ALTERED EXPRESSION OF THE T CELL RECEPTOR-CD3 COMPLEX IN SYSTEMIC LUPUS ERYTHEMATOSUS

SLE T cells may play a key role in autoantibody production in SLE B cells. In addition, accumulating evidence has shown that SLE T cells participate in the attack on target cells or tissues through the overproduction of pro-inflammatory cytokines or an increase in cell-to-cell adhesion. Thus, the functional abnormality of SLE T cells appears to be pivotal to an understanding of SLE pathogenesis. Accumulating evidence suggests that potential defects may reside in the proximal signal transduction around the TCR-CD3 complex. We have demonstrated that the expression of TCR ζ chain is significantly decreased in peripheral blood T cells from SLE patients. To explore the mechanism of defective expression of TCR ζ chain, we examined mRNA of TCR ζ, and found that two alternatively spliced variants such as exon 7 (-) and short 3'-UTR are detected in SLE. We review the possible role of the TCR ζ defects in autoimmunity and discuss how the splicing variants lead to downregulated protein expression of TCR ζ chain.     

MITOCHONDRIAL DYSFUNCTION IN T CELLS OF PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS

Activation, proliferation, or programmed cell death of T lymphocytes are dependent on controlled reactive oxygen intermediates (ROI) production and ATP synthesis in mitochondria. The mitochondrial transmembrane potential (ΔΨ_m) also plays a decisive role in cell survival by controlling activity of redox-sensitive caspases. T lymphocytes of patients with systemic lupus erythematosus (SLE) exhibit mitochondrial hyperpolarization, increased ROI production, diminished intracellular glutathione levels, cytoplasmic alkalinization, and ATP depletion that mediate enhanced spontaneous and diminished activation-induced apoptosis and sensitize lupus T cells to necrosis. These redox and metabolic checkpoints represent novel targets for pharmacological intervention in SLE.     

蛋白酪氨酸激酶的酶联免疫吸附测定方法

通过对多种影响因素的摸索，建立了蛋白酪氨酸激酶（ＰＴＫ）的固相ＥＬＩＳＡ测定方法，并对该方法进行了改进和标准化处理。研究表明ＥＬＩＳＡ法具有较好的可重复性、特异性与灵敏度，操作简便安全，是一种切实可行的好方法。     

TRANSLATIONAL CONTROL IN T LYMPHOCYTES

Translational control is an important but relatively unappreciated mechanism that regulates levels of protein products. In addition to a global translational control that regulates the cell's response to external stimuli such as growth factors, cytokines, stress, and viral infections, selective translational control has recently been demonstrated to affect many genes related to growth and apoptotic processes. Translational infidelity has recently been suggested as a new mechanism of T cell dysregulation in SLE.

This review discusses current data on translational control of T cell biology and the central aspect of translational control in the signalling pathway leading to T cell proliferation, apoptotic response, and cytokine production. The utility for global analysis by genomics to study translational control of T cell gene expression is also discussed.     

依赖于环腺苷酸蛋白激酶的单克隆抗体制备及其在正常和恶性细胞内的免疫细胞化学定位观察

用依赖于环腺苷酸(cAMP)的蛋白激酶(蛋白激酶A,PKA)免疫LOU/C大鼠,分离出脾细胞与大鼠骨髓瘤细胞系IR 983 F融合,经HAT培养液筛选和ELISA检测,获得1株分泌抗PKA单克隆抗体的大鼠杂交瘤细胞系B5 A8。免疫扩散实验表明,该单克隆抗体亚类为IgG_1。免疫印迹实验显示,此单克隆抗体可识别蛋白激酶A的52～56 kd多肽。利用经亲和层析法纯化的单克隆抗体,采用免疫荧光细胞化学方法检测人正常成纤维细胞、人胃癌细胞系和小鼠艾氏腹水癌细胞内的蛋白激酶A定位,结果显示蛋白激酶A主要定位于细胞质,与细胞骨架的分布一致。同步化的胃癌细胞系蟹白激酶A在G_2期进入胞核,经外源性cAMP处理后的胃癌细胞蛋白激酶A向核周围积聚。本文对蛋白激酶A在细胞内分布的变化进行了讨论。     

