抗APO—1单抗促进SEB活化的淋巴细胞凋亡及其与胞内游离Ca^ …

为探讨ＡＰＯ－１单抗处理的金黄色葡萄球菌肠毒菌Ｂ活化的淋巴细胞胞浆中游离Ｃａ６２＋浓度的变化，用形态学观察，流式细胞光度术观测了鼠抗人ＡＰＯ－１单克隆抗体对ＳＥＢ活化不同天数的人外周血淋巴细胞凋亡的诱导作用，同时采用Ｆｕｒａ－２／ＡＭ荧光探针检测ＡＰＯ－１单抗处理的淋巴细胞浆内游离Ｃａ＾２＋浓度。     

SRBC膜提取物对猪PBMNC第二信使的影响

胰酶水解绵羊红细胞（ＳＲＢＣ）释放膜表面活性蛋白组分Ⅲ（ＴＲＦ－Ⅲ），单独作用可使猪外周血单个核细胞（ＰＢＭＮＣ）胞内ｃＡＭＰ水平升高，以１００μｇ／ｍｌ浓度刺激达最高峰，由对照组０．９４±０．１４ｐｍｏｌ／Ｌ升高到２．７５±０．２５ｐｍｏｌ／Ｌ（Ｐ＜０．０１）。如果ＰＢＭＮＣ事先与腺苷酸环化酶（ＡＣａｓｅ）抑制剂ＬｉC１孵育后再以ＴＲＦ－Ⅲ或ＰＡＨ刺激。ｃＡＭＰ增高受到抑制（Ｐ＜０．０１）；而ＥＤＴＡ－２Ｎａ（一种磷酸二酯酶ＰＤＥ抑制剂）对此ｃＡＭＰ升高无影响。结果提示，此ｃＡＭＰ升高主要是通过活化ＡＣａｓｅ水解ＡＴＰ生成ｃＡＭＰ，而不像是抑制ＰＤＥ减少ｃＡＭＰ降解引起的。ＴＲＦ－Ⅲ诱导猪ＰＢＭＮＣ胞内Ｃａ~(２＋)浓度升高，以１００μｇ／ｍｌ刺激２分钟升高最多，由对照组的２４２±７ｎｍｏｌ／Ｌ升高到３２３±１５ｎｍｏｌ／Ｌ（Ｐ＜０．０１）。以ＥＧ－ＴＡ除去胞外Ｃａ~(２＋)再以ＴＲＦ－Ⅲ或ＰＡＨ刺激，仅观察到小范围［Ｃａ~(２＋)］ｉ升高。看来这一过程包括了刺激胞内Ｃａ~(２＋)释放和胞外Ｃａ~(２＋)内流两种方式。以上结果证明，ＴＲＦ－Ⅲ对淋巴细胞功能影响与细胞内第二信使有关。     

抗钙调素抗体与人淋巴细胞凋亡

钙调素（ｃａｌｍｏｄｕｌｉｎ,ＣａＭ）是真核细胞中的Ｃａ２＋结合蛋白。Ｃａ２＋／ＣａＭ复合物在多个位点上参与细胞周期调控,促进细胞增殖与分化。抗ＣａＭ抗体可穿入淋巴细胞并干扰其功能,在自身免疫病发病机制中可能起作用［１］。本实验用抗ＣａＭ抗体与人淋巴细胞作用,并以抗Ｓｍ抗体为对照,研究其与细胞凋亡的关系。１　材料和方法１－１　抗体　兔抗ＣａＭ抗体和鼠抗ＣａＭ单抗（自制）。抗ＣａＭ阳性血清、抗Ｓｍ阳性血清分别筛选自甲亢和ＳＬＥ患者。抗ＣａＭ水平设定参照文献［２］进行。１－２　淋巴细胞分离与培养　常…     

