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

慢性移植物抗宿主反应中自身抗体产生的机制分析

• 批准号: 01570364
• 负责人: AOKI Ichiro
• 金额: $ 1.22万
• 依托单位: Yokohama City University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1989
• 资助国家: 日本
• 起止时间: 1989 至 1991
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-01570364/
• 关键词: Graft-versus-host reaction; B cell activation; Autoantibody; Glomerulonephrites; IgG; Ultrastructure; Interstitial pneumonia; Mouse; 移植片対宿主反応; 急速凍影・ディ-ブエッチング法; FK506; mesangiolysis; BALF; 細胞回転; 癌遺伝子; 自己免疫; 免疫複合体; myc遺伝子; raf遺伝子

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

本实验旨在探讨慢性移植物抗宿主反应（GVHR）诱导自身免疫的机制。(1)采用ELISA斑点试验研究GVHR产生自身抗体的机制。常规抗体（抗TNP、抗OVA）产生细胞显示被GVHR以及自身抗体（抗DNA、iTA等）产生细胞激活。提示慢性GVIIR中自身抗体的产生可能与多克隆B细胞活化有关。(2)电镜下见肾小球内电子致密物先沉积于系膜基质，后沉积于与免疫复合物相容的肾小球上皮下。未观察到亚内皮层沉积物。免疫荧光研究显示IgG沉积在毛细血管壁和IgM沉积在系膜早期的过程中。一些肾小球表现出节段性系膜溶解，表明改变的系膜细胞在其中发挥作用 ...更多信息 肾小球改变的发展，连同血清自身抗体滴度的升高，提示在该模型系统中自身抗体促进肾小球病变。(3)在BDF_1和（BALB/cx A）F_1（CAF_1）小鼠受体中，通过注射其亲本或B10诱导GVIIR。D2来源的脾细胞。肾小球肾炎的发病率与自身抗体的产生相关。所有接受DBA/2脾细胞的BDF_1小鼠（称为DBA/2->BDF_1）和33%接受BALB/c脾细胞的CAF_1小鼠（BALB/c->CAF_1）发生肾小球肾炎。然而，在其他组合（B10. D_2->BDF_1，A/J->CAF_1）无明显肾小球病变。用ELISA法检测抗体，发现肾脏疾病组IgG类抗体水平明显升高，包括自身抗体（抗DNA、抗MRBC和NTA）和常规抗体（抗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，这表明IgM到IgG类转换在GVHR中肾小球肾炎的发展中是重要的。似乎还涉及其他因素。BALB/C->CAF_1的IgG类抗体水平与DBA/2->BDF_1相当，但只有33%的小鼠发生了严重的肾小球肾炎。(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细胞在转移后3周出现短暂增殖。Mye和Raf基因表达增强，3周达高峰。这与ELISA斑点法检测B细胞增殖的结果一致。细胞动力学分析显示，移植后4周，G2+M期细胞增殖增加。因此，认为myc和raf活性与B细胞活性相关。这些癌基因的活性被认为与淋巴细胞恶性肿瘤和自发性自身免疫模型小鼠的研究中发现的淋巴细胞异常增殖有关。由于GVHR中增殖的B细胞是内在正常的，本研究结果似乎表明体内证据表明myc和raf基因参与B细胞增殖。(5)光镜下见DBA/2->BDF_1肺泡壁有单核细胞浸润，提示有轻度间质性肺炎。支气管肺泡灌洗液中淋巴细胞和血管紧张素转换酶活性增高与此一致。DBA/2->BDF_1的肺组织病理学改变可作为间质性肺炎的模型。少

项目成果

Ishii N,Ishii H,Ono H,Horiuchi Y,Nakajima H and Aoki I.: "Genetic Control of Nickel Sulfate Delayed-Type Hypersensitivity." Jouranal of Investigative Dermatology. 94. 673-676 (1990)

Ishii N、Ishii H、Ono H、Horiuchi Y、Nakajima H 和 Aoki I.：“硫酸镍迟发型超敏反应的基因控制”。

大谷 方子、その他: "乳児期早期における皮膚限局性組織球増殖症の2症例." 病理と臨床. 8. 1549-1553 (1990)

Masako Otani 等人：“婴儿早期皮肤组织细胞增多症的两例。” 8. 1549-1553 (1990)。

Aoki A, Ishigatsubo Y, Hagiwara E, Shirai A, Narita M, Matsunaga K, Tani K, Okubo T, Otani M, Miyagi Y, Aoki I, Misugi K, Okuda K.: "Comparative study of serum antibody titer and antibody-producing cells in chronic GVHD (graft-versus-host disease) mice."

Aoki A、Ishigatsubo Y、Hagiwara E、Shirai A、Narita M、Matsunaga K、Tani K、Okubo T、Otani M、Miyagi Y、Aoki I、Misugi K、Okuda K.：“血清抗体滴度和抗体的比较研究-

Otani M, Aoki I, Nakatani Y, Misugi K, Ehara M, Iwamoto M, Fujiwara Y.: "Obesevation of PAM stained human renal biopsy specimen by scanning electron microscopy." J. Clin Electron Microscopy. 23. 5-6 (1990)

Otani M、Aoki I、Nakatani Y、Misugi K、Ehara M、Iwamoto M、Fujiwara Y.：“通过扫描电子显微镜观察 PAM 染色的人肾活检标本。”

Ishii N, Nakajima H, Aoki I, shimoda N, Mori T.: "Genetic Control of Delayed Type Hypersensitivity to Mycobacterium Leprae Antigen." Jap. J Leprosy. 59. 154-161 (1990)

Ishii N、Nakajima H、Aoki I、shimoda N、Mori T.：“麻风分枝杆菌抗原迟发型超敏反应的基因控制”。