临床表现及实验室检查数据的相关性。结果:SLE活动组和稳定组CD19+PD-L1+B细胞百分率均低于健康对照组,活动组又低于稳定组,差异均有统计学意义(均P<0.05)。狼疮肾炎患者CD19+PD-L1+B细胞百分率低于无狼疮肾炎患者(P<0.05)。SLE患者CD19+PD-L1+B细胞百分率与SLEDAI评分、尿蛋白定量、呈负相关,与C3呈正相关。SLE患者中抗dsDNA抗体、抗Sm抗体、抗U1snRNP抗体、抗核小体抗体阳性组外周血B细胞PD-L1表达水平均低于对应阴性组,且均有统计学意义(均P<0.05)。结论:SLE患者外周血CD19+B细胞表达PD-L1下降,与病情活动性和抗体产生有很好的相关性。     

Serum levels of soluble programmed death-1 (sPD-1) and soluble programmed death ligand 1(sPD-L1) in systemic lupus erythematosus: Association with activity and severity

The programmed cell death-1 (PD-1)/programmed cell death ligand 1 (PD-L1) pathway is an important host immunosuppression mechanism. Soluble PD-1 (sPD-1) and PD-L1 (sPD-L1) expression regulates co-inhibitory signals in autoimmune disorders. Here, we evaluated whether serum sPD-1 and sPD-L1 are involved in immune dysfunction and assessed its relationship with SLE. Blood samples were obtained from 130 patients with SLE and 44 healthy controls. Serum sPD-1 and sPD-L1 were tested by enzyme-linked immunosorbent assay (ELISA). Relevant immune parameters were analysed. Both serum sPD-1 and sPD-L1 were significantly higher in the SLE patients than in the controls. A series of severe disease clinical manifestations and laboratory features such as presence of decreased complement component 3, complement component 4 and SLEDAI >8 were associated with elevated sPD-1 and sPD-L1. Our study suggests that abnormal sPD-1 and sPD-L1 expression may be involved in the imbalance of immune regulation in SLE.     

In vivo anti-inflammatory activities of novel cytokine IL-38 in Murphy Roths Large (MRL)/lpr mice

The newly named interleukin (IL)-36 subfamily member IL-38 has been shown to exert anti-inflammatory activity. However, the in vivo immunomodulatory activity of IL-38 was poorly investigated in systemic lupus erythematosus (SLE). We have investigated the expression of CD4⁺IL-17⁺ Th17, CD4⁺IFN-γ⁺ Th1 and CD3⁺CD4⁻CD8⁻ double negative (DN) T cells and the related immunopathological mechanisms in female MRL/lpr mice model of spontaneous lupus-like disease, with or without IL-38 treatment. Intravenous administration of murine recombinant IL-38 into MRL/lpr mice can ameliorate the lupus-like clinical symptoms including proteinuria, leukocyteuria and skin lesions. A remission of histopathology characteristics of skin and nephritis was also observed upon IL-38 treatment. Accordingly, IL-38 receptor was expressed on the cell surface of both CD4⁺ Th and CD19⁺ B lymphocytes. The splenic Th17 and DN T lymphocytes, the average mRNA level of epigenetically regulated gene expression of Th17 cells, and serum concentrations of IL-17 and IL-22 were significantly decreased upon the treatment of IL-38 (all p < 0.05). The in vivo results suggest that IL-38 can ameliorate skin inflammation and nephritis in SLE mice probably via suppressing the formation of inflammatory cytokines such as IL-17 and IL-22, and pathogenic DN T cells. These findings may provide a biochemical basis for further investigation of the therapeutic mechanisms of IL-38 for the treatment of autoimmune-mediated inflammation.     

Matrix metalloproteinase-9 and its natural inhibitor TIMP-1 expressed or secreted by peripheral blood mononuclear cells from patients with systemic lupus erythematosus

Matrix metalloproteinase-9 (MMP-9) was involved in inflammation and immune system dysfunctions. Besides immunologic abnormalities, systemic lupus erythematosus (SLE) also presents chronic inflammatory components. Therefore, a role of MMP-9 in SLE pathology might be supposed. To verify this hypothesis, SLE patients and healthy donors were compared for the MMP-9 and MMP-9 mRNA levels in peripheral blood mononuclear cells (PBMCs), the spontaneous secretion of MMP-9 and TIMP-1 and the MMP-9 activity. Thus, we found that fresh PBMCs from SLE patients expressed a significantly higher activity of MMP-9 and spontaneously released higher levels of MMP-9, as compared to healthy donors, while the secreted TIMP-1 level was the same for both groups. When the patients were sub-grouped based on disease status, the most increased pro-MMP-9 activity inside the PBMCs was identified for relapse SLE sub-group. A similar observation for SLE patients with positive serum fibrinogen was found. Following culture, the PBMCs from remission SLE patients secreted significantly higher MMP-9 level, than the PBMCs from relapse SLE patients. PBMCs from relapse SLE patients secreted the highest levels of TIMP-1, although this difference was not statistically significant. Taken together, these observations suggested the multiple roles of MMP-9 and TIMP-1 in progress of inflammation and tissue damage and/or in repair, depending on clinical stages of SLE.     