重组人红细胞生成素单克隆抗体的制备

本文应用常规淋巴细胞杂交瘤技术制备了４株能稳定分泌抗人重组红细胞生成素（ｒＨｕＥＰＯ）单克隆抗体（ＭｃＡｂ）的小鼠杂交瘤细胞系ＢⅡ１Ｂ５、ＤⅡ６Ｂ９、ＭⅡ１Ｈ４和ＧＩ３Ｅ７。用鼠单克隆抗体分型试剂盒鉴定，其分泌的ＭｃＡｂ的类分别是ＩｇＭ、ＩｇＭ、ＩｇＧ１和ＩｇＧ２ａ。间接ＥＬＩＳＡ法测定细胞上清的效价为１×１０－２～１．２５×１０－４，腹水效价为１×１０－２～１×１０－８。培养上清经ＥＬＩＳＡ鉴定，与ＩＬ－２、ＧＭ－ＣＳＦ、ＩＦＮ－α等细胞因子均无交叉反应，只与ｒＨｕＥＰＯ特异性结合。     

粘附分子对CD3介导的胸腺细胞[Ca^2＋]i应答的影响

ＬＦＡ－１和ＩＣＡＭ－１广泛表达于各胸腺细胞亚群，但ＩＣＡＭ－１在ＰＮＡ￣＋细胞的表达下调。本文报道：用抗ＬＦＡ－１／ＩＣＡＭ－１和抗ＣＤ３单抗，分析了粘附分子ＬＦＡ－１／ＩＣＡＭ－１对抗ＣＤ３诱导的胸腺细胞［Ｃａ￣（２＋）］ｉ应答的影响。结果显示，可溶性抗ＬＦＡ－１／ＩＣＡＭ－１可抑制ＣｏｎＡ刺激的胸腺细胞增殖，且以抗ＬＦＡ－１抗体的作用更为显著，在ＣｏｎＡ或抗ＣＤ３诱导的胸腺细胞［Ｃａ￣（２＋）］ｉ应答中，抗ＬＦＡ－１单抗可明显抑制［Ｃａ￣（２＋）ｉ升高。但如果用二抗交联ＣＤ３和ＬＦＡ－１，胸腺细胞［Ｃａ￣（２＋）ｉ则显著高于单独交联ＣＤ３时的水平（Ｐ＜０．０１），而ＣＤ３与ＩＣＡＭ－ｌ交联却无此效应，此外，仅交联ＬＦＡ－１或ＩＣＡＭ－１也无诱导［Ｃａ￣（２＋）］ｉ应答的作用。提示在ＬＦＡ－ｌ与ＩＣＡＭ－１介导的胸腺细胞与胸腺基质细胞相互作用中，ＬＦＡ－１可为ＴＣＲ／ＣＤ３途径介导的胸腺细胞活化提供复合刺激信号。     

抗IL-2单抗诱导SLE患者外周血活化T淋巴细胞凋亡的研究

采用流式细胞仪定量研究了抗白介素２单抗（ＩＬ ２ＭｃＡｂ）诱导７０例系统性红斑狼疮（ＳＬＥ）患者外周血活化Ｔ淋巴细胞凋亡的百分率,并与正常人对照组相比较。结果表明：ＩＬ ２ＭｃＡｂ能诱导ＳＬＥ患者及正常人外周血活化Ｔ淋巴细胞凋亡,且ＳＬＥ患者明显高于正常人（Ｐ＜００１）,活动期高于非活动期（Ｐ＜００１）;ＩＬ ２ＭｃＡｂ对ＳＬＥ患者外周血活化Ｔ淋巴细胞凋亡的调控呈剂量效应和时间效应。提示：从抗ＩＬ ２ＭｃＡｂ能诱导ＳＬＥ患者外周血活化Ｔ淋巴细胞凋亡,可间接地证明ＩＬ ２在ＳＬＥ疾病过程通过抑制外周血活化Ｔ淋巴细胞凋亡的作用而参与ＳＬＥ免疫病理损伤。     

