BXSB小鼠外周B淋巴细胞异常增生的研究

系统性红斑狼疮（ＳＬＥ）突出的免疫学异常表现是Ｂ淋巴细胞的异常增生与活化，其发生机制至今不明，本文以遗传性自发性ＳＬＥ模型ＢＸＳＢ小鼠为对象，采用体内ＢｒｄＵ标记法及ＨＵ增殖细胞删除法研究了ＢＸＳＢ小鼠脾脏Ｂ细胞的增殖动力学。结果表明，与正常对照相比，ＢＸＳＢ小鼠脾脏Ｂ细胞更新速率明显增高。提示Ｂ细胞更新加速可能是ＢＸＳＢ小鼠外周Ｂ细胞异常增生的原因。     

用细胞酶联免疫吸附法筛选分泌抗人B细胞分化抗原单抗的杂交瘤

用活化的人Ｂ细胞株３Ｄ５细胞免疫ＢＡＬＢ／Ｃ小鼠，取小鼠脾脏细胞与ＳＰ２／０细胞融合，融合后细胞置甲基纤维素半固体培养基生长。以０．５％甲醛处理的３Ｄ５细胞和ＣＥＭ细胞包被酶细胞反应阳性而与ＣＥＭ细胞反应阴性的克隆。再经间接免疫荧光染色后，用流式细胞仪复测以上所得３３个阳性克隆，结果３Ｄ５阳性ＣＥＭ阴性者２７例，占８１．８２％，表明ＣＥＬＩＳＡ法是一个粗筛抗人Ｂ细胞分化抗原的简便有效方法。     

狼疮性BXSB小鼠B细胞增殖反应异常的初步探讨

系统性红斑狼疮 (ＳＬＥ )小鼠模型是研究人ＳＬＥ发病机制的主要工具。本室曾发现狼疮性ＢＸＳＢ小鼠Ｂ细胞增生异常升高 ,更新速度明显加快[1] 。另一方面 ,ＢＸＳＢ小鼠血清ＩｇＧ2ａ和ＩｇＧ3水平及相应亚类的自身抗体显著升高[2 ,3 ] ,而一般认为ＩｇＧ类型抗体负反馈性抑制Ｂ细胞的功能是机体免疫调节的重要机制之一。本实验初步探讨了正常的 2月龄ＢＡＬＢ/ｃ小鼠和发病的 4月龄雌雄ＢＸＳＢ小鼠Ｂ细胞体外增殖反应状态以及ＩｇＧ类型的兔抗小鼠Ｉｇ (ＲａＭＩｇ )对此的调节作用。1　材料和方法1 1　动物　ＢＸＳＢ小鼠由北医大三院…     

系统性红斑狼疮患者CD28表达及其意义

ＣＤ２８是Ｔ细胞激活中重要的共刺激分子。为了解Ｂ７－Ｃ礤刺激途径在系统性红斑狼疮（ＳＬＥ）中的作用，我们对３０例期ＳＬＥ２外周血Ｔ细胞ＣＤ２８的表达进行了检测，并分析了其激活后凋亡情况。结果表明，ＳＬＥ组的ＣＤ２８Ｔ细胞低于正常对照组，在抗ＣＤ３单抗刺激后ＣＤ２８细胞凋亡率增加。这提示ＳＬＥ中Ｂ７－ＣＤ２８共刺激途径介导的ＡＩＣＤ可能导致ＳＬＥ中的Ｔ细胞淋巴细胞贫血症。     

SLE病人外周血Bcl—2／JH基因重排检测的意义

应用聚合酶链反应（ＰＣＲ）方法检测３１例ＳＬＥ病人外周血单个核细胞（ＰＢＭＣ）Ｂｃｌ－２／ＪＨ基因重排现象和流式细胞仪间接双标记法分析其Ｔ（ＣＤ３）、Ｂ（ＣＤ１９）细胞Ｂｃｌ－２蛋白的表达。结果显示，ＳＬＥ病人Ｔ细胞Ｂｃｌ－２蛋白表达明显高于正常人（４２．９５％±２８．４７％对比９．９４％±４．９６％，Ｐ＝０．０００４），尤其以活动期ＳＬＥ病人为明显，而Ｂ细胞Ｂｃｌ－２蛋白表达与正常人之间并无统计学差异（７９．２１％±１０．６９％对比８１．９６％±６．９７％；Ｐ＝０．４６０２）。７例ＳＬＥ病人具有典型的Ｂｃｌ－２／ＪＨ基因重排（占２２．５８％），且均为ＳＬＥ活动期病人，其Ｔ细胞Ｂｃｌ－２蛋白表达明显高于无基因重排的ＳＬＥ病人，其Ｂ细胞Ｂｃｌ－２表达并无差异（Ｐ＞０．３９０５）。说明Ｂｃｌ－２／ＪＨ基因重排现象可见于ＳＬＥ，并与Ｔ细胞Ｂｃｌ－２蛋白高表达有关，表明细胞凋亡抑制基因Ｂｃｌ－２在ＳＬＥ发病机制中具有重要作用。     

