Malipiero U, Heuss C, Schlapbach R, Tschopp J, Gerber U, Fontana A
Clinical Immunology Department of Internal Medicine, University Hospital Zürich, Zürich, Switzerland.
Eur J Immunol. 1999 Oct;29(10):3053-62. doi: 10.1002/(SICI)1521-4141(199910)29:10<3053::AID-IMMU3053>3.0.CO;2-I.
The mechanisms underlying neurotoxicity mediated by cytotoxic T lymphocytes (CTL) and their secretory granule proteins perforin and granzymes remain unclear. We evaluated the possible role of the neurotransmitter glutamate in cell death observed in differentiated neurons exposed to CTL-derived granules. Excitotoxicity induced by excessive concentrations of extracellular glutamate is associated with a rise in intracellular calcium and can lead to generation of NO through the activation of glutamatergic N-methyl-D-aspartate (NMDA) receptors. Consistent with an involvement of glutamate, we found that cell death in mature cerebral granule cells was inhibited by 65-80% by two NMDA receptor blockers (MK-801 and D-2-amino-5-phosphonovaleric acid) or a NO synthase blocker (N(G)-nitro-L-arginine methylester). Furthermore, neurons treated with secretory granules responded with a biphasic rise in the intracellular calcium concentration ([Ca2+]i). Whereas MK-801 did not interfere with the immediate rise of [Ca2+]i, the second wave of calcium accumulation starting at 40 min was delayed by 20 min and reduced in amplitude in the presence of MK-801. In immature, NMDA receptor-negative neurons, MK-801 prevented neither the cytotoxicity nor the calcium influx observed 5 min after addition of cytotoxic granules. The demonstration that NMDA receptors and NO are involved in granule-mediated killing of mature neurons opens new avenues in the treatment of neuronal cell death in CTL-mediated diseases such as viral encephalitis.
细胞毒性T淋巴细胞（CTL）及其分泌颗粒蛋白穿孔素和颗粒酶介导神经毒性的潜在机制仍不清楚。我们评估了神经递质谷氨酸在暴露于CTL衍生颗粒的分化神经元中观察到的细胞死亡中的可能作用。细胞外谷氨酸浓度过高诱导的兴奋毒性与细胞内钙升高有关，并可通过激活谷氨酸能N-甲基-D-天冬氨酸（NMDA）受体导致一氧化氮的生成。与谷氨酸的参与一致，我们发现两种NMDA受体阻滞剂（MK-801和D-2-氨基-5-磷酸戊酸）或一种一氧化氮合酶阻滞剂（N（G）-硝基-L-精氨酸甲酯）可使成熟脑颗粒细胞中的细胞死亡受到65-80%的抑制。此外，用分泌颗粒处理的神经元细胞内钙浓度（[Ca2+]i）呈双相升高。虽然MK-801不干扰[Ca2+]i的立即升高，但在MK-801存在的情况下，从40分钟开始的第二波钙积累延迟了20分钟，且幅度减小。在未成熟的、NMDA受体阴性的神经元中，MK-801既不能阻止细胞毒性，也不能阻止在加入细胞毒性颗粒5分钟后观察到钙内流。NMDA受体和一氧化氮参与颗粒介导的成熟神经元杀伤的证明为治疗CTL介导疾病（如病毒性脑炎）中的神经元细胞死亡开辟了新途径。
