Independent roles for IL-2 and GATA-3 in stimulating naive CD4+ T cells to generate a Th2-inducing cytokine environment

T cell receptor (TCR) signaling plays an important role in early interleukin (IL)-4 production by naive CD4+ T cells. This "antigen-stimulated" early IL-4 is sufficient for in vitro Th2 differentiation. Here, we provide evidence that early IL-4 production by naive CD4+ T cells stimulated with cognate peptide requires TCR-induced early GATA-3 expression and IL-2 receptor signaling, both of which are controlled by the degree of activation of extracellular signal-regulated kinase (ERK). Stimulation of naive CD4+ T cells from TCR transgenic mice with low concentrations of peptide-induced IL-2-dependent STAT5 phosphorylation, IL-4-independent early GATA-3 expression, and IL-4 production. Neutralization of IL-2 abolished early IL-4 production without affecting early GATA-3 expression. In addition, naive CD4+ T cells from GATA-3 conditional KO mice failed to produce early IL-4 in response to TCR/CD28 stimulation. Stimulation with high concentrations of peptide abrogated early GATA-3 expression and IL-2-dependent STAT5 phosphorylation, and resulted in the failure to produce early IL-4. This high concentration-mediated suppression of early IL-4 production was reversed by blockade of the ERK pathway. A MEK inhibition rescued early GATA-3 expression and responsiveness to IL-2; these cells were now capable of producing early IL-4 and undergoing subsequent Th2 differentiation.     

Impaired Interleukin 4 Signaling in T Helper Type 1 Cells

Cluster of differentation (CD)4⁺ T helper cells (Th)1s fail to produce interleukin (IL)-4. Even if restimulated in the presence of IL-4, a condition that induces IL-4–producing capacity in naive CD4⁺ T cells, Th1s fail to become IL-4 producers. We report that Th1 cells have a major impairment in IL-4 signaling. When compared to both Th2s and naive T cells, they display a striking diminution in phosphorylation of Stat6. They also show reduced phosphorylation of Janus kinase (JAK)-3 and insulin receptor substrate (IRS)-2 when compared to Th2s. Stat6 and JAK-3 are present in equivalent amounts in Th1s and Th2s, but IRS-2 protein levels are much lower in Th1s than in Th2s. Altered sensitivity to IL-4, the major inducer of the Th2 phenotype, may explain the stability of the Th1 state.     

环孢菌素A对慢性再生障碍性贫血患者T-bet、GATA-3、相关信号分子、细胞因子及Th1/Th2的影响

本研究探讨转录因子T-bet、GATA-3及相关信号通路在慢性再生障碍性贫血(CAA)免疫发病中的作用,从Th细胞失衡、转录因子及相关信号通路水平研究环孢菌素(CsA)治疗慢性AA的免疫调节机理。采用实时荧光定量PCR(real-time FQ-PCR)检测慢性AA患者治疗前和CsA治疗6月后外周血单个核细胞(PBMNC)T-bet、GATA-3及STAT4、STAT6mRNA表达;采用流式细胞术、酶联免疫吸附法(ELISA)检测慢性AA患者治疗前和CsA治疗6月后外周血Th1、Th2比例及PBMNC培养上清IFN-γ、IL-12、IL-4水平,并与正常人进行比较。结果表明:慢性AA患者PBMNC T-bet、STAT4mRNA表达、T-bet/GATA-3比值、Th1比例、Th1/Th2比值、PBMNC培养上清IFN-γ、IL-12表达均明显高于正常组(p0.01),经CsA治疗6月后,患者T-bet、STAT4表达、T-bet/GATA-3比值、Th1比例、IFN-γ、IL-12表达均有所下降,但T-bet、STAT4、T-bet/GATA-3比值、Th1比例、IFN-γ表达仍未达到正常水平,GATA-3、STAT6mRNA表达、Th2比例、IL-4表达在治疗前后与正常人比较均无明显差异(p0.05)。结论:IFN-γ/T-bet、IL-12/STAT4通路的异常活化及Th平衡向Th1型偏移在AA免疫异常的发病过程中起到关键的作用;CsA能通过下调IFN-γ/T-bet、IL-12/STAT4通路的异常活化及纠正Th1过度极化而减轻AA异常亢进的细胞免疫,解除造血抑制。     

GATA-3 induces T helper cell type 2 (Th2) cytokine expression and chromatin remodeling in committed Th1 cells

