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

肠道神经系统(ENS)能够独立于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壁中的其他细胞有很强的相互作用，例如肥大细胞，它们高密度地位于固有层和粘膜下层，与神经细胞关系密切。肥大细胞脱颗粒导致组胺、蛋白水解酶、二十烷类化合物等介质的释放，以及其他刺激分泌运动的粘膜下神经元。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胞浆钙离子浓度的增加。与肥大细胞共培养的实验表明，组胺受体在肥大细胞脱颗粒的反应中起中心作用。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的调节。组胺H1和H2受体，即大鼠粘膜下神经元的主要组胺受体亚型，是通过什么信号机制兴奋肠道神经系统这一部分的。全细胞膜片钳记录将阐明微电极穿孔过程中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。由于肥大细胞不仅被认为与食物过敏有关，而且被认为在炎症性肠病中也扮演着重要的角色，因此肥大细胞与粘膜下神经元的相互作用将在与RBL-2H3细胞(一种与肥大细胞相当的永久细胞系)的共培养实验中以及在用促炎症细胞因子预处理后的粘膜固有层准备中(以研究神经元与粘膜肥大细胞的相互作用)中进行研究。肥大细胞脱颗粒引起的神经元内细胞内钙离子浓度的变化可作为这些实验的功能读出。在已经对卵蛋白致敏的动物的制剂中，将测试当动物预先用三硝基苯磺酸(TNBS)处理时，体外由Ussing小室中的抗原暴露引起的离子转运或细胞旁通透性的变化是否发生改变，即结肠炎是否会加剧肠道中的过敏反应。此外，还应研究肥大细胞和肠神经元之间的串扰是否受胆碱能受体的调节，如文献中的数据所建议的那样。

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.