超抗原SEB和D—氨基半乳糖对小鼠肝细胞的作用观察

用超抗原葡萄球菌Ｂ型肠毒素（ＳＥＢ）、Ｄ－氨基半乳糖（Ｄ－ＧａｌＮ）及两者合用腹腔注射ＢＡＬＢ／ｃ小鼠，于２、６、１２、２４ｈ取肝脏和血标本，从形态学和生化指标来研究肝细胞的死亡模式；同时还检测了小鼠２４ｈ死亡率。结果发现单用ＳＥＢ可诱导肝细胞凋亡，其机制可能与ＴＮＦ、ＩＦＮ－γ等细胞因子的产生和作用有关。单用Ｄ－ＧａｌＮ可同时引起肝细胞凋亡和变性，ＳＥＢ和Ｄ－ＧａｌＮ合用２、６ｈ见肝细胞凋亡，１２ｈ后以肝细胞坏死为主，小鼠２４ｈ死亡率达５０％。因而，推测凋亡与坏死间存在一定的联系，在急性肝坏死的发病机制中大量肝细胞凋亡可能是坏死的前奏     

系统性红斑狼疮外周血淋巴细胞凋亡和循环中自身核抗原的关系

系统性红斑狼疮（ＳＬＥ）是一种针对自身细胞核抗原的免疫性疾病，其核抗原中最主要的包括核小体复合物，组蛋白和ＤＮＡ，细胞程序性死亡或凋亡的特征是将细胞核染色质裂解的寡聚核小体，然后将其释放到细胞外空间，这些核小体是否作为免疫原驱动自身免疫反应：ＳＬＥ患者外周血淋巴细胞凋亡是否与ＳＬＥ的发病有关，本文就ＳＬＥ细胞凋亡与自身抗原来源之间的关系作一综述。     

SLE患者外周血B细胞表型的变化

ＳＬＥ患者外周血Ｂ细胞表型的变化梁再赋张士发赵慕丽王壮志顾绍裘ＳＬＥ患者有多种免疫异常，Ｂ细胞高度活化而产生的大量自身抗体是ＳＬＥ发病机理中最为关键的因素，以抗核抗体与ＤＮＡ结合所形成的免疫复合物导致了广泛的组织病理损伤。为了探索Ｂ细胞过度活化的原因...     

系统性红斑狼疮外周血淋巴细胞凋亡和循环中自身核抗原的关系

系统性红斑狼疮（ＳＬＥ） 是一种针对自身细胞核抗原的免疫性疾病，其核抗原中最主要的包括核小体复合物、组蛋白和ＤＮＡ。细胞程序性死亡或凋亡的特征是将细胞核染色质裂解为寡聚核小体，然后将其释放到细胞外空间。这些核小体是否作为免疫原驱动自身免疫反应；ＳＬＥ患者外周血淋巴细胞凋亡是否与ＳＬＥ的发病有关，本文就ＳＬＥ细胞凋亡与自身抗原来源之间的关系作一综述。     

超抗原金黄色葡萄球菌肠毒素B诱导小鼠免疫细胞凋亡

为探讨超抗原金黄色葡萄球菌肠毒素Ｂ对小鼠免疫细胞调亡的影响，采用形态学观察及ＤＮＡ断裂指标检测了ＳＥＢ诱导小鼠免疫细胞凋亡的剂量和效应关系。结果表明，１２．５μｇ，２５ｇ和２５μｇ和５０μｇＳＥＢ均要引起部分胸腺细胞、脾细胞和肠系膜淋巴结细胞出现核固缩、核断裂及凋亡小体形成等形态学变化。上述免疫细胞ＮＤＡ琼脂糖电泳可见典型的“梯状”带。而对照组免疫未见上述明显变化。本实验表明超抗原ＳＥＢ可诱导小鼠     

