天花粉蛋白对T淋巴细胞功能的调节作用

天花粉蛋白是从中药天花粉中提取纯化的一种活性蛋白,具有引产、抗肿瘤、抗病毒等多种生物学活性。近年来的研究发现,天花粉蛋白对免疫系统具有调节作用。本文从细胞增殖、细胞分化、细胞凋亡三个方面就其对T淋巴细胞功能的调节作用作一综述。     

天花粉蛋白合成肽通过诱导CD8抑制性T细胞治疗实验性自身反应性脑脊髓炎

为了探讨天花粉蛋白合成肽(M-Tk)治疗实验性自身反应性脑脊髓炎(EAE)的可能性及其作用机制,应用自身抗原MBP衍生肽MOG35-55免疫C57BL/6小鼠成功地诱发EAE后,以流式细胞术测定小鼠淋巴结细胞及脾细胞T亚群及细胞因子的表达,并通过临床评分观察M-Tk治疗EAE的有效性,包括以HE和LFB染色观察髓鞘病变。结果发现,M-Tk可抑制MOG35-55特异性T细胞的增殖反应,选择性诱导CD8+CD28-T调节细胞的扩增,明显提高IL-10的分泌和降低IFN-γ的产生,并有效改善EAE小鼠神经功能评分。采用M-Tk诱导的CD8 T细胞作体内输注可取得相似甚至更好的治疗效果(P>0.01)。提示M-Tk在体内诱导产生CD8+CD28-抑制性T细胞并上调IL-10分泌降低IFN-γ产生,是对EAE取得疗效的关键。     

天花粉蛋白诱发CD8阳性细胞参与的人体免疫抑制

已证明0.05～5μg/ml 剂量范围内的天花粉蛋白对同种异体抗原、可溶性抗原、致分裂原诱发的人体淋巴细胞体外增殖的强烈抑制作用,与细胞裂解无关。本文提供的下列证据进一步表明,天花粉蛋白诱发的免疫低反应性属于免疫抑制:①经该蛋白脉冲处理过的 PBMC 照射后加到不含天花粉的培养组合中可抑制新鲜PBMC的增殖;②天花粉蛋白脉冲处理过的PBMC培养上清液显示抑制功能;③去除CD8阳性细胞抑制缓解;回加 CD8阳性细胞抑制再现,提示有CD8抑制性T细胞参与反应。     

天花粉蛋白合成肽通过激活抑制性CD8~+ T细胞诱导免疫抑制

为了探讨天花粉蛋白合成肽(M-Tk)诱导免疫抑制的机制,应用小鼠针对可溶性抗原OVA的增殖系统,检测M-Tk处理后对淋巴细胞增殖的抑制作用和细胞因子格局的变化,以及M-Tk激活的T淋巴细胞亚群的表型及功能。结果表明,M-Tk可诱导一群CD8+ T抑制性细胞,其表型为CD8+CD28-CTLA4+,采用双层非接触共培养系统发现,该群抑制性细胞主要依赖于细胞因子IL-10、IL-4和细胞间接触发挥免疫抑制作用。     

天花粉蛋白对T细胞活化的抑制作用与信息传导

目的 观察天花粉蛋白经由ＴＣＲ／ＣＤ３介导的信号传递途径抑制Ｔ细胞活化增殖的效应方法 用各种不同的刺激信号来激活外周血淋巴细胞，然后观察天花粉蛋白对其增殖抑制的影响。接着测定天花粉蛋白对ＣＤ３单抗刺激后Ｔ淋巴细胞内一些信号传导物质变化的影响。结果 天花粉蛋白抑制经由ＴＣＲ／ＣＤ３信号传导途径诱发的淋巴细胞增殖。进而发现细胞内游离钙离子的升高，蛋白激酶Ｃ的转位和细胞内蛋白酪氨酸磷酸化受天花粉蛋白的抑     

抗CD3,CD28单抗对人T淋巴细胞共刺作用的影响因素

以抗ＣＤ３和抗ＣＤ２８作为Ｔ细胞激活的第一和第二信号，观察二者不同浓度，不同作用顺序和不同作用时间对人Ｔ淋巴细胞增殖反应的影响。结果显示单一的抗ＣＤ３和抗ＣＤ２８不能刺激Ｔ细胞增殖，只有同时域在一定时间内接受二者的共同刺激才能促使细胞增殖。     