ALTERED SIGNALING TRIGGERED BY LIGATION OF THE TCR/CD3 RECEPTOR IN T LYMPHOCYTES FROM PATIENTS WITH SYSTEMIC LUPUS ERYTHEMATOSUS: THE ROAD FROM ANERGY TO AUTOIMMUNITY

Some of the signaling phenotypic abnormalities observed in peripheral blood T cells from patients with systemic lupus erythematosus resemble those seen in cells anergized by stimulation through the TCR/CD3 complex, in the absence of costimulatory signals, or by stimulation with altered ligands. The potential association of this “anergic” phenotype and the triggering of autoimmune responses in human lupus is discussed.     

蛋白激酶C亚型在原代培养神经元生长中的作用

目的　探讨PKC不同亚型在原代培养脊髓神经元突起生长中的作用。　方法　采用免疫组织化学技术研究蛋白激酶C(PKC) 4种亚型 (α、βⅠ、βⅡ和ε)在胎鼠脊髓原代培养神经元突起生长过程中的表达与分布 ,借助计算机图像分析系统同步测定突起长度和胞体面积。　结果　原代培养神经元生长过程中 ,7d前随着PKC 4种亚型的表达逐渐增加 ,神经元胞体逐渐长大及突起逐渐延长 ;7d以后无论在胞体还是突起中 ,PKC 4种亚型的表达均下降 ,突起开始萎缩 ,胞体内出现空泡。相关分析发现 βⅡ亚型与突起生长关系最密切 (r =0 73,P <0 0 1)。 　结论　PKC可能参与了神经元的生长过程 ;βⅡ型PKC在培养脊髓神经元突起生长中的作用较为重要     

固定剂和蛋白激酶C活性对蛙骨骼肌纤维三联体超微结构的影响

用透射电镜研究了不同固定方法和蛋白激酶Ｃ（ＰＫＣ）活性对蛙骨骼肌细胞三联体超微结构，特别是对接头足（以下简称为足）形态和数量的影响。结果表明，常规戊二醛固定和含苦味酸的多聚甲醛固定对三联体的超微结构无明显影响，足呆以分为柱、不完整足、空心柱、桥和非典型足等５类、各类足的比例及数量也相似。肌纤维用ＰＫＣ激动剂和抑制剂作用后，三联体的横小管和肌浆网形态无改变，而各种形态足的比较有变化。但是，这些变化与     

Intrinsic hyporesponsiveness of invariant natural killer T cells precedes the onset of lupus

Patients with systemic lupus erythematosus (SLE) display reduced numbers and functions of invariant natural killer T (iNK T) cells, which are restored upon treatment with corticosteroids and rituximab. It is unclear whether the iNK T cell insufficiency is a consequence of disease or is a primary abnormality that precedes the onset of disease. To address this, we analysed iNK T cell function at different stages of disease development using the genetically lupus‐susceptible NZB × NZW F₁ (BWF₁) model. We found that iNK T cell in‐vivo cytokine responses to an iNK T cell ligand α‐galactosylceramide (α‐GalCer) were lower in BWF₁ mice than in non‐autoimmune BALB/c and major histocompatibility complex (MHC)‐matched NZB × N/B10.PL F₁ mice, although iNK T cell numbers in the periphery were unchanged in BWF₁ mice compared to control mice. Such iNK T cell hyporesponsiveness in BWF₁ mice was detected at a young age long before the animals exhibited any sign of autoimmunity. In‐vivo activation of iNK T cells is known to transactivate other immune cells. Such transactivated T and B cell activation markers and/or cytokine responses were also lower in BWF₁ mice than in BALB/c controls. Finally, we show that iNK T cell responses were markedly deficient in the NZB parent but not in NZW parent of BWF₁ mice, suggesting that BWF₁ might inherit the iNK T cell defect from NZB mice. Thus, iNK T cells are functionally insufficient in lupus‐prone BWF₁ mice. Such iNK T cell insufficiency precedes the onset of disease and may play a pathogenic role during early stages of disease development in SLE.     

