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

通过一系列实验分析慢性移植物抗宿主反应（GVHR）诱导自身免疫的机制。(1)采用ELISA斑点法探讨GVHR产生自身抗体的机制。常规抗体（抗tnp、抗ova）产生细胞和自身抗体（抗dna、iTA等）产生细胞均被GVHR激活。因此，提示自身抗体的产生可能与慢性GVIIR的多克隆B细胞活化有关。(2)肾小球病变的电镜检查显示，电子致密沉积首先在系膜基质中，然后在与免疫复合物相容的上皮下肾小球。未观察到内皮下沉积。免疫荧光研究显示IgG沉积于毛细血管壁，IgM沉积于系膜。一些肾小球出现节段性肾小球系膜溶解，提示系膜细胞改变在肾小球改变的发展中起作用，同时血清自身抗体滴度升高提示自身抗体促进了该模型系统的肾小球病变。(3) BDF_1和（BALB/cx A）F_1（CAF_1）小鼠受体通过注射亲本或B10诱导GVIIR。d2级脾细胞。然后将肾小球肾炎的发生率与自身抗体的产生相关联。所有接受DBA/2脾细胞（DBA/2->BDF_1）的BDF_1小鼠和33%接受BALB/c脾细胞（BALB/c->CAF_1）的CAF_1小鼠发生肾小球肾炎。然而，在其他组合中(B10。D2->BDF_1、A/J->CAF_1)未见明显肾小球病变。ELISA抗体分析显示肾脏疾病组igg类抗体显著多克隆升高，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，未观察到显著的IgG类抗体产生。因此，提示IgM到IgG类转换在GVHR肾小球肾炎的发展中起重要作用。其他因素似乎也参与其中。尽管IgG类抗体产生水平与DBA/2->BDF_1相当，但只有33%的BALB/C->CAF_1发生肾小球肾炎，其中100%发生严重肾小球肾炎。(4)分析Myc和Raf基因表达，并与B细胞活性和细胞动力学进行比较。ELISA斑点试验显示B细胞瞬时增殖，在转移后3周达到高峰。Mye和Raf基因表达增加，并在转移后3周达到高峰。这与ELISA斑点试验显示的B细胞增殖情况一致。细胞动力学分析显示，移植后4周G2+M期增殖细胞增多。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，认为这些癌基因活性与淋巴细胞异常增殖有关。由于增殖性B细胞在GVHR中本质上是正常的，目前的研究结果似乎表明myc和raf基因参与B细胞增殖的体内证据。(5)显微镜检查示DBA/2->BDF_1肺泡壁单核细胞浸润，提示轻度间质性肺炎。支气管肺泡灌洗液中淋巴细胞和血管紧张素转换酶活性的增加与这一概念一致。DBA/2->BDF_1的肺病理改变提示其为间质性肺炎模型。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