锌7—金属硫蛋白对过氧化氢引起NIH3T3细胞增殖的抑制作用

为了研究锌7-金属硫蛋白（Zn7-metallothionein,Zn7-MT）对细胞内外过氧化氢引起NIH3T3细胞增殖的影响,本文采用了MTT细胞计数法,^3H-TdR掺入实验,细胞周期时相测定等方法。结果显示低浓度的甲萘醌和氧化氢促进了NIH3T3细胞增殖,使其MTT计数增加,细胞周期S期比例增高,DNA合成率增加。锌7-金属硫蛋白本身对NIH3T3细胞增殖没有影响,但它可以抑制甲萘醌和过氧化氢对NIH3T3细胞促增殖作用。提示锌7-金属硫蛋白对预防由活性氧诱导的成纤维细胞增殖所引起的疾病可能具有重要作用。     

胆固醇缺乏抑制Jurkat T淋巴细胞增殖及分子机制研究

人蛋白酪氨酸激酶信号传递系统及细胞周期调控这一角度来探讨胆固醇缺乏抑制Ｊｕｒｋａｔ细胞增殖的分子机制。方法采用接免疫荧光染色，流式细胞久仰分析技术及免疫细胞化学染色等方法做相关分析。结果经去脂血清培养基培养的Ｊｕｒｋａｔ细胞加ｌｏｖａｓｔａｔｉｎ处理３ｄ后，＾３Ｈ－ＴｄＲ掺入率明显下降，细胞增殖受抑细胞受阻于Ｇ０／Ｇ１期，ＰＴＫ活性及细胞ｃｙｃｌｉｎＤ１、ＣＤＫ４蛋白的表达明显降低，加入ＬＤＬ可以     

抗 CD3 、 CD28 单抗对人 T 淋巴细胞共刺激作用的影响因素

以抗 Ｃ Ｄ３ 和抗 Ｃ Ｄ２８ 作为 Ｔ 细胞激活的第一和第二信号, 观察二者不同浓度, 不同作用顺序和不同作用时间对人 Ｔ淋巴细胞增殖反应的影响。结果显示单一的抗 Ｃ Ｄ３ 和抗 Ｃ Ｄ２８ 不能刺激 Ｔ 细胞增殖, 只有同时或在一定时间内接受二者的共同刺激才能促使 Ｔ 细胞增殖。单独接受抗 Ｃ Ｄ３ ２４ｈ 或抗 Ｃ Ｄ２８ １６ｈ 后, Ｔ 细胞对相应的另一信号刺激呈低反应性, 这一现象可能与 Ｔ细胞耐受及自身免疫病的发生有关。     

角膜上皮细胞的免疫学特性

目的：探讨角膜上皮细胞刺激淋巴细胞增生的能力和共同刺激通路分子在角膜上皮细胞的表达。方法：体外共同培养人角膜上皮细胞和外周血单个核细胞，免疫组织化学染色方法测定HLAⅡ类抗原、CD40、CD154、CD80和CD86的表达．同时对比角膜移植排斥反应的移植片。荧光辅助细胞筛选、分析、测定CD69的上调。结果：HLAⅡ类抗原在角膜上皮细胞表达，γ-干扰素能够升高共同刺激通路分子CD40和CD80的表达；而且HLAⅡ类抗原和CD40均在角膜移植排斥反应的移植片上被检测出。T淋巴细胞在共同培养系统中被上皮细胞激活。结论：人角膜上皮细胞参与角膜移植排斥反应过程，移植免疫排斥反应被上皮细胞所诱导和初始化，但最终反应作用于内皮细胞。     

树突状细胞研究中激光扫描共聚焦显微镜的应用进展

免疫应答是机体重要的防御功能 ,抗原呈递细胞 (Antigenpresentingcells ,APC)是启动特异性免疫应答的关键。树突状细胞 (Dendriticcells,DC)是体内最强的一类APC ,参与免疫应答的双重调控作用。激光扫描共聚焦显微镜 (Laserscanningconfocalmicroscope ,LSCM)由于具有高分辨率、高灵敏度、“光学切片”、三维重建、动态分析等特点 ,为基础医学与临床医学的研究提供了有效手段 ,使DC的研究更加细致和深入。本文就LSCM在DC的形态学、免疫荧光定位、功能学方面的应用最新进展作一综述。     