Special regulatory T cell review: How I became a T suppressor/regulatory cell maven

I have briefly reviewed the factors that motivated me to change my views about the existence and importance of suppressor/regulatory T cells and to devote the majority of my laboratory efforts to this newly revitalized area of immunologic research. I am optimistic that manipulation of regulatory T-cell function will shortly be applicable to the clinic.     

The p38 mitogen‐activated protein kinase cascade modulates T helper type 17 differentiation and functionality in multiple sclerosis

The p38 mitogen‐activated protein kinase cascade is required for the induction of a T helper type 17 (Th17) ‐mediated autoimmune response, which underlies the development and progression of several autoimmune diseases, such as experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis (MS). However, the contribution of p38 phosphorylation to human Th cell differentiation has not been clarified. Here we demonstrate that the p38 signalling pathway is implicated in the generation of Th17 lymphocytes from human CD4⁺ CD27⁺ CD45RA⁺ naive T cells, both in healthy donors and in patients affected by the relapsing–remitting form of MS. Our data also indicate that p38 activation is essential for interleukin‐17 release from central memory lymphocytes and committed Th17 cell clones. Furthermore, CD4⁺ T cells isolated from individuals with relapsing–remitting MS display an altered responsiveness of the p38 cascade, resulting in increased p38 phosphorylation upon stimulation. These findings suggest that the p38 signalling pathway, by modulating the Th17 differentiation and response, is involved in the pathogenesis of MS, and open new perspectives for the use of p38 inhibitors in the treatment of Th17‐mediated autoimmune diseases.     

T cell receptor induced intracellular redistribution of type I protein kinase A

The productive activation of CD4(+) T lymphocytes, leading to proliferation and cytokine secretion, requires precise temporal regulation of intracellular cyclic AMP concentrations. The major effector molecule activated by cyclic AMP in mammalian cells is the cyclic AMP-dependent protein kinase A (PKA). The type I PKA isozyme mediates the inhibitory effects of cyclic AMP on T-cell activation. Using laser scanning confocal microscopy, we demonstrated that the regulation of PKA type I activity involves spatial redistribution of PKA type I molecules following T-cell receptor (TCR) stimulation. In resting T cells, PKA type I was located in membrane proximal regions and distributed equally across the cell. Shortly after antigen engagement, T cells and antigen-presenting cells formed an area of intense contact, known as the immunological synapse. TCR concentrated at the synapse, whereas PKA type I molecules redistributed to the opposite cell pole within 10 min after T-cell stimulation. Type I PKA redistribution was solely dependent on TCR signalling, because we observed the same temporal and spatial distribution after antibody-mediated cross-linking of the TCR-associated CD3 complex. Segregation of TCR and PKA type I molecules was maintained for at least 20 min. Thirty minutes after stimulation, PKA type I partially colocalized with the TCR. After 60 min, PKA type I distribution again approached the resting state. Considering that initial TCR signals lead to increases in intracellular cyclic AMP, PKA type I molecules may be targeted towards localized cyclic AMP accumulations or transported away from these areas, depending on the requirements of the cellular response.     