Dnase1‐deficient mice spontaneously develop a systemic lupus erythematosus‐like disease

Systemic lupus erythematosus (SLE) is an autoimmune disease that has high morbidity and can result in multi‐organ damage. SLE is characterized by dysregulated activation of T‐ and B‐lymphocytes and the production of autoantibodies directed against nuclear components. The endonuclease deoxyribonuclease 1 (DNase1) is abundant in blood and a subset of SLE patients have mutations in DNASE1. Furthermore, a report showed that Dnase1‐deficient mice develop an SLE‐like disease, but these mice also carry a deletion of the gene adjacent to Dnase1, which encodes the chaperone TRAP1/HSP75. We generated a murine strain deficient in Dnase1 with an intact Trap1 gene to examine if a lack of DNase1 is responsible for the development of a spontaneous SLE‐like disease. We show that the Dnase1‐deficient mice do indeed develop an SLE‐like phenotype with elevated autoantibody production by 9 months and kidney damage by 12 months. Notably, this model recapitulates the female bias seen in human SLE patients since female Dnase1‐deficient mice produced the highest concentrations of autoantibodies and had more severe kidney damage than males. Since there is currently no cure for SLE the protective role of DNase1 as demonstrated in our study remains of great therapeutic interest.     

Systemic IFN-alpha drives kidney nephritis in B6.Sle123 mice

The impact of IFN-alpha secretion on disease progression was assessed by comparing phenotypic changes in the lupus-prone B6.Sle1Sle2Sle3 (B6.Sle123) strain and the parental C57BL/6 (B6) congenic partner using an adenovirus (ADV) expression vector containing a recombinant IFN-alpha gene cassette (IFN-ADV). A comprehensive comparison of cell lineage composition and activation in young B6 and B6.Sle123 mice revealed a variety of cellular alterations in the presence and absence of systemic IFN-alpha. Most IFN-alpha-induced phenotypes were similar in B6 and B6.Sle123 mice; however, B6.Sle123 mice uniquely exhibited increased B1 and plasma cells after IFN-alpha exposure, although both strains had an overall loss of mature B cells in the bone marrow, spleen and periphery. Although most of the cellular effects of IFN-alpha were identical in both strains, severe glomerulonephritis occurred only in B6.Sle123 mice. Mice injected with IFN-ADV showed an increase in immune complex deposition in the kidney, together with an unexpected decrease in serum anti-nuclear antibody levels. In summary, the predominant impact of systemic IFN-alpha in this murine model is an exacerbation of mechanisms mediating end organ damage.     

The Yaa gene-mediated acceleration of murine lupus: Yaa− T cells from non-autoimmune mice collaborate with Yaa+ B cells to produce lupus autoantibodies in vivo

The BXSB Y chromosome-linked mutant gene, Yaa, promotes autoimmune responses in mice predisposed to a lupus-like autoimmune disease. We have previously shown that a cognate interaction of T cells with B cells expressing the Yaa gene appears to be responsible for the accelerated production of autoantibodies. To investigate whether T cells that provide help for autoantibody production by Yaa⁺ B cells need to express the Yaa gene, we have made radiation bone marrow chimeras containing two sets of T and B cells from mice with or without the Yaa gene and differing by the Thy-1 and Igh allotypes. We then determined autoantibody production following the selective elimination of T cells of Yaa⁺ origin by treating mice with allele-specific anti-Thy-1 monoclonal antibody. Our results demonstrated that the selective production of autoantibodies by Yaa⁺ B cells in Yaa⁺-Yaa^? double bone marrow chimeras can be mediated as efficiently by T cells from non-autoimmune mice lacking the Yaa gene as by T cells from autoimmune mice bearing the Yaa gene. This indicates that T cells from non-autoimmune Yaa^? mice are capable of providing help for autoimmune responses by collaborating with Yaa⁺ B cells. These data thus strongly suggest that the Yaa gene defect is not functionally expressed in T cells, but only in B cells, and contrast with parallel experiments in the lpr model, in which defects of the Fas antigen in both T and B cells are crucial for the lpr gene-mediated promotion of autoantibody production.