Cellular and molecular studies on the transmembrane mechanism of neuroimmuno-interaction

为了阐明神经递质在免疫系统中的调节机制和神经免疫相互作用，我们研究了人类T淋巴细胞中毒蕈碱乙酰胆碱（mACh）受体及其跨膜调控。在克隆的人T淋巴细胞Jurkat细胞中，毒蕈碱激动剂oxotremorine-M（Oxo-M）引起细胞内Ca^2+浓度（[Ca^2+] i）的短暂升高。此外，Oxo-M还能促进1，4，5-三磷酸肌醇（IP_3）的生成，但不影响cAMP的水平。Oxo-M诱导的[Ca^<2+>]_i升高不受培养基中Ca^<2+>缺失和百日咳、霍乱或肉毒毒素预处理的影响。而Gpp（NH）p和IP_3则引起毛地黄皂苷透化细胞内[Ca^<2+>]_i升高。[ ^3H]QNB结合Jurkat细胞的Scatchard分析表明：K_D=14 nM，Bmax=45，000位点/细胞。北方印迹分析显示，表达m3型mACh受体mRNA。这些结果表明， 关于我们 人T淋巴细胞中存在mACh受体，该受体激活后，通过细菌毒素不敏感的GTP结合蛋白（可能是Gq样GTP结合蛋白）、磷脂酶C（PLC）激活和IP_3形成，使细胞内Ca ^2+从胞内Ca^2+库中动员出来。为了进一步阐明M_3-mACh受体在免疫系统中的生理功能，我们进一步研究了M_3-mACh受体激活对人外周血T淋巴细胞产生白细胞介素-2（IL-2）的影响，并在电生理检测系统中鉴定了一种新的IL-2受体。受体似乎通过大脑中的GTP结合蛋白激活PLC。MPP^+是一种对多巴胺（DA）神经元具有选择性的神经毒素，可引起帕金森样综合征，它可降低DA神经元模型PC 12细胞中DA含量。IL-6抑制MPP^+诱导的DA含量下降。IL-6还增强酪氨酸羟化酶mRNA的表达，表明IL-6对中枢神经系统中的神经元具有保护作用。少

To clarify the regulatory mechanism of neurotransmitter in the immune system and the neuroimmune interaction, we have been investigated the presence of muscarinic acetylcholine (mACh) receptors and the transmembrane control in human T lymphocytes. In Jurkat cells which is a cloned human T lymphocyte, muscarinic agonist oxotremorine-M (Oxo-M) caused a transient rise of intracellular concentration of Ca^<2+> ([Ca^<2+>]_i). In addition, Oxo-M stimulated a formation of inositol-1, 4, 5-trisphosphate (IP_3) but did not affect the cyclic AMP level. Oxo-M-induced [Ca^<2+>]_i rise was not affected by omission of Ca^<2+> in the medium and by pretreatment with pertussis, cholera or botulinus toxin. While, Gpp (NH) p and IP_3 caused [Ca^<2+>]_i in digitonin-permeabilized cells. Scatchard analysis of [ ^3H]QNB binding in Jurkat cells indicated : K_D=14 nM and Bmax=45, 000 sites/cell. In addition, Northern blot analysis showed to be expressed m3 type of mACh receptor mRNA. These results suggest tha … More t M_3-mACh receptors exist in human T lymphocytes and the activation of the receptors causes Ca^<2+> mobilization from intracellular Ca^<2+> stores via bacterial toxin-insensitive GTP-binding proteins (probably Gq-like GTP-binding proteins), phospholipase C (PLC) activation and IP_3 formation. Furthermore, to clarify the physiological function of M_3-mACh receptors in the immune system, we then examined effects of M_3-mACh receptor activation on interleukin-2 (IL-2) production from human peripheral blood T lymphocytes.In electrophysiological assay system using Xenopus oocytes into which rat brain mRNA were microinjected, we identified novel type of IL-2 receptors. The receptors seem to activate PLC via GTP-binding proteins in the brain. We are now planning the cDNA cloning of the novel IL-2 receptors.MPP^+, a selective neurotoxin to dopamine (DA) neurons, which causes Parkinsonism-like syndromes, decreased the DA content in PC12 cells, a DA neuron-model. IL-6 inhibited MPP^+-induced decrease in the DA content. IL-6 also enhanced the expression of tyrosine hydroxylase mRNA, suggesting that IL-6 exerts protective effects to neurons in the central nervous system. Less