Sato T, van Dixhoorn M G, Schroeijers W E, van Es L A, Daha M R
Department of Nephrology, University Hospital Leiden, The Netherlands.
Kidney Int. 1997 Jan;51(1):173-81. doi: 10.1038/ki.1997.21.
Apoptosis of glomerular cells (GMC) has been observed in the early phase as well as the resolution phase of Thy-1 nephritis. Recently, we and others reported that IgG2a (ER4G) and IgG1 (OX7) monoclonal mouse anti-Thy-1 antibodies (anti-Thy-1 MoAb) are able to induce apoptosis of rat GMC in vivo. The purpose of this study was to investigate whether cross-linking of Thy-1 would influence the degree of apoptosis in cultured rat GMC using monomeric and dimeric IgA anti-Thy-1 MoAb. IgA anti-Thy-1 MoAb (ER4A) was generated by class switching of the IgG producing ER4 (ER4G) hybridoma. The ER4A clone spontaneously produces monomeric (m-ER4A) and dimeric IgA anti-Thy-1 MoAb *di-ER4A). Unaltered epitope specificity of ER4A was confirmed by blocking experiments of the binding of fluorescence labeled ER4G to cultured rat GMC with unlabeled ER4A on FACS. For the experiments of apoptosis, quiescent rat GMC were incubated for eight hours with medium alone or with medium in the presence of 10 micrograms/ml of m-ER4A, di-ER4A or control IgA MoAb of corresponding sizes. Apoptosis was assessed by morphological studies, agarose gel electrophoresis and quantitative FACS analyses using terminal deoxynucleotidyl transferase (TDT) method and the annexin V method. The TDT method detects specific-DNA nicking in apoptosis. The annexin V method detects early membrane changes during apoptosis. In morphological studies, cells incubated with m-ER4A and di-ER4A showed typical apoptotic features such as nuclear condensation and fragmentation. DNA isolated from the cells incubated with di-ER4A was cleaved into a distinctive ladder pattern compatible with apoptosis. In contrast, both medium alone and control IgA MoAb did not reveal detectable changes in morphological studies and agarose gel electrophoresis. In quantitative analyses by FACS using the TDT method and the annexin method, both m-ER4A and di-ER4A induced significantly higher percentages of apoptosis in rat GMC as compared to the controls. Furthermore, di-ER4A was considerably more efficient than m-ER4A in inducing apoptosis possibly through additional cross-linking of Thy-1 on the cell surface. This notion was confirmed by experiments, in which the addition of goat anti-mouse kappa antibodies enhanced apoptosis of rat GMC pre-sensitized with m-ER4A. Taken together, our results indicate that apoptosis of rat GMC by anti-Thy-1 antibodies is enhanced by cross-linking of Thy-1 on the cell surface. These studies are of importance for our understanding of mechanisms that may play a role in glomerular diseases.
在Thy-1肾炎的早期阶段以及消退阶段均观察到肾小球细胞（GMC）凋亡。最近，我们和其他人报道，IgG2a（ER4G）和IgG1（OX7）单克隆小鼠抗Thy-1抗体（抗Thy-1 MoAb）能够在体内诱导大鼠GMC凋亡。本研究的目的是使用单体和二聚体IgA抗Thy-1 MoAb研究Thy-1的交联是否会影响培养的大鼠GMC的凋亡程度。IgA抗Thy-1 MoAb（ER4A）是由产生IgG的ER4（ER4G）杂交瘤通过类别转换产生的。ER4A克隆自发产生单体（m-ER4A）和二聚体IgA抗Thy-1 MoAb（di-ER4A）。通过在FACS上用未标记的ER4A阻断荧光标记的ER4G与培养的大鼠GMC的结合的阻断实验，证实了ER4A的表位特异性未改变。对于凋亡实验，将静止的大鼠GMC单独用培养基或在存在10微克/毫升的m-ER4A、di-ER4A或相应大小的对照IgA MoAb的培养基中孵育8小时。通过形态学研究、琼脂糖凝胶电泳以及使用末端脱氧核苷酸转移酶（TDT）方法和膜联蛋白V方法的定量FACS分析来评估凋亡。TDT方法检测凋亡中的特异性DNA切口。膜联蛋白V方法检测凋亡期间的早期膜变化。在形态学研究中，用m-ER4A和di-ER4A孵育的细胞显示出典型的凋亡特征，如核浓缩和碎片化。从用di-ER4A孵育的细胞中分离的DNA被切割成与凋亡相容的独特梯状模式。相比之下，单独的培养基和对照IgA MoAb在形态学研究和琼脂糖凝胶电泳中均未显示可检测到的变化。在使用TDT方法和膜联蛋白方法的FACS定量分析中,与对照相比，m-ER4A和di-ER4A均诱导大鼠GMC中显著更高百分比的凋亡。此外，di-ER4A在诱导凋亡方面比m-ER4A效率高得多，可能是通过细胞表面Thy-1的额外交联。这一观点通过实验得到证实，在该实验中，添加山羊抗小鼠κ抗体增强了用m-ER4A预致敏的大鼠GMC的凋亡。综上所述，我们的结果表明，抗Thy-1抗体诱导的大鼠GMC凋亡通过细胞表面Thy-1的交联而增强。这些研究对于我们理解可能在肾小球疾病中起作用的机制具有重要意义。
