Gouin E, Segain J P, Saï P
Laboratory of Cellular and Molecular Immuno-Endocrinology, INRA/ENVN, Nantes, France.
Diabetes Metab. 1996 Dec;22(6):439-50.
Our group previously reported an assay for the study of lymphocyte adhesion to insulin-producing cells in which xenogeneic rat insulinoma (RIN) cells were used as targets. The present study found an increased number of RIN-cytoadherent lymphocytes in 63 patients with Type 1 diabetes compared with 150 control subjects and in 211 NOD mice compared with 104 BALB/c mice (p < 0.001). Proteins concentrated from spontaneous RIN cell culture supernatants inhibited increased RIN-adhesion of NOD splenocytes or lymphocytes from diabetic patients (p < 0.001). In addition, increased RIN binding was dose-dependently abolished by RIN membrane extracts. The fact that RIN binding was inhibited by proteins from both membrane and the culture supernatant from RIN cells suggests that soluble inhibitory proteins were spontaneously released into the supernatant from a hydrophobic membrane-bound form. This tended to be confirmed since inhibition obtained with both preparations involved a 55-75 kDa HPLC protein fraction. The possibility that the membrane form of the inhibitory protein was anchored by a glycosylphosphatidylinositol (GPI) tail was evaluated. When RIN cells were treated with PI-PLC, their ability to bind lymphocytes from diabetic patients or NOD splenocytes decreased (p < 0.001) to control levels. Co-incubation with the 55-75 kDa fraction of proteins cleaved from RIN cells by PI-PLC also lowered the number of RIN-adherent NOD splenocytes to control levels. SDS-PAGE and IEF analyses of the 55-75 kDa inhibitory fraction from RIN cell supernatant revealed a major band with Mr 66 kDa and PI5.4, which may correspond to a protein with similar characteristics noted on 2-D electrophoresis of proteins cleaved from RIN cells by PI-PLC. Specific labelling of GPI moieties with 3H-ethanolamine, 3H-glucosamine, or 14C-glucosamine, as well as conversion of the hydrophobic Triton-X114 solubilised form into a hydrophilic form after PI-PLC treatment, confirmed the presence of a GPI anchor in this approximately 66 kDa RIN protein, which could thus be the molecule inhibiting adhesion in the system. Our data suggest that GPI-proteins from insulin-producing cells may influence the immune system both in their membrane-anchored and soluble forms. When considering the binding model, in which beta cells were tumoral and xenogeneic to diabetic lymphocytes, this potential influence of GPI-proteins suggests possible implications in situations of lymphocyte-beta cell interaction, i.e. anti-beta cell autoimmunity, immune reaction against insulinomas, and reaction against islet xenografts.
我们小组之前报道了一项用于研究淋巴细胞与胰岛素生成细胞黏附的检测方法，该方法中使用异种大鼠胰岛素瘤（RIN）细胞作为靶细胞。本研究发现，与150名对照受试者相比，63例1型糖尿病患者体内RIN细胞黏附的淋巴细胞数量增加；与104只BALB/c小鼠相比，211只非肥胖糖尿病（NOD）小鼠体内RIN细胞黏附的淋巴细胞数量增加（p<0.001）。从自发的RIN细胞培养上清液中浓缩得到的蛋白质可抑制NOD脾细胞或糖尿病患者淋巴细胞与RIN细胞黏附的增加（p<0.001）。此外，RIN膜提取物可剂量依赖性地消除RIN结合的增加。RIN细胞的膜蛋白和培养上清液中的蛋白质均能抑制RIN结合，这一事实表明可溶性抑制蛋白是从疏水的膜结合形式自发释放到上清液中的。这一点倾向于得到证实，因为两种制剂产生的抑制作用都涉及一个55 - 75 kDa的高效液相色谱蛋白质组分。对抑制蛋白的膜形式是否由糖基磷脂酰肌醇（GPI）尾锚定的可能性进行了评估。用磷脂酰肌醇特异性磷脂酶C（PI - PLC）处理RIN细胞后，其与糖尿病患者淋巴细胞或NOD脾细胞结合的能力下降（p<0.001）至对照水平。与经PI - PLC从RIN细胞裂解得到的55 - 75 kDa蛋白质组分共同孵育，也可使黏附于RIN细胞的NOD脾细胞数量降至对照水平。对RIN细胞上清液中55 - 75 kDa抑制性组分进行十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）和等电聚焦（IEF）分析，发现一条主要条带，其相对分子质量（Mr）为66 kDa，等电点（PI）为5.4，这可能对应于经PI - PLC从RIN细胞裂解得到的蛋白质在二维电泳中显示出的具有相似特征的一种蛋白质。用3H - 乙醇胺、3H - 葡糖胺或14C - 葡糖胺对GPI部分进行特异性标记，以及在PI - PLC处理后将疏水的Triton - X114溶解形式转化为亲水形式，均证实了这种约66 kDa的RIN蛋白中存在GPI锚定，因此它可能是该系统中抑制黏附的分子。我们的数据表明，来自胰岛素生成细胞的GPI蛋白可能以其膜锚定形式和可溶性形式影响免疫系统。考虑到β细胞是肿瘤性的且与糖尿病淋巴细胞是异种的这种结合模型，GPI蛋白的这种潜在影响提示在淋巴细胞 - β细胞相互作用的情况下可能具有重要意义，即抗β细胞自身免疫、针对胰岛素瘤的免疫反应以及针对胰岛异种移植的反应。