The power and the promise of restimulation-induced cell death in human immune diseases

Summary: Controlled expansion and contraction of lymphocytes both during and after an adaptive immune response are imperative to sustain a healthy immune system. Both extrinsic and intrinsic pathways of lymphocyte apoptosis are programmed to eliminate cells at the proper time to ensure immune homeostasis. Genetic disorders of apoptosis described in mice and humans have established Fas and Bim as critical pro-apoptotic molecules responsible for T-cell death in response to T-cell receptor restimulation and cytokine withdrawal, respectively. Emerging evidence prompts revision of this classic paradigm, especially for our understanding of restimulation-induced cell death (RICD) and its physiological purpose. Recent work indicates that RICD employs both Fas and Bim for T-cell deletion, dispelling the notion that these molecules are assigned to mutually exclusive apoptotic pathways. Furthermore, new mouse model data combined with our discovery of defective RICD in X-linked lymphoproliferative disease (XLP) patient T cells suggest that RICD is essential for precluding excess T-cell accumulation and associated immunopathology during the course of certain infections. Here, we review how these advances offer a refreshing new perspective on the phenomenon of T-cell apoptosis induced through antigen restimulation, including its relevance to immune homeostasis and potential for therapeutic interventions.     

人直肠腺癌血管内皮细胞局部粘附激活酶的表达

癌细胞发生血管转移,首先涉及癌细胞与血管内皮细胞的相互作用。关于血管内皮细胞与肿瘤细胞相互作用的分子机制,国外一些学者采用体外培养血管内皮细胞进行研究,发现与一些粘附分子有关,但作用途径尚不清楚。研究采用直肠癌病人癌周直肠组织和癌转移淋巴结,研究癌浸润转移的血管内皮细胞的胞内信号传导激活途径,为探讨恶性肿瘤血管浸润转移机理提供理论资料。信号传递通路中令人瞩目的是ｐｐ＾１２５ＦＡＫ,它与Ｖｉｎｃｕｌｉｎ,ｔａｌｉｎ等一起分布于细胞粘附斑外,分子量１２５ＫＤ,故称为局部粘附激活酶（ｆｏｃａｌ ａｄｈｅｓｉｏｎ ｋｉｎａｓｅ ｐｐ＾１２５ＦＡＫ）。应用冰冻切片免疫组织化学法,结果发现癌细胞发生血管转移时,血管内皮细胞ｐｐ＾１２５ＦＡＫ呈阳性,免疫反应产物位于内皮细胞胞质内,说明癌细胞的血管转移与血管转移与血管内皮细胞     

蛋白激酶B在佛波酯诱导人胃癌细胞系凋亡中的作用

目的探讨佛波酯(TPA)诱导胃癌细胞系凋亡过程中蛋白激酶B(PKB)的作用。方法通过BrdU处理,流式细胞术检测以及DAPI染色,荧光显微镜观察分析TPA对胃癌BGC-823细胞的影响;免疫印迹法检测TPA对PKB蛋白表达水平、磷酸化的影响;核浆分离获得核浆蛋白,用于免疫印迹法检测TPA对胃癌细胞内PKB和其Ser 473位点磷酸化的核浆表达影响,以及激光扫描共焦显微镜观察TPA是否改变胃癌细胞内PKB的分布。结果TPA诱导BGC-823细胞凋亡;TPA下调BGC-823细胞内PKB蛋白表达,并且与TPA作用时间和TPA浓度呈正相关,与PP2A降解作用无关;TPA抑制PKB的Ser 473位点磷酸化,而对其Thr 308位点磷酸化没有明显影响;TPA下调细胞核内PKB的表达以及Ser 473位点的磷酸化;TPA并不改变PKB在胃癌细胞内的分布。结论PKB的抑制作用可能参与TPA诱导胃癌细胞凋亡过程;由TPA诱导的胃癌细胞凋亡可能部分是由于细胞核内PKB蛋白表达和Ser 473位点磷酸化下降所致。     