OKT3＋rhIL－2激活胎儿脾细胞的研究

观察了１８～２８周龄胎儿脾单个核细胞（ＦＳＭＣ）在体外对ＯＫＴ３＋ｒｈＩＬ－２联合刺激的反应性，结果发现：ＯＫＴ３单独能活化ＦＳＭＣ，最适浓度ＯＫＴ３与ｒｈＩＬ－２联合对ＦＳＭＣ有强协同刺激作用。ＯＫＴ３刺激ＦＳＭＣ后明显促进ＩＬ－２Ｒ表达，表明ＯＫＴ３对ＦＳＭＣ的括化作用与ＩＬ－２／ＩＬ－２Ｒ途径相关。ＯＫＴ３＋ｒｈＩＬ－２协同诱导的ＦＳＭｃ能产生ＮＫ和ＬＡＫ活性，且ＬＭ活住较单用ｒｈＩＬ－２诱导者强，间接免疫荧光染色ＦＡＣＳ分析显示，ＯＫＴ３＋ｒｈｌＬ－２协同激活的ＦＳＭＣ主要是ＣＤ８￣＋Ｔ细胞。结果表明ＦＳＭＣ与成人ＰＢＭＣ－样能被ＯＫＴ３活化。     

Epstein—Barr病毒潜伏膜蛋白2A重组痘苗病毒转染DC及特异性CTL的体外诱导

通过ＥＢ病毒ＬＭＰ２Ａ重组痘苗病毒转染的ＤＣＳ体外诱导ＬＭＰ２Ａ特异性ＣＴＬ,并通过ＧＭ－ＣＳＦ、ＩＬ－４和ＴＮＦ－ａ培养体系,我们诱导出了人外周血单核细胞来源的ＤＣ。同时选用在鼻咽癌患者中表达的ＥＢ病毒潜伏蛋白之一ＬＭＰ２Ａ作为靶基因,利用重组痘苗病毒转染诱导的ＤＣＳ。ＤＣＳ与自体ＰＢＭＣＳ混合培养,在ＩＬ－２的刺激作用下获得特异性ＣＴＬ。结果如下：１．人外周血单核细胞经ＧＭ－ＣＳＦ、ＩＬ－４、ＴＮＦ－ａ的混合培养,１０天可获得成熟的功能性ＤＣＳ。ＦＡＣＳ检测显示ＤＣ表面相对特异性标志ＣＤ８３…     

系统性红斑狼疮患者B淋巴细胞EBV-LMP1和ZEBRA的表达研究

目的：探讨ＥＢＶ－ＬＭＰ１和ＺＥＢＲＡ在系统性红斑狼疮患者（ＳＬＥ）的表达情况。方法：间接荧光免疫标记，流式细胞仪检测。结果：ＳＬＥ患者Ｂ淋巴细胞中ＥＢＶ－ＬＭＰ１和ＺＥＢＲＡ的表达显著高于正常对照组（Ｐ＜０．０１）。活动期患者ＣＤ２３＋细胞ＥＢＶ－ＬＭＰ１和ＺＥＢＲＡ的表达率均高于ＣＤ１９＋细胞（Ｐ＜０．０１）。非活动期患者ＣＤ２３＋细胞ＥＢＶ－ＬＭＰ１表达也高于ＣＤ１９＋细胞（Ｐ＜０．０１）。但ＥＢＶ－ＺＥＢＲＡ表达在两亚群间差异没有统计学意义（Ｐ＞０．０５）。结论：朋病毒参与了ＳＬＥ的发病机制，病毒主要以潜伏期状态存在于患者中，病毒复制促进病情发展，检测Ｂ淋巴细胞ＥＢＶ－ＬＭＰ１和ＺＥＢＲＡ的表达率，有助于病情活动指标的判断。     

胸腺基质细胞的抗原提呈作用

目的 研究胸腺基质细胞的抗原提呈能力。方法 应用ＯＶＡ－特异的、受Ｉ－Ａ＾ｄ分子识别限制的辅助Ｔ细胞杂交瘤（３ＤＯ．１８．３）识别提呈的ＯＶＡ的ＣＮＢｒ水解片段而被活化后产生ＩＬ－２,测定ＩＬ－２活性来分析胸腺基质细胞的抗原提呈作用。结果ＩＦＮ－γ能促进ＭＴＥＣＩ和ＭＴＳＣ４表达Ｉ－Ａ＾ｄ分子,并促进ＭＴＳＣ４表达Ｂ７－１分子。经ＩＦＮ－γ作用后,ＭＴＥＣ１和ＭＴＳＣ４均有抗原提呈能力,ＭＴＳＣ     