狼疮性BXSB小鼠脾脏淋巴细胞增殖与凋亡的初步分析

为了比较全面准确地了解系统性红斑狼疮 (SLE )BXSB小鼠的发病过程中 ,淋巴细胞增殖与凋亡的动力学变化及其机制。采用细胞双色荧光染色的标记技术 ,检测了脾脏淋巴细胞中的增殖细胞和凋亡细胞的百分率 ,并且测定了巨噬细胞吞噬凋亡细胞的能力。结果发现 ,发病的雄性BXSB小鼠和雌性BXSB小鼠脾脏中增殖的CD4 + T淋巴细胞和B淋巴细胞百分率显著高于对照C5 7小鼠 ,而凋亡的B淋巴细胞的百分率显著低于对照C5 7小鼠 ;但是 ,雌雄BXSB小鼠和对照C5 7小鼠巨噬细胞吞噬凋亡细胞的吞噬指数相同。本研究结果表明 ,在BXSB小鼠的SLE发病过程中 ,淋巴细胞的增殖速度异常升高、而凋亡速度下降 ,可能与其脾脏肿大有关 ;而且淋巴细胞的增殖与凋亡的失衡与巨噬细胞的功能无关 ,可能与淋巴细胞内在的异常有关。     

The Role of the Yaa Gene in Lupus Syndrome

The BXSB/MpJ (BXSB) murine strain (H-2^b) spontaneously develops an autoimmune syndrome with features of systemic lupus erythematosus (SLE) that affects males much earlier than females. A mutant gene located on the BXSB Y chromosome, designated Yaa (Y chromosome-linked autoimmune acceleration), is responsible for the acceleration of the disease observed in male BXSB mice. Studies on H-2 congenic and I-E transgenic mice have clearly demonstrated that the MHC class II genes play a crucial role in the development or protection of SLE. However, the MHC effect can be completely masked by the presence of the Yaa gene in mice with certain genetic backgrounds. It is intriguing that the Yaa gene effect is selective on autoimmune responses, varying in different lupus-prone mice. Studies on immune responses against foreign antigens have shown that the Yaa gene potentiates immune responses only against antigens to which mice are genetically (H-2-linked) low-responding, but not high-responding. Thus, the selective immune enhancing activity of the Yaa gene may be related to differences in the capacity of T helper cells specific for given antigens. Moreover, studies on Yaa⁺Yaa^? bone marrow cell chimeric mice have suggested that a specific cognate interaction of T helper cells with Yaa⁺ B cells is responsible for a selective enhancing effect of immune responses to foreign antigens as well as autoantigens. It is significant that unlike the lpr mutation, whose abnormality is associated with the capacity of the Fas antigen to mediate apoptosis, the Yaa gene by itself is unable to induce significant autoimmune responses in mice without apparent SLE background. This suggests that-the molecular defect of the Yaa gene is likely to differ from that of the lpr gene, and that the Yaa gene effect requires the abnormal autosomal genome present in lupus-prone mice. Based on these findings, a possible molecular nature of the Yaa gene abnormality will be discussed.     

Expanded macrophage precursor populations in BXSB mice: possible reason for the increasing monocytosis in male mice.

The BXSB mouse spontaneously develops an autoimmune disease that resembles human systemic lupus erythematosus (SLE). During their lifetime, male BXSB mice show an increasing monocytosis in the peripheral blood as opposed to their female littermates. This monocytosis is unique among autoimmune-prone mice. To test the hypothesis that alterations at the stem cell level may be responsible for this monocytosis, myeloid bone marrow precursor cells were examined in both male and female BXSB mice from 4 to 40 weeks of age. The number of M-CSF responding stem cells (CFU-M) and the number of GM-CSF responding stem cells (CFU-GM) were higher than in all other inbred mouse strains tested. In addition, male BXSB mice developed a progressive increase of CFU-M and CFU-GM in the bone marrow during their lifetime, which paralleled the peripheral blood monocytosis. The monocytosis in male BXSB mice is the result of a further expansion of the strain-specific high number of macrophage precursors by intrinsic factors, which may be attributed to the influence of the Yaa factor. The sex-specific expanded mononuclear phagocyte system may promote the autoimmune process and may be one reason for the dramatic course of murine SLE in male BXSB mice.     

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.     

脱氢表雄酮对狼疮性BXSB小鼠脾脏淋巴细胞凋亡的影响

目的 研究脱氢表雄酮(DHEA)对BXSB狼疮性小鼠脾脏淋巴细胞凋亡的影响,并探讨其作用机理.方法 运用流式细胞术、免疫双荧光染色法检测脾脏淋巴细胞凋亡.结果 当DHEA的浓度为10-8mol/L时,淋巴细胞凋亡率降低,与对照组比较有差异有统计学意义(P＜0.05),细胞坏死率差异无统计学意义(P＞0.05);DHEA与西药组或中药组联合应用对淋巴细胞凋亡抑制作用更加显著,差异有统计学意义(P＜0.01).结论 脱氢表雄酮可明显抑制BXSB小鼠脾脏淋巴细胞凋亡率.     

