Nagata N, Miyashima S, Taketani S, Toki J, Hosaka N, Tomita T, Fujishima H, Tokunaga R, Ikehara S
First Department of Pathology, Kansai Medical University, Osaka, Japan.
Lab Invest. 1994 Nov;71(5):765-72.
Autoantibodies such as anti-DNA and antimyeloperoxidase (MPO) antibodies have been shown to cause glomerulonephritis in experimental animal models. To analyze pathogenic autoantibodies, we developed hybridomas from spleen cells of nontreated FGS mice, in which focal segmental glomerular sclerosis develops spontaneously.
Reactivity and specificity of a monoclonal antibody (FG1H5) were examined using enzyme-linked immunosorbent assay and cryosections of mouse organs as substrates. Immunoprecipitation was performed to analyze reactive antigens. Hybridoma cells were injected ip into severe combined immunodeficiency (SCID) mice to examine their nephritogenicity in vivo.
The binding of FG1H5 to single-stranded DNA (ssDNA) was inhibited by ssDNA and also MPO. The binding of FG1H5 to MPO was weak, not inhibited by MPO, and markedly enhanced by the presence of ssDNA. This marked enhancement of the binding to MPO was abolished by DNase I-treatment of the mixture of FG1H5 and ssDNA. When MPO was introduced into ssDNA-coated wells, the binding of FG1H5 to ssDNA was inhibited. On the other hand, when ssDNA was introduced into MPO-coated wells, the binding of FG1H5 to MPO was markedly enhanced. Inhibition tests using double-stranded DNA revealed that FG1H5 is specific for ssDNA. Histologic examination of FG1H5-reactive antigen using SCID mouse kidney showed positive stainings in the nucleus and glomerulus (mainly the mesangium). These positive stainings were abolished after the incubation of FG1H5 with ssDNA. The DNase I treatment of kidney sections markedly reduced the nuclear staining, but the staining of the glomerulus was preserved. Immunoprecipitation of a soluble fraction of SCID mouse kidney with FG1H5 revealed that FG1H5-reactive antigen in the glomerulus is an approximately 28-kilodalton molecule. When FG1H5 hybridoma cells were injected ip into SCID mice, the mice showed glomerulonephritis with the increases in mesangial cells and matrix as well as immunoglobulin M deposition mainly in the mesangium.
Data demonstrate that FG1H5 binds strongly and specifically to ssDNA (but weakly and nonspecifically to MPO), and that ssDNA and MPO bind to each other. One monoclonal antibody reacts with both the nucleus and glomerulus (mainly the mesangium), and glomerular staining is not caused by nonspecific DNA binding. FG1H5, which binds to ssDNA, can induce glomerulonephritis, probably because of a direct crossreactivity to glomerular components.
抗DNA和抗髓过氧化物酶（MPO）抗体等自身抗体已在实验动物模型中被证明可导致肾小球肾炎。为了分析致病性自身抗体，我们从未经处理的FGS小鼠的脾细胞中制备杂交瘤，FGS小鼠会自发发生局灶节段性肾小球硬化。
使用酶联免疫吸附测定法，并以小鼠器官的冰冻切片为底物，检测单克隆抗体（FG1H5）的反应性和特异性。进行免疫沉淀以分析反应性抗原。将杂交瘤细胞腹腔注射到严重联合免疫缺陷（SCID）小鼠中，以检测其在体内的致肾炎性。
FG1H5与单链DNA（ssDNA）的结合受到ssDNA以及MPO的抑制。FG1H5与MPO的结合较弱，不受MPO抑制，且在ssDNA存在时显著增强。用DNA酶I处理FG1H5和ssDNA的混合物后，与MPO结合的这种显著增强被消除。当将MPO引入包被有ssDNA的孔中时，FG1H5与ssDNA 的结合受到抑制。另一方面，当将ssDNA引入包被有MPO的孔中时，FG1H5与MPO的结合显著增强。使用双链DNA的抑制试验表明，FG1H5对ssDNA具有特异性。使用SCID小鼠肾脏对FG1H5反应性抗原进行组织学检查，结果显示在细胞核和肾小球（主要是系膜）中有阳性染色。FG1H5与ssDNA孵育后，这些阳性染色消失。对肾脏切片进行DNA酶I处理可显著减少细胞核染色，但肾小球染色得以保留。用FG1H5对SCID小鼠肾脏的可溶性部分进行免疫沉淀，结果显示肾小球中的FG1H5反应性抗原是一种约28千道尔顿的分子。当将FG1H5杂交瘤细胞腹腔注射到SCID小鼠中时，小鼠出现肾小球肾炎，系膜细胞和基质增加，免疫球蛋白M沉积主要在系膜中。
数据表明FG1H5与ssDNA强烈且特异性结合（但与MPO结合较弱且非特异性），并且ssDNA和MPO相互结合。一种单克隆抗体与细胞核和肾小球（主要是系膜）均发生反应，肾小球染色并非由非特异性DNA结合引起。与ssDNA结合的FG1H5可能由于与肾小球成分的直接交叉反应性而诱导肾小球肾炎。
