Zhou J, Cox N R, Ewald S J, Morrison N E, Basker H J
Department of Internal Medicine, St. Luke's Hospital, St. Louis, MO 63017, USA.
Vet Immunol Immunopathol. 1998 Nov 6;66(1):25-42. doi: 10.1016/s0165-2427(98)00180-9.
Cats with inherited GM1 gangliosidosis (GM1 mutant cats) have premature thymic involution characterized by decreased total thymocytes primarily affecting the CD4+ CD8+ subpopulation. While GM1 mutant cats have increased cell surface GM1 gangliosides, as determined by cholera toxin B binding, on both thymocytes and peripheral lymph node cells only thymocytes show increased apoptosis. To determine if GM1 gangliosides can increase the occurrence of apoptosis in feline thymocytes directly, we added exogenous GM1 ganglioside (GM1) to feline thymocyte primary cultures and compared the results to apoptotic changes seen in untreated cells or in cells treated with dexamethasone (Dex), a known inducer of thymocyte apoptosis in other species. Incorporation of exogenous GM1 into thymocyte cytoplasmic membranes was confirmed by flow cytometric analyses of cholera toxin B labelling. Apoptosis in feline thymocytes was analyzed by electron microscopy, spectrophotometric evaluation of DNA fragmentation, flow cytometric enumeration of apoptotic nuclei, and gel electrophoretic analysis of degraded DNA. Alterations in percentages of thymocyte immunophenotype following GM1 incorporation were determined by flow cytometric analyses of labelled cell surface markers for feline CD4 and CD8. Because in vitro addition of GM1 gangliosides has been reported in other species to decrease surface expression of CD4 on both thymocytes and peripheral lymphocytes, we evaluated GM1-associated down-regulation of CD4 on the surface of feline thymocytes and peripheral lymph node cells by flow cytometry. Additionally, we compared the apoptotic response of the more mature peripheral lymph node cells to the less mature thymocytes. Our results indicate that incorporation of exogenous GM1 into feline thymocyte cell membranes produces a dose-dependent increase of apoptotic cell death. Although, CD4 expression on both feline thymocyte and lymph node cell membranes was abruptly decreased after introducing exogenous GM1, enhanced apoptotic death was observed only in thymocytes, not in lymph node cells at the same GM1 concentration. Enhancement of thymocyte apoptosis appears to be age-related since cells derived from cats <3 months of age were more vulnerable than those from cats >3 months of age.
患有遗传性GM1神经节苷脂贮积症的猫（GM1突变猫）存在胸腺过早退化，其特征是总胸腺细胞减少，主要影响CD4+CD8+亚群。虽然通过霍乱毒素B结合测定，GM1突变猫的胸腺细胞和外周淋巴结细胞上的细胞表面GM1神经节苷脂有所增加，但只有胸腺细胞显示出凋亡增加。为了直接确定GM1神经节苷脂是否能增加猫胸腺细胞凋亡的发生率，我们将外源性GM1神经节苷脂（GM1）添加到猫胸腺细胞原代培养物中，并将结果与未处理细胞或用已知的其他物种胸腺细胞凋亡诱导剂地塞米松（Dex）处理的细胞中的凋亡变化进行比较。通过霍乱毒素B标记的流式细胞术分析证实了外源性GM1掺入胸腺细胞胞质膜。通过电子显微镜、DNA片段化的分光光度评估、凋亡细胞核的流式细胞术计数以及降解DNA的凝胶电泳分析来分析猫胸腺细胞中的凋亡。通过对猫CD4和CD8标记细胞表面标志物的流式细胞术分析来确定GM1掺入后胸腺细胞免疫表型百分比的变化。因为在其他物种中已报道体外添加GM1神经节苷脂会降低胸腺细胞和外周淋巴细胞表面CD4的表达，所以我们通过流式细胞术评估了GM1相关的猫胸腺细胞和外周淋巴结细胞表面CD4的下调。此外，我们比较了更成熟的外周淋巴结细胞与不太成熟的胸腺细胞的凋亡反应。我们的结果表明，将外源性GM1掺入猫胸腺细胞膜会导致凋亡细胞死亡呈剂量依赖性增加。虽然在引入外源性GM1后，猫胸腺细胞和淋巴结细胞膜上的CD4表达均突然下降，但在相同GM1浓度下，仅在胸腺细胞中观察到凋亡死亡增强，而在淋巴结细胞中未观察到。胸腺细胞凋亡的增强似乎与年龄有关，因为来自小于3个月龄猫的细胞比来自大于3个月龄猫的细胞更易受影响。
