Koutouzov S, Cabrespines A, Amoura Z, Chabre H, Lotton C, Bach J F
INSERM U 25, Hôpital Necker, Paris, France.
Eur J Immunol. 1996 Feb;26(2):472-86. doi: 10.1002/eji.1830260230.
In the present study, we sought evidence for a surface nucleosome receptor in the fibroblastic cell line CV-1, and questioned whether anti-double-stranded (ds) DNA and/or anti-histone autoantibodies could recognize and influence the fate of cell surface-bound nucleosomes. 125I-labeled mononucleosomes were shown to bind to the cell layer in a specific, concentration-dependent and a saturable manner. Scatchard analysis revealed the presence of two binding sites: a high-affinity site with a Kd of approximately 7nM and a low-affinity site (Kd approximately 400 nM) with a high capacity of 9 x 10(7) sites. Visualization of bound mononucleosomes by fluorescence revealed staining on both the cell surface and the extracellular matrix (ECM). Purified mononucleosome-derived ds DNA (180-200 bp) was found to complete for binding of 125I-mononucleosomes on the low-affinity site, to stain exclusively the ECM in immunofluorescence, and to precipitate three specific proteins of 43, 180 and 240 kDa from 125-I-labeled cell lysates. Nucleosomes were found to precipitate not only the 180-kDa ds DNA-reactive component, but also a unique protein of 50 kDa, suggesting that this protein is a cell surface receptor for nucleosomes on these fibroblasts. Once bound on the cell surface, mononucleosomes were recognized and secondarily complexed by lupus anti-ds DNA or anti-histone antibodies (i.e. anti-nucleosome antibodies), thus forming immune complexes in situ. The presence of these complexing auto-antibodies was found dramatically to enhance the kinetics of mononucleosome internalization. Following the internalization of the nucleosome-anti-nucleosome complexes by immunofluorescence, we observed the formation of vesicles at the edge of the cells by 5-10 min which moved toward the perinuclear region by 20-30 min. By means of double-fluorescence labeling and proteolytic treatment, these fluorescent vesicles were shown to be in the cytoplasm, suggesting true endocytosis of nucleosome-anti-nucleosome immune complexes. As shown by confocal microscopy, at no stage of this endocytic process was there any indication that coated pits or coated vesicles participated. Co-distribution of the endocytic vesicles with regions rich in actin filaments and inhibition of endocytosis of nucleosome-anti-nucleosome complexes by disruption of the microfilament network with cytochalasin D suggest a mechanism mediated by the cytoskeleton. Taken together, our data provide evidence for the presence of a surface nucleosome receptor. We also show that anti-ds DNA and anti-histone antibodies can form nucleosome-anti-nucleosome immune complexes in situ at the cell surface, and thus dramatically enhance the kinetics of nucleosome endocytosis.
在本研究中，我们在成纤维细胞系CV-1中寻找表面核小体受体的证据，并质疑抗双链（ds）DNA和/或抗组蛋白自身抗体是否能够识别并影响细胞表面结合核小体的命运。结果显示，125I标记的单核小体以特异性、浓度依赖性和饱和性方式与细胞层结合。Scatchard分析揭示存在两个结合位点：一个高亲和力位点，解离常数（Kd）约为7nM；一个低亲和力位点（Kd约为400nM），结合容量高，为9×10⁷个位点。通过荧光观察结合的单核小体，发现细胞表面和细胞外基质（ECM）均有染色。纯化的单核小体衍生的ds DNA（180 - 200bp）可竞争125I - 单核小体在低亲和力位点的结合，在免疫荧光中仅使ECM染色，并从125I标记的细胞裂解物中沉淀出三种特异性蛋白质，分子量分别为43kDa、180kDa和240kDa。发现核小体不仅沉淀出180kDa的ds DNA反应性成分，还沉淀出一种独特的50kDa蛋白质，提示该蛋白质是这些成纤维细胞上核小体的细胞表面受体。单核小体一旦结合到细胞表面，就会被狼疮抗ds DNA或抗组蛋白抗体（即抗核小体抗体）识别并继而形成复合物，从而在原位形成免疫复合物。发现这些形成复合物的自身抗体的存在显著增强了单核小体内化的动力学。通过免疫荧光观察核小体 - 抗核小体复合物的内化过程，我们在5 - 10分钟时观察到细胞边缘形成囊泡，这些囊泡在20 - 30分钟时向核周区域移动。通过双荧光标记和蛋白酶处理，这些荧光囊泡显示位于细胞质中，提示核小体 - 抗核小体免疫复合物发生了真正的内吞作用。共聚焦显微镜显示，在这个内吞过程的任何阶段都没有迹象表明有被膜小窝或被膜囊泡参与。内吞囊泡与富含肌动蛋白丝的区域共分布，以及用细胞松弛素D破坏微丝网络抑制核小体 - 抗核小体复合物的内吞作用，提示这是一种由细胞骨架介导的机制。综上所述，我们的数据为表面核小体受体的存在提供了证据。我们还表明，抗ds DNA和抗组蛋白抗体可在细胞表面原位形成核小体 - 抗核小体免疫复合物，从而显著增强核小体内吞的动力学。
