Analysis of Mechanisms underlying Autoantibody production in Chronic Graft-versus-host Reaction

对慢性移植物抗宿主反应(GVHR)诱导自身免疫的机制进行了一系列的实验研究：(1)采用EL ISA斑点试验研究GVHR产生自身抗体的机制。常规抗体(ANT-TNP、抗OVA)产生细胞被GVHR激活，自身抗体(抗DNA、Ita等)产生细胞也被激活。因此，慢性GVIIR时自身抗体的产生可能与多克隆B细胞的激活有关。(2)肾小球病变的电子显微镜检查显示，电子致密沉积首先在系膜基质中，然后在与免疫复合物肾小球相容的肾小球系膜下。未见内皮下沉积。免疫荧光研究显示，在这一过程的早期，免疫球蛋白G沉积在毛细血管壁，免疫球蛋白M沉积在系膜。部分肾小球呈节段性系膜溶解，提示系膜细胞改变在…中起一定作用。(3)BDF_1和(BALB/CxA)F_1(CAF_1)小鼠注射其亲本或B10可诱导GVIIR。D2基因缺失的脾细胞。然后将肾小球肾炎的描述与自身抗体的产生联系起来。所有接受DBA/2脾细胞(DBA/2-&GT；BDF_1)和接受BALB/c脾细胞(BALB/c-&GT；CAF_1)的CAF_1小鼠均出现肾小球肾炎。然而，在其他组合中(B10.D2-&GT；BDF_1，A/J-&GT；CaF_1)未见明显的肾小球样改变。用酶联免疫吸附试验对抗体进行分析，发现肾脏疾病组的多克隆抗体水平显著升高，包括自身抗体(抗DNA、抗MRBC和NTA)和常规抗体(antl-TNP-KLII)。在未发生肾小球肾炎的组中，未观察到明显的免疫球蛋白抗体产生。提示Ig M向Ig G转换在GVHR肾小球肾炎的发生发展中起重要作用。其他因素似乎也参与其中。仅有33%的BALB/C-&GT；CAF_1发生肾小球肾炎，尽管其产生的抗体水平与DBA/2-&GT；BDF_1相似，其中100%表现为严重的肾炎。(4)Myc和Raf基因的表达与B细胞活性和细胞动力学进行比较。EL ISA斑点检测显示B细胞呈一过性增殖，在移植后3周达高峰。MYE和Raf基因表达增强，在移植后3周达高峰。这一结果与EL ISA斑点分析显示的B细胞增殖情况相一致。细胞动力学分析显示，移植后4周，处于G2+M期的增殖细胞增多。因此，Myc和RAF活性被认为与B细胞活性相关。通过对淋巴母细胞恶性肿瘤和自发自身免疫模型小鼠的研究，认为这些癌基因的活性与淋巴母细胞的异常增殖有关。由于GVHR中增殖的B细胞本质上是正常的，目前的发现似乎表明Myc和RAF基因参与了B细胞的增殖。(5)显微镜检查显示DBA/2-&GT；BDF_1的肺泡壁有单个核细胞的浸润，提示存在轻度的间质性肺炎。支气管肺泡灌洗液中淋巴细胞和安慰素转换酶活性的升高与这一观点一致。DBA/2-&GT；BDF_1的肺组织病理改变可能是间质性肺炎的一个模型。较少

Several series of the experiments were conducted to analyze the mechanisms underlying autoimmunity Induced by chronic graft-versus-host reaction (GVHR).(1) ELISA spot assay was employed to investigate the mechanisms of autoantibody production by GVHR. Conventional antibody (ant-TNP, anti-OVA) producing cells were shown to be activated by GVHR as well as autoantibody (anti-DNA, iTA, etc) producing cells. Thus, It was suggested that autoantibody production might be associated with polyclonal B cell activation in chronic GVIIR.(2) Electron microscopical examination on the glomerular lesion demonstrated electron dense deposits first In the mesangial matrix, then in the subepithellum compatible with Immune complex glomerulonephrltls. Subendothellal deposits were not observed. Immunofluorescent study revealed IgG deposition In the capillary wall and IgM in the mesanglum early In the process. Some glomerull showed segmental mesangiolysis, suggesting that altered mesangial cells have a role In … More the development of glomerular change, which together with rise In serum autoantibody titer suggest that autoantibodies promote the glomerular lesions In this model system.(3) GVIIR was induced In BDF_1 and (BALB/cx A)F_1(CAF_1) murine recipients by Injection of their parental or B10. D2-derlved spleen cells. The lncridences of glomerulonephritis and autoantibody production were then correlated. All BDF_1 mice that received DBA/2 spleen cells (termed DBA/2->BDF_1) and 33% of CAF_1 mice that received BALB/c spleen cells (BALB/c->CAF_1) developed glomerulonephritis. However, In other combinations (B10. D2->BDF_1, A/J->CAF_1) no significant glomerular leslons were observed. Analysis of antibodies by ELISA has revealed that the groups with renal disease showed significant polyclonal elevaton of IgG-class antibodies, including autoantibodies (anti-DNA, anti-MRBC and NTA) and conventional antibody (antl-TNP-KLII). No significant IgG class antibody production was observed In the groups that did not develop glomerulonephritis. Thus, it was suggested that an IgM to IgG class switch is important In the development of glomerulonephritis In GVHR. Other factors appear to be involved. Only 33% of BALB/C->CAF_1 developed glomerulonephritis even though a level of IgG class antibody production was comparable to that observed In DBA/2->BDF_1 In which 100% showed severe glomerulonephritis.(4) Myc and Raf gene expression was analysed and compared with B cell activity and cell kinetics. ELISA spot assay revealed transient B cell proliferation with a peak at 3 week after transfer. Mye and Raf genes showed the Increased expression wlth the peak at 3 weeks after transfer. This profile was consistent with that of B cell prollferation revealed by ELISA spot assay. Cell kinetics analysis revealed that proliferative cells In G2+M phase increased 4 weeks after transfer. Thus, the myc and raf activity has been considered to be correlated to the B cell activity. These oncogene activity Is considered to be correlated with abnormal lymphold cell proliferation from the lnvestigations on lymphold cell malignancy and spontaneous autoimmune model mouse. Since proliferative B cells In GVHR Is Intrinsically normal, the present findings seem to indicate In vivo evidence of Involvement of myc and raf gene In B cell proliferation.(5) Microscopical examination revealed mononuclear cell Infiltration In pulmonary alveolar wall of DBA/2->BDF_1, suggesting the presence of mild degree of interstitial pneumonlae. Increase of lymphocytes and angotensin converting enzyme activity In bronchloalveolar lavage fluid was consistent with this notion. Pathological change in the lungs of DBA/2->BDF_1 was suggested to be a model of interstitial pneumonia. Less