Committed T helper type 1 (Th1) and Th2 effector cells, resulting from chronic antigenic stimulation in interleukin (IL)-12 and IL-4, are implicated in the pathology of autoimmune and allergic diseases. Committed Th1 cells cannot be induced to change their cytokine profiles in response to antigenic stimulation and Th2 cytokine-inducing conditions. Here, we report that ectopic expression of GATA-3 induced Th2-specific cytokine expression not only in developing Th1 cells but also in otherwise irreversibly committed Th1 cells and a Th1 clone, HDK1. Moreover, cAMP, an inhibitor of cytokine production by Th1 cells, markedly augmented Th2 cytokine production in GATA-3-expressing Th1 cells. Ectopic expression of GATA-3 in developing Th1 cells, but not in Th1 clone HDK1, induced endogenous GATA-3, suggesting an autoregulatory mechanism for maintenance of GATA-3 expression in Th2 cells. Structure-function analyses of GATA-3 revealed that the NH(2)-terminal transactivation domain and the COOH-terminal zinc finger domain of GATA-3 were critical, whereas the NH(2)-terminal zinc finger domain was dispensable for the induction of IL-4. Both zinc fingers, however, were required for IL-5 induction. A Th2-specific DNaseI-hypersensitive site of the IL-4 locus was detected in GATA-3-expressing Th1 cells. Thus, GATA-3 can change the phenotype of committed Th1 cells, previously considered to be irreversible.     

三七皂甙对造血细胞GATA-1和GATA-2转录调控蛋白的诱导作用

目的　观察三七总皂甙 (PNS)对锌指结构GATA族转录调控蛋白的诱导作用 ,探讨PNS在造血细胞内的信号传递途径。方法　选择恰当浓度的PNS作用于正常人骨髓单个核细胞、HL 6 0、K5 6 2、CHRF 2 88和Meg 0 1细胞 ,2周后提取核蛋白 ,用GATA 1和GATA 2抗体行Western印迹杂交 ,检测GATA 1和GATA 2的表达情况。用3 2 P标记的GATA双链寡核苷酸探针行电泳带移动阻滞试验 (EMSA)和抗体胶移动实验 ,检测GATA DNA复合物及亚型。结果　PNS能够促进人骨髓红系、粒系祖细胞增殖。Western印迹杂交显示PNS诱导K5 6 2、CHRF 2 88和Meg 0 1细胞的GA TA 1和GATA 2蛋白表达量增高 ,分别是未经处理细胞的 1 .5～ 2 .8倍和 2 .0～ 3.1倍 ;EMSA结果表明PNS诱导三株细胞的GATA DNA结合复合物条带密度明显增高 ;HL 6 0细胞在PNS处理前后均未显示GATA活性。抗体胶移动实验证明PNS诱导的GATA DNA复合物的主要成分为GATA 1和GATA 2。免疫沉淀显示PNS诱导的GATA 1和GATA 2蛋白均处于磷酸化的功能激活状态。结论　PNS可通过诱导GATA 1和GATA 2蛋白合成增加 ,与相关基因上游调控区的启动子和 (或 )增强子结合的活性增高 ,而调控与造血细胞增殖、分化相关基因的表达     

转录因子GATA—2在早期造身过程中的作用

转录因子ＧＡＴＡ－２是ＧＡＴＡ家族的一成员,以其锌指结构结合于「（Ｔ／Ａ（ＧＡＴＡ）Ａ／Ｇ」的共同ＤＮＡ序列结构。近期,通过敲除小鼠ＧＡＴＡ－２基因的实验证明了ＧＡＴＡ－２在造血细胞发育中的重要地位。ＧＡＴＡ－２基因断裂导致全部造血祖细胞的减少;相反,强制表达ＧＡＴＡ－２则阻止正常造血。ＧＡＴＡ－２的调节作用发挥于早期胚胎时期并与其它的ＧＡＴＡ转录因子共同作用于粒系、红系、巨核系和肥大细胞系等的增     

Interferon gamma enhances both in vitro and in vivo priming of CD4+ T cells for IL-4 production

Classical studies have demonstrated that in vitro priming of naive CD4 T cells to become T helper (Th)2 cells is strikingly dependent on interleukin (IL)-4, whereas priming for interferon (IFN)gamma production is IL-12/IFNgamma-dependent. Therefore, it was quite surprising when we noted that priming of naive C57BL/6 CD4(+) cells to become IL-4 producers was substantially inhibited by the addition of anti-IFNgamma antibodies. This was true using immobilized anti-CD3 and anti-CD28 antibodies or soluble anti-CD3/anti-CD28 and antigen-presenting cells in the presence or absence of added IL-4. Priming of CD4 T cells from IFNgamma(-/-) C57BL/6 mice with immobilized anti-CD3 and anti-CD28 resulted in limited production of IL-4, even with the addition of 1,000 U/ml of IL-4. Titrating IFNgamma into such cultures showed a striking increase in the proportion of T cells that secreted IL-4 upon challenge; this effect was completely IL-4-dependent in that it was blocked with anti-IL-4 antibody. Thus, IFNgamma plays an unanticipated but substantial role in Th2 priming, although it is an important Th1 cytokine, and under certain circumstances a Th1 inducer.     

