HMG proteins : their intra-cellular trafficking and the role in inflammatory diseases

我们发现89%的自身免疫性肝炎患者存在抗HMG1/HMG2抗体。为了确定循环自身抗体识别的B细胞表位，我们进行了免疫印迹试验，其中抗原是HMG1和HMG2的一组重组片段多肽。用自身免疫性肝炎患者的抗体阳性血清。我们的结果表明，这些表位位于两个HMG盒之间的连接区。我们还研究了抗HMG1/HMG2抗体选择性染色中性粒细胞的机制。从患者血清中亲和纯化的抗HMG1/HMG2抗体仅与中性粒细胞反应，而与淋巴细胞、Hep-2或HL60细胞(早幼粒白血病细胞株)不反应。然而，在HL60用全反式维甲酸诱导粒细胞分化后，这些细胞对抗体产生反应。在免疫印迹中，抗体在中性粒细胞和分化的HL60细胞中显示出明显的条带，这与淋巴细胞和未处理的HL60细胞中的不同。Northern blotting显示HL60细胞分化过程中HMG1和HMG2的mRNA表达无明显差异。这些结果表明，中性粒细胞中HMG1/HGM2分子的表位被揭示或修饰，以在粒细胞分化过程中与抗体发生反应。我们确定连接区域是主要的表位，已知对HMG蛋白的核定位是必要的。因此，该区域抗原性的改变可能会影响成熟中性粒细胞的核定位信号和转录效率。

We found the existence of anti-HMG1/HMG2 antibodies in 89% of patients with autoimmune hepatitis. These antibodies seemed to be useful clinical markers in the disease based on the highest prevalence and the correlation with disease activity.To determine the B-cell epitopes recognized by circulating autoantibodies, we performed western blotting, in which antigens were a set of recombinant fragment peptides of HMG1 and HMG2. Antibody-positive sera from patients with autoimmune hepatitis were used. Our results indicated that the epitopes are located within a linker region between the two HMG boxes.We also investigated the mechanism of the selective staining of neutrophil by anti-HMG1/HMG2 antibodies. Affinity-purified anti-HMG1/HMG2 antibodies from patient serum reacted only with neutrophils, but not with lymphocytes, HEp-2 or HL60 cells(a promyelocytic leukemia cell line) in indirect immunofluorescence. After HL60 were treated with all-trans retinoic acid to induce granulocyte differentiation, however, these cells became reactive to the antibodies. In western blotting, the antibodies showed a distinct pattern of bands in neutrophils and differentiated HL60 cells, which was different from those in lymphocytes and untreated HL60 cells. Northern blotting revealed no significant difference in mRNA of HMG1 and HMG2 during differentiation of HL60 cells. These results indicated that the epitopes of HMG1/HGM2 molecules in neutrophils are disclosed or modified to become reactive to the antibodies during granulocyte differentiation.The linker region, which we determined to be the major epitope, is known to be necessary for the nuclear localization of HMG proteins. It seems therefore that the change of antigenicity of the region may affect the nuclear localization signal and the efficiency of transcription in mature neutrophils.