APO—1／Fas及其配体介导超抗原SEB诱导的淋巴细胞凋亡

为了探讨超抗原诱导的淋巴细胞凋亡机制．本实验采用超抗原金黄色葡萄球菌肠毒素B（SEB）处理人外周血单个核细胞,采用流式细胞术检测细胞DNA断裂的百分率;同时动态观察了细胞表面APO-1、FasL的表达及细胞内Bcl－2蛋白的表达。结果表明,SEB不但可引起人外周血淋巴细胞凋亡,而且可诱导淋巴细胞表达APO-1和FasL。细胞凋亡的百分率与细胞表面APO-1和FasL的表达呈正相关（r＝0．71,r＝0．98）,与细胞内Bcl－2表达呈负相关（r＝－0．72）。由此可见,APO－1／Fas及其配体参与了超抗原SEB诱导的淋巴细胞凋亡。     

人CD137单抗诱导不同状态T细胞增殖和凋亡的研究

为了研究一种新的T细胞共刺激分子—CD137对不同状态T细胞增殖和凋亡的双重调节作用。采用3 H TdR掺入法测定T细胞增殖 ,用流式细胞仪测定细胞凋亡。结果显示 :(1)CD137单抗可与T细胞表面的CD137抗原结合 ,明显增强PHA刺激T细胞增殖 ,使T细胞增殖指数较PHA单独作用高 2～ 3倍 ,但CD137单抗单独不能刺激T细胞增殖 ;(2 )对于慢性活化的T细胞 ,CD137单抗可协同PHA诱导T细胞凋亡 ,使T细胞凋亡率从PHA单独作用的 19 2 0 %增加到 36 31% ,CD137单抗单独并不能诱导慢性活化T细胞凋亡。CD137单抗一方面可协同PHA刺激静止状态T细胞的增殖 ,另一方面可协同PHA诱导慢性活化T细胞凋亡 ,对T细胞起双重调节作用。     

抗CD28＋B7．1单抗共刺激T细胞诱导肝癌细胞凋亡的第二信使系统研究

目的：探讨T淋巴细胞活化、增殖、诱导肝癌细胞凋亡过程中第二信使系统的调控机制。方法：用CD28＋B7．1（CD80）单克隆抗体共刺激T淋巴细胞诱导肝癌细胞（BLE－7402）凋亡,测定不同诱导时间T细胞内cAMP,cGMP和Ca^2 的浓度改变及肝癌细胞凋亡情况。结果：活化的T淋巴细胞中,cAMP浓度在初期短暂升高,继而快速下降,作用于肝癌细胞后逐步回升至1－2倍,而细胞内cGMP的浓度急剧升高6－7倍,Ca^2 浓度也显著增加2－3倍,且均在第4天达最高值,与癌细胞的凋亡呈正相关,结论：T淋巴细胞内第二信使系统cAMP、cGMP和Ca^2 的水平与细胞的活化增殖、杀伤效应密切相关。     

A co-stimulatory role for CD28 in the activation of CD4+ T lymphocytes by staphylococcal enterotoxin B.