Mast cell IL-4 expression is regulated by Ikaros and influences encephalitogenic Th1 responses in EAE

When exposed to a pathogen, a naive CD4(+) T cell is forced to make a cell fate decision that leads to a polarized population of Th1 IFN-gamma- or Th2 IL-4- producing cells. Although IL-4 has traditionally been considered a factor that promotes Th2 cell differentiation, recent evidence has demonstrated that the site and timing of IL-4 expression in an immune response determines its ultimate effects on CD4(+) T cell fate. Using a mast cell (MC) reconstitution model, we demonstrate that MC-derived IL-4 promoted Th1 responses in vivo. Furthermore, MCs from genetically disparate mouse strains varied in their potential for IL-4 expression. Independent of the activation mode, MCs from Th1-prone C57BL/6 mice exhibited a more robust Il4 response than did the Th2-prone strain Balb/c. The hierarchy of IL-4 expression potential was directly associated with the degree of basal chromatin accessibility at cis-regulatory elements conserved noncoding sequence-1 and V(A) enhancer within the Th2 locus. GATA1/2 and Ikaros, factors with opposing roles in chromatin remodeling, acted at these sites. We propose that GATA and Ikaros proteins coordinately fine-tune accessibility at the Il4 locus during development to variably regulate IL-4 expression. These events likely contribute to the genetically determined heterogeneity in Th1 responses that underlie susceptibility to many diseases.     

转录因子T-bet、GATA-3、FoxP3及CD4~+CD25~+调节性T细胞在儿童过敏性紫癜发病机制中的作用

本研究旨在探讨转录因子T-bet、GATA-3和CD4+CD25+调节性T细胞及其转录因子FoxP3在儿童过敏性紫癜(HSP)发病机制中的作用。2009年2月-2010年2月在本院收治的46例急性期HSP患儿(HSP组)及30例健康对照儿童(对照组)纳入研究。采用SYBR GreenⅠ实时荧光定量PCR方法检测外周血单个核细胞T-bet、GATA-3及FoxP3 mRNA的表达。运用流式细胞术检测外周血中T淋巴细胞亚群CD4+CD25+的表达。结果表明,HSP组患儿GATA-3 mRNA相对表达水平(964.30±655.18)显著高于对照组儿童GATA-3 mRNA相对表达水平(78.09±57.20,P<0.01)。HSP组患儿T-bet mRNA(53.98±35.79)、FoxP3 mRNA(32.17±23.04)和CD4+CD25+(5.34±2.51)相对表达水平低于对照组儿童T-bet mRNA(181.56±96.90)、FoxP3 mRNA(147.91±99.15)和CD4+CD25+(7.85±1.97)相对表达水平(P<0.01)。结论:HSP患儿急性期存在Th1特异性转录因子T-betmRNA表达下调,Th2特异性转录因子GATA-3 mRNA表达上调。HSP患儿急性期存在CD4+CD25+调节性T细胞及其特异性转录因子FoxP3 mRNA表达下调,调节性T细胞的减少及由此引发的免疫抑制效应不足可能是HSP急性期免疫失衡的重要原因之一。本研究为从调节性T细胞及其调控的分子机制角度进一步阐明儿童HSP的发病机制提供了实验依据。     

Toll-like receptor-dependent production of IL-12p40 causes chronic enterocolitis in myeloid cell-specific Stat3-deficient mice

Stat3 plays an essential role in IL-10 signaling pathways. A myeloid cell-specific deletion of Stat3 resulted in inflammatory cytokine production and development of chronic enterocolitis with enhanced Th1 responses in mice. In this study, we analyzed the mechanism by which a Stat3 deficiency in myeloid cells led to the induction of chronic enterocolitis in vivo. Even in the absence of Stat1, which is essential for IFN-gamma signaling pathways, Stat3 mutant mice developed chronic enterocolitis. TNF-alpha/Stat3 double-mutant mice developed severe chronic enterocolitis with enhanced Th1 cell development. IL-12p40/Stat3 double-mutant mice, however, showed normal Th1 responses and no inflammatory change in the colon. RAG2/Stat3 double-mutant mice did not develop enterocolitis, either. These findings indicate that overproduction of IL-12p40, which induces potent Th1 responses, is essential for the development of chronic enterocolitis in Stat3 mutant mice. Furthermore, enterocolitis was significantly improved and IFN-gamma production by T cells was reduced in TLR4/Stat3 double-mutant mice, indicating that TLR4-mediated recognition of microbial components triggers aberrant IL-12p40 production by myeloid cells, leading to the development of enterocolitis. Thus, this study clearly established a sequential innate and acquired immune mechanism for the development of Th1-dependent enterocolitis.