Mechanisms and modulation of neuro-immune interactions: Cross-talk between mast cells and enteric neurons

肠神经系统（ENS）能够独立于大脑或脊髓调节胃肠道的几乎所有功能。自主神经系统的这一部分与胃肠道壁上的其他细胞有很强的相互作用，如肥大细胞，肥大细胞高密度地位于固有层和粘膜下层，靠近神经细胞。肥大细胞的脱粒诱导释放介质，如组胺、蛋白酶、类二十烷酸和其他刺激粘膜下分泌运动神经元。其结果是诱导上皮阴离子分泌，临床上导致分泌性腹泻。从以前对分离的粘膜下神经元进行的实验中，我们知道这些神经元表达几种类型的组胺受体。这些受体的激活导致膜的去极化和细胞质Ca2+浓度的增加。肥大细胞共培养实验表明，组胺受体在ENS对肥大细胞脱颗粒的反应中起主要作用。该项目的目的是阐明这种神经-免疫相互作用的机制和潜在调节。组胺H1和H2受体，即大鼠粘膜下神经元中功能上占优势的组胺受体亚型，是通过何种信号机制激发这部分肠神经系统的神经元的？全细胞膜片钳记录将阐明在微电极插入期间观察到的组胺引起的膜去极化的离子机制和信号转导途径。由于肥大细胞被认为不仅参与食物过敏，而且还被认为在炎症性肠病中发挥重要作用，因此将在与RBL-2H3细胞（一种永久性肥大细胞等效细胞系）共培养实验中，以及在用促炎细胞因子预处理后的粘膜下固有层制备（研究神经元与粘膜肥大细胞的相互作用）中，研究肥大细胞与粘膜下神经元的相互作用。肥大细胞脱颗粒诱导的神经元内胞质Ca2+浓度的变化可作为这些实验的功能读出。在对卵清蛋白致敏的动物制备的制剂中，当动物预先用三硝苯磺酸（TNBS）处理时，将测试在Ussing室中暴露抗原诱导的体外离子转运或细胞旁通透性的变化是否会改变，即结肠炎是否会加重肠道中的过敏反应。此外，肥大细胞与肠神经元之间的串扰是否如文献所述受胆碱能受体调节，还有待进一步研究。

The enteric nervous system (ENS) is able to regulate nearly all functions of the gastrointestinal tract independent from the brain or the spinal cord. There is a strong interaction of this part of the autonomous nervous system with other cells in the gastrointestinal wall such as e.g. mast cells, which are located in high densitiy within the lamina propria and the submucosa in close vicinitiy to nerve cells. A degranulation of mast cells induces the release of mediators like histamine, proteases, eicosanoids and others stimulating secretomotor submucosal neurons. The consequence is the induction of epithelial anion secretion, clinically leading to secretory diarrhea. From previous experiments performed at isolated submucosal neurons it is known that these neurons express several types of histamine receptors. Activation of these receptors leads to depolarization of the membrane and an increase in the cytosolic Ca2+ concentration. Coculture experiments with mast cells show that histamine receptors are centrally involved in the response of the ENS to mast cell degranulation.The aim of the project is to elucidate the mechanisms and the potential modulation of this neuro-immune-interaction. It shall be clarified by which signaling mechanisms histamine H1 and H2, receptors, i.e. the functionally predominant histamine receptor subtypes of rat submucosal neurons, excite neurons in this part of the enteric nervous system. Whole-cell patch-clamp recordings shall elucidate the ionic mechanisms and the signal transduction pathways underlying the membrane depolarization evoked by histamine observed during microelectrode impalements. As mast cells are assumed not only to be involved in food allergy, but are also thought to play a significant role e.g. for inflammatory bowel disease, the cross-talk of mast cells with submucosal neurons will be studied in coculture experiments with RBL-2H3 cells (a permanent mast cell-equivalent cell line) as well as in submucosa-lamina propria-preparations (to study neuronal interaction with mucosal mast cells) after pretreatment with proinflammatory cytokins. Changes in the cytosolic Ca2+ concentration within the neurons induced by mast cell degranulation serves as functional read-out for these experiments. At preparations from animals which have been sensitized against ovalbumin it will be tested whether changes in ion transport or paracellular permeability induced ex vivo by antigen exposure in Ussing chambers is altered, when the animals have been pretreaded with TNBS (trinitrobenzenesulfonic acid), i.e. whether a colitis aggravates allergic responses in the gut. Furthermore, it shall be investigated whether the cross-talk between mast cells and enteric neurons is modulated by cholinergic receptors as suggested by data from the literature.