In this study we investigated the differential effect of the co-stimulatory receptor ligand molecules CD2/LFA-3, LFA-1/ICAM-1, and CD28/B7 on microbial superantigen mediated activation of CD4+ T cells. Highly purified CD4+ T cells, depleted of antigen presenting cells (APCs), do not proliferate in response to the superantigen, staphylococcal enterotoxin B (SEB). However, CD4+ T cells do respond to SEB in the presence of the LFA-3, ICAM-1, and B7 positive erythroleukemic cell line K562, murine L cells, human B7 transfected L cells or CD28 mAb. The K562 plus SEB induced response can be inhibited by combinations of mAbs to CD2 and LFA-1, and to LFA-3, ICAM-1, and B7. Addition of CD28 mAb to the CD2 and LFA-1 inhibited cultures could restore the response. Furthermore, soluble CD28 mAb alone is able to synergize with SEB to induce a proliferative CD4+ T cell response. CD4+ T cells depleted of APCs could also be activated by a pool of four mAbs directed to the V beta 5, V beta 6, V beta 8, and V beta 12 region of the TCR when a co-stimulatory signal was provided by the CD28 mAb, while the V beta mAbs alone or in combination are unable to activate CD4+ T cells in the absence of APCs. In contrast, addition of soluble mAbs to CD2 and LFA-1 molecules failed to co-stimulate SEB activated CD4+ T lymphocytes. The kinetics of the different modes of activation are distinct. SEB induced proliferation is most efficient in the presence of autologous APCs with maximal proliferation at a log4 lower SEB concentration than when CD28 mAbs were used. SEB plus K562 activation peaks on day 7, while SEB plus CD28 mAb induced proliferative responses do not peak until day 9. Thus, superantigen mediated activation of CD4+ T cells requires co-stimulatory signals, among which CD28 has distinct and unique effects.     

SEB-induced T cell apoptosis in atopic patients--correlation to clinical status and skin colonization by Staphylococcus aureus

INTRODUCTION: We asked whether in atopic dermatitis (AD) increased T cell apoptosis in staphylococcal enterotoxin B (SEB)-activated cultures of peripheral blood mononuclear cells (PBMCs) is characteristic of the exacerbation of the disease or connected with skin colonization by Staphylococcus aureus. MATERIAL/METHODS: The clinical status of the patients was evaluated using the SCORAD index. The number of bacteria colonizing patients' skin lesions was determined by the cfu method. Mononuclear cells isolated from peripheral blood were stimulated by SEB and the apoptosis of CD3+ cells in culture was determined by flow cytometry using the monoclonal antibody APO2.7. The cytokine production in the culture supernatants was determined by ELISA and Cytometric Bead Array kits. RESULTS: T cell apoptosis was increased, while the production of interferon (IFN)-gamma was reduced in cultures of PBMCs of AD patients during exacerbation. The proportion of CD3+ APO2.7+ cells positively correlated with the density of S. aureus recovered from skin lesions, but not with SCORAD index. By contrast, SCORAD index, but not S. aureus density, negatively correlated with IFN- gamma production. Furthermore it was found that the presence of S. aureus on uninvolved skin distinguishes a group of severe cases with high serum IgE level, increased T cell apoptosis, and reduced production of tumor necrosis factor alpha in SEB- -stimulated cultures. CONCLUSIONS: Among AD patients the increased activation-induced T cell apoptosis observed in SEB- -stimulated cultures is related to skin colonization by S. aureus. The presence of bacteria on uninvolved skin is a feature of a distinct group of AD patients.     

SEB诱导的CD4+ T细胞无能、凋亡及MHC-I类分子表达下调

目的：探讨超抗原金黄色葡萄球菌肠毒素（SEB）体外诱导外周T细胞免疫耐受的作用机制。方法：采用SEB体外刺激C57BL/6J（B6）小鼠的脾细胞后，以MTT比色法检测脾细胞的增殖，并用PI染色后以流式细胞术（FCM）分析不同时间段处于S期，G0-G1期的细胞及无能T细胞的凋亡，测定T细胞亚群及MHC-I（H-2K^b)表达的变化。用琼脂糖凝胶电泳，观察不同时间段凋亡T细胞的DNA特征。结果：部分去除CD8^ T细胞后。SEB可刺激B6小鼠脾细胞中CD4^ T细胞大量增殖，在SEB刺激后第3天，CD4^ T细胞中处于S期的比率最大，此后开始下降；而处于G0-G1期的CD4^ T细胞变化则相反，在初次刺激后第3天，增殖的CD4^ T细胞出现无能，FCM检测及用琼脂糖凝胶电泳检查DNAladder证实，在第7天，无能CD4^ T细胞出现凋亡，且凋亡细胞的比率逐渐增多，不因加入抗CD3抗体或CoN A而逆转，在SEB刺激后，CD4^ T细胞表面MHC-I类分子（H-2K^b)的表达，随细胞无能的出现而明显下调。结论：SEB诱导的T细胞免疫耐受，可能与CD4^ T细胞的无能，凋亡及细胞表面分子MHC-I的表达下调有关。     