SLE患者T细胞TCR/CD3复合物介导的信号转导研究

为证明SLE患者T细胞功能异常是否与其生物化学信号转导异常有关。用CD3单抗与羊抗鼠二抗IgG相交联刺激T细胞并用Thapsigargin和EGTA干预后 ,用分别粘附细胞仪连续观察 10minT细胞 [Ca2 + ]i的变化 ,并评价 [Ca2 + ]i反应与CD3分子、InsP3 生成量和患者临床特征的相关性。结果显示 :正常人和SLE患者T细胞 [Ca2 + ]i反应的基准值相似 (P =0 10 5 ) ;SLE患者高峰值、平台值T细胞的 [Ca2 + ]i反应明显高于正常对照 (P <0 0 0 1,P <0 0 0 1) ;加入Thapsigargin后二者 [Ca2 + ]i反应无显著差异 ,而加入EGTA后二者 [Ca2 + ]i反应有显著差异 ;二者的T细胞CD3阳性率和InsP3 生成量无差异 (P =0 6 6 5 ,P =0 5 37) ;且 [Ca2 + ]i异常反应与患者的疾病活动性无关。这些结果提示 :SLE患者T细胞TCR/CD3介导的信号转导途径存在异常 ;SLE患者T细胞功能异常可能是因细胞内生物化学信号途径异常所致。     

Regulatory T Cells in Lupus

Naturally occurring, CD4⁺CD25⁺ regulatory T cells that are exported from the thymus early in life play an important role in controlling organ-specific autoimmune diseases, but they may not be critical for suppressing systemic autoimmunity in lupus. On the other hand, lupus-prone subjects appear to be deficient in generation of adaptive T-regulatory cells that can be induced by various means. We review autoantigen-specific therapeutic approaches that induce such regulatory T cells. Of particular interest are TGF-β producing CD4⁺CD25⁺ and CD8⁺ regulatory T cells that are induced by low dose tolerance therapy of lupus-prone mice with nucleosomal histone peptide epitopes, administered subcutaneously in subnanomolar doses. These regulatory T cells are not only efficient in suppressing autoantigen recognition and autoantibody production, but they also inhibit migration/accumulation of pathogenic autoimmune cells in the target organ, such as the kidneys of mice prone to develop lupus nephritis. We discuss why and under what conditions such therapeutic approaches would be beneficial in lupus patients and lupus-prone subjects.     

TAK1 is indispensable for development of T cells and prevention of colitis by the generation of regulatory T cells

Transforming growth factor (TGF)-beta-activating kinase 1 (TAK1) is critical for Toll-like receptor- and tumor necrosis factor-mediated cellular responses. In B cells, TAK1 is essential for the activation of mitogen-activated protein kinases (MAPKs), but not nuclear factor-kappaB (NF-kappaB), in antigen receptor signaling. In this study, we generate T cell-specific TAK1-deficient (Lck(Cre/(+))Tak1(flox/flox)) mice and show that TAK1 is indispensable for the maintenance of peripheral CD4 and CD8 T cells. In thymocytes, TAK1 is essential for TCR-mediated activation of both NF-kappaB and MAPKs. Additionally, Lck(Cre/(+))Tak1(flox/flox) mice developed colitis as they aged. In these mice, accumulations of activated/memory T cells as well as B cells were observed. Development of regulatory T (Treg) cells in thymus was abrogated in Lck(Cre/(+))Tak1(flox/flox) mice, suggesting that the loss of Treg cells is the cause of the disease. Together, the results show that TAK1, by controlling the generation of central Treg cells, is important for preventing spontaneously developing colitis.     

Ly49A expression on T cells alters T cell selection

Ly49 receptors are inhibitory receptors expressed on subsets of both NK cells and NK1.1(+) T cells. The function of these receptors on NK cells is believed to be important in maintaining self-tolerance, yet their role on T cells is unclear. In this report we investigated how an Ly49A transgene alters T and NK cell development in an in vivo environment, where a ligand for Ly49A is expressed. Ly49A transgenic mice that co-expressed an MHC ligand for Ly49A, H-2D(d), developed a severe inflammatory disorder that resulted in death within the first weeks of age. T cells expressing forbidden TCR V(beta) chains were found both in the thymus and periphery of transgenic mice, while non-transgenic littermates had successfully deleted these T cell subsets. These data indicate that the expression of Ly49A on T cells could alter T cell selection and allow survival of potentially self-reactive T cells.