Vlase H, Weiss M, Graves P N, Davies T F
Department of Medicine, Mount Sinai School of Medicine, New York, NY, USA.
Clin Exp Immunol. 1998 Jul;113(1):111-8. doi: 10.1046/j.1365-2249.1998.00622.x.
The thyrotropin receptor (TSHR) is the major autoantigen of human Graves' disease. In order to define the antigenicity of the TSHR in a defined model, we examined the immune response of BALB/c mice to immunization with a new bioactive, recombinant preparation of the ectodomain of the murine TSHR (mTSHR-ecd). Mice (n = 10) were immunized with 25-50 microg of insect cell expressed, purified and refolded, mTSHR-ecd in alum adjuvant containing pertussis toxin, on days 0, 21, 36, 50 and 70. Control mice received wild-type baculovirus-infected insect cell protein lysate, in a similar way. After 28 days, murine serum contained high titres of antibodies specific to mTSH-ecd and their titres continued to increase over 90 days. Antibody epitope mapping, using 26 peptides spanning the human TSHR-ecd, showed that a variety of regions of the ectodomain were antigenic. The earliest epitope included aa 22-41, but later two regions of reactivity were noted clustered towards the mid portion and carboxyl terminus of the ectodomain. The murine TSHR autoantibodies (TSHR-Abs) inhibited up to 78% of the binding of labelled TSH to native TSHR, demonstrating the presence of antibodies capable of blocking the native TSHR. We showed that these TSHR antibodies acted, in vitro, as TSH blocking antibodies, inhibiting TSH-induced generation of cyclic AMP in chinese hamster ovary (CHO) cells transfected with the hTSHR. Hence, the antibody response to mTSHR-ecd was potentially antagonistic in its influence on the TSHR. Assessment of thyroid function in the immunized mice showed a fall in serum total T3 by 90 days and markedly elevated murine TSH levels (from 64.0 to 239.6 ng/ml), confirming the onset of thyroid failure. However, thyroid histology remained grossly normal. These data demonstrate that mTSHR-ecd is a potent antigen with three major immunogenic regions. The induced mTSHR-Abs blocked TSH action in vivo and reduced murine thyroid function.
促甲状腺激素受体（TSHR）是人类格雷夫斯病的主要自身抗原。为了在特定模型中确定TSHR的抗原性，我们检测了BALB/c小鼠对用一种新的具有生物活性的、重组的鼠促甲状腺激素受体胞外域（mTSHR-ecd）制剂免疫的免疫反应。在第0、21、36、50和70天，用25 - 50微克在含有百日咳毒素的明矾佐剂中表达、纯化和复性的昆虫细胞来源的mTSHR-ecd免疫小鼠（n = 10）。对照小鼠以类似方式接受野生型杆状病毒感染的昆虫细胞蛋白裂解物。28天后，小鼠血清中含有高滴度的针对mTSH-ecd的特异性抗体，并且其滴度在90天内持续升高。使用跨越人TSHR-ecd的26种肽进行抗体表位作图显示，胞外域的多个区域具有抗原性。最早的表位包括第22 - 41位氨基酸，但后来注意到两个反应区域聚集在胞外域的中部和羧基末端附近。鼠促甲状腺激素受体自身抗体（TSHR-Abs）可抑制高达78%的标记促甲状腺激素与天然TSHR的结合，表明存在能够阻断天然TSHR的抗体。我们表明，这些TSHR抗体在体外作为促甲状腺激素阻断抗体起作用，抑制促甲状腺激素诱导的在中国仓鼠卵巢（CHO）细胞中转染人TSHR后环磷酸腺苷的生成。因此，对mTSHR-ecd的抗体反应对TSHR的影响可能是拮抗的。对免疫小鼠甲状腺功能的评估显示，到90天时血清总T3下降，鼠促甲状腺激素水平显著升高（从64.0到239.6纳克/毫升），证实甲状腺功能减退的发生。然而，甲状腺组织学大体上仍正常。这些数据表明，mTSHR-ecd是一种具有三个主要免疫原性区域的强效抗原。诱导产生的mTSHR-Abs在体内阻断促甲状腺激素作用并降低鼠甲状腺功能。