TCRVβ基因亚家族的优势取用和PTK信号传导途径的激活及体外对肝癌细胞凋亡的诱导

目的：研究特异识别肝癌细胞株肿瘤相关抗原的TCRVβ基因亚家族的优势取用及对肝癌细胞凋亡的诱导。方法：流式细胞术检测淋巴细胞表型,RT-PCR和Southem印迹分析TCRVβ基因亚家族表达水平,Westem blot检测PTK含量,透射电镜观察凋亡细胞超微结构。结果：McAb共刺激T细胞与肝癌细胞混合培养后,T细胞表面CD3和CD8分子表达量明显升高,而CD4玩明显变化,TCRVβ6选择性扩增,表达水平由5%升高至13%-25%,且在第4天达到高峰,同时相应的PTK信号传导途径被激活,其含理由11%升至58%,亦在第4,5天达到高峰,以抗CD3 CD28,抗CD28 CD80,抗CD2 CD58共刺激的淋巴细胞均在体外诱导了肝癌细胞的凋亡。结论：TCRVβ7为特异的肿瘤抗原识别受体,TCR-CD4复合物与抗原结合后激活PTK信号传导途径。并诱导了肝癌细胞的凋亡。     

超抗原SEB诱导的免疫耐受与细胞因子无关

目的 研究超抗原金黄色葡萄球菌肠毒素B(staphylococcal enterotoxin B,SEB)诱导的耐受效应和耐受调节机制.方法 收集SEB活化10天的细胞,充分洗涤后做为效应细胞,分别与刀豆蛋白(ConA)、脂多糖(LPS)和白介素-2(IL-2)共同培养,用MTT方法测定细胞的耐受性应答反应.正常淋巴细胞在与ConA、LPS、IL-2共同培养的同时添加效应细胞,用MTF方法测定效应细胞的抑制性应答反应功能.流式细胞技术解析耐受性效应细胞类型.结果 效应细胞对ConA、LPS和IL-2的应答反应能力明显降低(P<0.01,n=3),但保持对ConA的应答反应能力.效应细胞抑制正常淋巴细胞与ConA、LPS和IL-2的应答反应(P<0.01,n=3),尤其抑制ConA和IL-2诱导的细胞增殖.SEB活化的效应细胞中CD8+NK1.1+、TcRVβ8+NK1.1+和CD4+NK1.1+NKT细胞以及TcRVβ8+T、CD8+T细胞数量明显增加(P<0.01和P<0.05,n=4).结论 超抗原SEB诱导的耐受性应答反应是效应细胞的直接作用,与细胞因子无关;这些效应细胞能抑制T淋巴细胞增殖,并保存识别ConA的受体功能.     

在小鼠异基因骨髓细胞移植中超抗原SEB诱导的耐受特征

目的:研究在异基因骨髓细胞移植中葡萄球菌肠毒素B(SEB)诱导的耐受强度和细胞学特征。方法:选用C57BL/J小鼠作为受体,BALB/c小鼠作为供体。所有受体小鼠在接受6×107骨髓细胞前均接受6.0Gy60Coγ射线的照射,随机分成3组:组1为只接受6.0Gy60Coγ射线照射的对照组(照射对照组,RI);组2为照射后注射生理盐水(移植对照组,Tran.);组3为照射后注射60μgSEB(SEB组)。180d后由组2和组3实验鼠获得两组C57BL/L-BALB/c嵌合体小鼠。用流式细胞术分析移植后30~180d受体小鼠体内CD4 T、CD8 T、CD3 /NK1.1 NKT淋巴细胞亚群的数量和MHCH-2Kb、H-2Kd抗原表达的百分率,用MLR方法测定嵌合体小鼠淋巴细胞对ConA和异源性抗原的反应性。结果:(1)接受大剂量骨髓细胞移植后,注射SEB和注射生理盐水的两组小鼠均可存活180d以上,SEB组小鼠呈现出BALB/c供体小鼠的颜色特征(白色);Tran.组小鼠呈现出其灰白色。(2)SEB组嵌合体小鼠对ConA的反应性明显低于RI组和Tran.组;对异源性抗原的应答高于Tran.组。(3)SEB组小鼠外周血中CD4 T细胞的数量在移植后30~60d明显下降,随后增加;CD8 T细胞的数量不变。CD3 /NK1.1 NKT细胞的数量,从接受移植后30d开始增加,随着时间的延长而递增,到180d达到5.71%。存活180d的嵌合体小鼠,体内供体小鼠特有的MHCH-2Kd抗原的表达率高达80.95%,自体MHCH-2Kb抗原表达的百分率只占1.45%。(4)与SEB组相反,Tran.组小鼠CD8 T细胞的数量持续下降;CD3 /NK1.1 NKT细胞的数量的增加只在移植后180d上升达到5.07%。结论:在异基因骨髓细胞移植中,SEB诱导的耐受性比单纯骨髓移植诱导的耐受性强。SEB诱导的耐受性与移植早期特异性CD4 T细胞数量的减少和NKT细胞数的持续增加有关。反应性的降低表现为T细胞对ConA反应性的下降。     

