De Giorgio Roberto, Bovara Monica, Barbara Giovanni, Canossa Marco, Sarnelli Giovanni, De Ponti Fabrizio, Stanghellini Vincenzo, Tonini Marcello, Cappello Silvia, Pagnotta Eleonora, Nobile-Orazio Eduardo, Corinaldesi Roberto
Department of Internal Medicine, University of Bologna, Italy.
Gastroenterology. 2003 Jul;125(1):70-9. doi: 10.1016/s0016-5085(03)00664-4.
BACKGROUND & AIMS: The role of autoimmunity underlying paraneoplastic gut dysmotility remains unsettled. Because anti-Hu antibodies may impair enteric neuronal function, we tested whether anti-HuD-positive sera from patients with paraneoplastic gut dysmotility or commercial anti-HuD antibodies activated the apoptotic cascade in a neuroblastoma cell line and cultured myenteric neurons.
Anti-HuD antibodies from patients with severe paraneoplastic gut dysmotility were characterized by immunofluorescence and immunoblot. SH-Sy5Y neuroblasts and cultured myenteric neurons were exposed to sera containing anti-HuD antibodies or 2 commercial anti-HuD antibodies. Cells were processed for terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) technique to evaluate apoptosis. Immunofluorescence was used to identify activated caspase-3 and apaf-1, along with microtubule-associated protein 2.
In SH-Sy5Y cells, the percentage of TUNEL-positive nuclei observed after exposure to anti-HuD-positive sera (32% +/- 7%) or anti-HuD antibodies (23% +/- 2%) was significantly greater than that of control sera or fetal calf serum (P < 0.001). The time-course analysis showed a significantly greater number of apoptotic neuroblastoma cells evoked by the 2 commercial anti-HuD antibodies at 24, 48, and 72 hours versus controls. The number of TUNEL-positive myenteric neurons exposed to anti-HuD antibodies (60% +/- 14%) was significantly greater than that of fetal calf serum (7% +/- 2%; P < 0.001). Apaf-1 and caspase-3 immunolabeling showed intense cytoplasmic staining in a significantly greater proportion of cells exposed to anti-HuD-positive sera or to commercial anti-HuD antibodies compared with controls.
Anti-HuD antibodies evoked neuronal apoptosis that may contribute to enteric nervous system impairment underlying paraneoplastic gut dysmotility. Apaf-1 activation suggests participation of a mitochondria-dependent apoptotic pathway.
自身免疫在副肿瘤性肠道运动障碍中所起的作用尚未明确。由于抗Hu抗体可能损害肠神经元功能，我们检测了副肿瘤性肠道运动障碍患者的抗HuD阳性血清或市售抗HuD抗体是否能激活神经母细胞瘤细胞系和培养的肌间神经丛神经元中的凋亡级联反应。
通过免疫荧光和免疫印迹对严重副肿瘤性肠道运动障碍患者的抗HuD抗体进行鉴定。将SH-Sy5Y神经母细胞和培养的肌间神经丛神经元暴露于含有抗HuD抗体的血清或两种市售抗HuD抗体中。对细胞进行末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸缺口末端标记（TUNEL）技术检测以评估细胞凋亡。免疫荧光用于鉴定活化的半胱天冬酶-3和凋亡蛋白酶激活因子-1，以及微管相关蛋白2。
在SH-Sy5Y细胞中，暴露于抗HuD阳性血清（32%±7%）或抗HuD抗体（23%±2%）后观察到的TUNEL阳性细胞核百分比显著高于对照血清或胎牛血清（P<0.001）。时间进程分析显示，与对照相比，两种市售抗HuD抗体在24、48和72小时时诱发的凋亡神经母细胞瘤细胞数量显著更多。暴露于抗HuD抗体的TUNEL阳性肌间神经丛神经元数量（60%±14%）显著高于胎牛血清组（7%±2%；P<0.001）。与对照相比，凋亡蛋白酶激活因子-1和半胱天冬酶-3免疫标记显示，暴露于抗HuD阳性血清或市售抗HuD抗体的细胞中，有显著更高比例的细胞出现强烈的细胞质染色。
抗HuD抗体诱发神经元凋亡，这可能导致副肿瘤性肠道运动障碍所潜在的肠神经系统损害。凋亡蛋白酶激活因子-1的激活提示线粒体依赖性凋亡途径的参与。