Multifocal structure of the T cell - dendritic cell synapse

The structure of immunological synapses formed between murine naive T cells and mature dendritic cells has been subjected to a quantitative analysis. Immunofluorescence images of synapses formed in the absence of antigen show a diffuse synaptic accumulation of CD3 and LFA-1. In electron microscopy, these antigen-free synapses present a number of tight appositions (cleft size approximately 15 nm), all along the synapse. These tight appositions cover a significantly larger surface fraction of antigen-dependent synapses. In immunofluorescence, antigen-dependent synapses show multiple patches of CD3 and LFA-1 with a variable overlap. A similar distribution is observed for PKCtheta and talin. A concentric organization characteristic of prototypical synapses is rarely observed, even when dendritic cells are paralyzed by cytoskeletal poisons. In T-DC synapses, the interaction surface is composed of several tens of submicronic contact spots, with no large-scale segregation of CD3 and LFA-1. As a comparison, in T-B synapses, a central cluster of CD3 is frequently observed by immunofluorescence, and electron microscopy reveals a central tight apposition. Our data show that it is inappropriate to consider the concentric structure as a "mature synapse" and multifocal structures as immature.     

CD83 on murine APC does not function as a costimulatory receptor for T cells

The transmembrane glycoprotein CD83 is rapidly upregulated on murine and human DC upon maturation and therefore a costimulatory function for T cell activation has been suggested. Studies employing human APC indeed showed that CD83 expression was positively correlated to the stimulatory capacity of the APC. Murine APC that were CD83 deficient however, did not display a reduced capacity to activate T cells. To elucidate this contradiction, we thoroughly compared the stimulatory capacity of CD83-overexpressing and CD83-deficient APC. Here we show that CD83 expression levels on APC did not affect the capacity of the APC to activate CD8(+) T cells. CD83 expression levels did not significantly affect CD4(+) T cell activation in vivo, but a weak positive correlation of CD83 expression with CD4(+) T cell activation was observed in vitro under suboptimal stimulation conditions. As CD83 expression also positively correlated with MHC-II but not with MHC-I expression, this differential stimulation specifically of CD4(+) T cells could be explained by a higher density of MHC-II peptide complexes on the APC surface. Taken together, our results strongly suggest that CD83 does not deliver crucial costimulatory signals to murine T cells.     

NPY and NPY receptors in airway structural and inflammatory cells in allergic asthma

Purpose

Neuropeptide Y (NPY) level is elevated in allergic asthmatic airways and activation of NPY receptor-1 (NPY-Y1) on antigen‐presenting cells (APCs) is essential for T cell priming. Paradoxically, NPY-Y1 modulates hyper-responsiveness in T cells, suggesting a bimodal role for NPY in APCs and T cells. Therefore, determination of the temporal and spatial expression pattern of NPY and its receptors in asthmatic airways is essential to further understand the role of NPY in allergic asthma.

Methods

Lungs were isolated from control and acute and chronic stages of OVA-sensitized and challenged mice (OVA). Stains, including H&E, PAS, and trichrome, were used to determine the severity of lung pathology. The expression patterns of NPY and NPY-Y receptors in the airways were determined using ELISA and immunofluorescence. Cytokine levels in the BALF were also measured.

Results

NPY levels were undetectable in the BALF of control mice, but significantly increased in the OVA group at day 80. Levels of IL-4, TGF-β1 and TGF-β2, significantly increased and peaked on day 45 and decreased on day 80 in the OVA group, exhibiting an inverse correlation with NPY levels. NPY expression was localized to macrophage-like cells in the peri-bronchial and peri-vascular areas in the lung tissue. NPY-Y1 and -Y5 receptors were constitutively expressed by both structural and inflammatory cells in the lung tissue.

Conclusions

NPY produced by activated macrophage-like cells may be involved in regulating cytokine production and cellular activities of immune cells in asthma. However, it remains unclear whether such an increase in NPY is a defensive/compensatory mechanism to modulate the effects of inflammatory cytokines.     

Immunohistochemical study of epidermal Langerhans cells and dermal dendritic cells in benign and malignant skin lesions characterized by a dermal lymphoid infiltrate consisting either of B-cells or T-cells