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.     

B cells from autoimmune BXSB mice are hyporesponsive to signals provided by CD4+ T cells

Male BXSB mice, unlike female BXSB mice, develop an early-onset, lupus-like disease characterized by high levels of anti-nuclear antibodies (Abs) and total Ig. It has recently been shown that the male BXSB mice contain an expanded population of large B cells which are hyperresponsive to stimulation by anti-CD40 mAb. The present study was undertaken to determine whether their potential for extra CD40 signaling enabled the B cells from male BXSB mice to hyper-respond to CD40L-expressing CD4+ T cells. In contrast to expectations, large B cells from male BXSB mice did not interact with CD4+ T cells in a positive manner; cultures of B cells from antigen (Ag)-primed male BXSB mice, unlike cultures of B cells from Ag-primed female mice, generated few antibody forming cells (AFC) following interaction with activated CD4+T cells. In addition, B cells from male BXSB mice, unlike B cells from female BXSB mice, failed to upregulate MHC class II molecules following interaction with activated CD4+ T cells. Subsequent experiments revealed that the inability of the B cells from the male mice to upregulate MHC class II molecules in response to T cell-mediated activation resided primarily in the population of large B cells. Large B cells from male BXSB mice were also defective in their ability to proliferate following stimulation with activated CD4+ T cells. Taken together, these findings demonstrated that similar to B cells in lupus patients, large B cells from male BXSB mice could function in a hyporesponsive manner, and that this hyporesponsiveness related to the inability of the B cells to interact in a positive manner with CD4+T cells.     

Male BXSB mice develop a thymic hormonal dysfunction with presence of intraepithelial crystalline inclusions

The thymus is morphologically abnormal in male BXSB mice with cortical involution densification of the epithelium and the presence of intraepithelial crystals. The thymic endocrine function in BXSB mice was appraised using a biological assay to measure the level of the zinc-dependent thymic hormone, thymulin, and an indirect immunofluorescence technique to evaluate the number of cells synthesizing the hormone within the thymus. Unlike the dramatically accelerated age-linked decline of thymulin production reported in females of other autoimmune strains (measured as early as 6 weeks of age), only male BXSB were affected, as compared to normal strains. The number of hormone-producing cells was significantly reduced in male BXSB thymuses, in parallel with this hormonal decrease. Thymulin inhibitory molecules were detected in male BXSB sera, as early as 8 weeks of age, as evaluated by their capacity to absorb in vitro and in vivo the biological activity of the hormone. These inhibitors are thymus dependent since they disappear after adult thymectomy. They are low MW molecules (less than 10 kDa), as previously found in normal aging mice, rather than autoantibodies, as evidenced in two autoimmune strains (B/W and db/db mice). These findings demonstrate that male BXSB mice develop thymic abnormalities very similar to those observed in other autoimmune strains. The presence of intrathymic crystals and of low MW inhibitors suggests the role of abnormal storage and the excretion of thymulin. This thymic dysfunction may play a role in the maintenance of B cell hyperactivity previously shown in BXSB males.     

Antibody production in autoimmune BXSB mice. I. CD40L-expressing B cells need fewer signals for polyclonal antibody synthesis.

Male, but not female, BXSB mice develop severe lupus associated with multiple immune system defects. It was recently shown that one immunological abnormality found in male BXSB mice encompasses B cell expression of CD40 ligand (CD40L) by an expanded population of large B cells. The present study was undertaken to determine how the CD40L-expressing large B cells in male BXSB mice compared with size-matched B cells from female mice in terms of their ability to secrete antibody. It was shown that the large B cells from female mice, similar to the small B cells from either male or female mice, required CD40 signalling, immunoglobulin cross-linking and cytokines for optimal antibody synthesis. In contrast, large B cells from male BXSB mice produced high levels of antibody when stimulated with only two of the three signals, and made significantly more total IgM and IgG, and anti-ssDNA antibody than size-matched B cells from female mice when stimulated with IL-4/IL-5 alone, IL-4/IL-5 plus low levels of anti-IgD-dextran, or IL-4/IL-5 plus anti-CD40 MoAb. The ability of the large B cells from male mice to produce antibody under suboptimal stimulatory conditions correlated with their expression of CD40L, and was inhibited by CD40-immunoglobulin. Taken together, these findings suggested that large CD40L-expressing B cells from male BXSB mice may be able to bypass a need for CD40 signalling from T cells, thus contributing to autoimmune disease by promoting antibody production in the absence of cognate T cell help.