CD4+ T cell-mediated cytotoxicity toward thyrocytes: the importance of Fas/Fas ligand interaction inducing apoptosis of thyrocytes and the inhibitory effect of thyroid-stimulating hormone

The accumulation of activated CD4+ T cells and antigen (Ag)-dependent cellular interactions between thyrocytes and CD4+ T cells have been determined in thyroid gland from patients with Graves' disease. The Fas/Fas ligand (FasL) interaction between antigen-presenting cells and T cells regulates the apoptosis of the former cells triggered by the latter cells. The inhibition of Fas-mediated apoptosis in thyrocytes could be a underlying mechanism of hyperplasia of thyrocytes in patients with Graves' disease. We investigated the potential role of Fas/FasL interaction between thyrocytes and CD4+ T cells in the induction of Fas-mediated apoptosis of the former cells induced by the latter cells. The presence of only a few specific T cells responsive to a putative autoantigen has hampered the investigation of specific T cell activation toward antigen-presenting cells (APCs). Therefore, we used a superantigen, staphylococcal enterotoxin B (SEB), to examine specific T cell activation toward thyrocytes in vitro since it stimulates a large proportion of T cells with particular Vbeta elements. Spontaneous apoptosis of thyrocytes in culture was not found even in the presence of various kinds of cytokines. In contrast, a clear induction of Fas-mediated apoptosis by anti-Fas IgM was determined in interferon-gamma (IFN-gamma)-stimulated thyrocytes. In addition, a significant cytotoxicity of purified CD4+ T cells toward IFN-gamma-stimulated thyrocytes in the presence of SEB was induced, and the addition of anti-HLA-DR and -DQ monoclonal antibodies (mAbs) or blockade of the Fas/FasL interaction reduced this cytotoxicity. FasL expression of CD4+ T cells cocultured with IFN-gamma-stimulated thyrocytes in the presence of SEB was clearly induced. Furthermore, the addition of mAbs against CD54 and CD58 inhibited both cytotoxicity and FasL expression of CD4+ T cells. The cytotoxicity of CD4+ T cells toward IFN-gamma-stimulated, SEB-pulsed thyrocytes was markedly inhibited when we used thyrocytes cultured with IFN-gamma in the presence of thyroid-stimulating hormone (TSH) as target cells. Our results suggest that 1) CD4+ T cells were activated by thyrocytes expressing MHC class II molecules in an SEB-dependent manner and then expressed FasL. 2) These activated FasL+ CD4+ T cells killed thyrocytes by interacting with Fas on thyrocytes and FasL on activated CD4+ T cells. The presence of costimulating molecules such as CD54 and CD58 on thyrocytes was also necessary to generate activated FasL+ CD4+ T cells. 3) Since the actions of thyroid stimulating antibody (TSAb) toward thyrocytes are similar to those of TSH, one goitrogenic activity of TSAb may, in part, be due to the inhibitory effect on Fas-mediated apoptosis of thyrocytes triggered by activated CD4+ T cells.