Summary Skin biopsies from 43 patients with a rather dense dermal lymphoid infiltrate of either inflammatory or neoplastic nature have been investigated. We studied the number, distribution and immunophenotype of epidermal Langerhans cells and dermal dendritic cells. As previously reported, differences in epidermal Langerhans cell and dermal dendritic cell numbers between skin biopsies with a B-cell infiltrate and skin biopsies with a T-cell infiltrate were found, dendritic cells being more numerous in the latter. The main finding of this study was an uneven distribution of epidermal Langerhans cells and dermal dendritic cells in skin biopsies with a T-cell infiltrate: in skin lesions with an inflammatory lymphoid infiltrate, small clusters of epidermal and dermal dendritic cells admixed with T-lymphocytes (predominantly T-helper/inducer cells) and small blood vessels were present at areas of exocytosis. In skin lesions with a neoplastic lymphoid infiltrate larger, more loosely arranged aggregates of dendritic cells and T-cells were seen. These cell aggregations composed of activated (inflammatory or neoplastic) T-cells and dendritic cells may represent the cutaneous homologue of the secondary T-nodule in the lymph node. Both types of cell aggregates may correspond to the dendritic cell-T cell clusters observed in in vitro induced immune responses.Presented at the XVIth International Congress of the International Academy of Pathology and 7th World Congress of Academic and Environmental Pathology in Vienna, 1986Aspirant of the NFWO (Nationaal Fonds voor Wetenschappelijk Onderzoek)     

On the mechanisms by which transforming growth factor-β2 alters antigen-presenting abilities of macrophages on T cell activation

Peritoneal exudate cells (PEC) incubated with antigen in the presence of transforming growth factor-(TGF)-β2 selectively suppress delayed hypersensitivity and IgG2a antibody production when injected intravenously into naive syngeneic recipients. In this study, we have examined in vitro the effects of TGF-β2 on the antigen presenting abilities of PEC to activate DO11.10 T cells that express a transgenic T cell receptor that recognizes ovalbumin peptide fragment 323–339 in the context of I-A^d. PEC were pretreated overnight with TGF-β2, washed extensively, then co-cultured with DO11.10 T cells in the presence of native OVA or P323–339. We found that TGF-β2-treated PEC induced the production of the T helper type 2 (Th2) cytokine, interleukin-4 (IL-4), but unlike untreated PEC, were unable to stimulate the Th1 cytokines, IL-2 and interferon-γ (IFN-γ). Furthermore, TGF-β2 was produced in an autocrine fashion by TGF-β2-treated PEC and was responsible for this shift to a Th2 response. This conclusion was supported by the following results. First, TGF-β2-treated PEC were found to express much more TGF-β1 and TGF-β2 mRNA than untreated PEC. Second, TGF-β2-treated PEC secreted large amounts of TGF-β including its mature form. Third, addition of neutralizing anti-TGF-β2 antibodies, but not neutralizing anti-TGF-β1 antibodies, restored the ability of antigen-pulsed, TGF-β2-pretreated PEC to stimulate DO11.10 T cells to secrete IL-2 and IFN-γ. These results indicate that antigen-presenting cells that encounter antigen in a TGF-β-enriched environment (e.g., in the eye) shift responding naive T cells toward Th2 responses by producing TGF-β during antigen presentation.     

Ia+ dendritic cells lining the dermo-epidermal junction in bullous skin disorders,associated with the deposition of immune complexes

Summary Skin biopsies from 34 patients, presenting with a variety of bullous skin disorders were investigated, using routine light microscopy and immunohistochemistry.In bullous skin diseases characterized by deposition of complement factors (CF) and/or immunoglobulins (Ig), a monolayer of OKIa ₁ ⁺ , OKT ₆ ^- , OKM ₁ ^- dendritic cells was found at the dermo-epidermal junction. Retrospectively, these cells were easily recognized on paraffin embedded, H & E stained material. In bullous skin disorders, showing no deposition of CF and/or Ig, this monolayer of dendritic cells was lacking.It is suggested that these OKIa ₁ ⁺ , OKT ₆ ^- , OKM ₁ ^- dendritic cells at the dermo-epidermal junction represent some type of antigen presenting cells, not corresponding to Langerhans cells, veiled cells or indeterminate cells.Aspirant NFWO (National Fonds voor Wetenschappelijk Onderzoek)     

B细胞在多发性硬化发病机制中的作用

多发性硬化(MS)是一种发生在中枢神经系统(CNS)内的慢性炎症性脱髓鞘疾病,具体病因及发病机制目前尚不明确.研究者认为,MS是在遗传易感性基础上,由环境因素及感染因素触发的髓鞘特异性CD4+T细胞攻击中枢神经系统髓鞘介导的自身免疫反应性疾病.然而近年研究表明,B细胞也参与了MS的发病及病程的调控,B细胞作为抗原递呈细胞(APCs)、产生促炎细胞因子和趋化因子以及分泌针对髓鞘和轴突的自身抗体都在MS发病机制中起重要作用.