Yabuzoe Atsushi, Shimizu Atushi, Nishifuji Koji, Momoi Yasuyuki, Ishiko Akira, Iwasaki Toshiroh
Laboratory of Veterinary Internal Medicine, Faculty of Agriculture, Tokyo University of Agriculture & Technology, Tokyo 183-8509, Japan.
Vet Immunol Immunopathol. 2009 Jan 15;127(1-2):57-64. doi: 10.1016/j.vetimm.2008.09.015. Epub 2008 Sep 24.
The target antigen of autoantibodies in human pemphigus foliaceus (PF) is a desmosomal cadherin, desmoglein 1 (Dsg1). It was demonstrated by immunoelectron microscopy (IEM) that the location of the binding sites of PF autoantibodies in the human epidermis was the extracellular regions of the desmosomes. Only a limited number of canine PF sera were shown to react with canine Dsg1, and the target proteins have not yet been identified. The purpose of this study was to demonstrate the ultrastructural binding site of canine PF autoantibodies to the canine skin by two kinds of IEM methods. Three canine PF sera, which were shown to react with the keratinocyte cell surface by immunofluorescence, were tested in this study. Using a technique of immunoprecipitation-immunoblotting, one out of the three canine PF sera were shown to react with recombinant canine Dsg1. By post-embedding IEM using cryofixation technique, one serum, which did not react with canine Dsg1 by immunoprecipitation-immunoblotting, bound broadly to the extra- and intracellular regions of the desmosomes of normal canine skin. By pre-embedding IEM using canine cultured keratinocytes (MCA-B1 cells), the autoantibodies of all three canine PF sera were identified to be bound to the cell-cell contact area of the adjacent cytoplasmic projections. When double stained with human PF serum and canine PF sera, the binding sites of both human and canine autoantibodies were co-localized on the MCA-B1 cells where the cytoplasmic projections contacted each other. Therefore, it may be concluded that the serum antibodies of canine PF targeted desmosomal proteins, regardless of whether or not they react with canine Dsg1 by immunoprecipitation-immunoblotting method.
人类落叶型天疱疮(PF)自身抗体的靶抗原是一种桥粒钙黏蛋白,桥粒芯糖蛋白1(Dsg1)。免疫电子显微镜(IEM)显示,PF自身抗体在人表皮中的结合位点位于桥粒的细胞外区域。仅有少数犬PF血清显示与犬Dsg1反应,且靶蛋白尚未确定。本研究的目的是通过两种IEM方法证明犬PF自身抗体与犬皮肤的超微结构结合位点。本研究检测了三种经免疫荧光显示与角质形成细胞表面反应的犬PF血清。使用免疫沉淀-免疫印迹技术,三种犬PF血清中有一种显示与重组犬Dsg1反应。通过使用冷冻固定技术的包埋后IEM,一种在免疫沉淀-免疫印迹中不与犬Dsg1反应的血清广泛结合于正常犬皮肤桥粒的细胞外和细胞内区域。通过使用犬培养角质形成细胞(MCA-B1细胞)的包埋前IEM,所有三种犬PF血清的自身抗体均被鉴定为结合于相邻细胞质突起的细胞-细胞接触区域。当用人PF血清和犬PF血清进行双重染色时,人和犬自身抗体的结合位点在细胞质突起相互接触的MCA-B1细胞上共定位。因此,可以得出结论,犬PF的血清抗体靶向桥粒蛋白,无论它们是否通过免疫沉淀-免疫印迹法与犬Dsg1反应。