Biosensor based approach to measure release of Nociceptin/Orphanin FQ from live single immune cells and consequences for immune cell function.

背景：经典阿片受体分为MOP（mu）、DOP（delta）和KOP（kappa）。除了这三种受体类型之外，还有第四种非经典类型，即伤害感受素/孤啡肽FQ（N/OFQ）或NOP的受体。肽N/OFQ由前体pre-pro-N/OFQ（ppNoc）产生。阿片类药物引起免疫抑制，但机制知之甚少;特别是对于N/OFQ-NOP系统。在这方面，我们迄今为止测试的所有免疫细胞都表达NOP的mRNA，但不表达N/OFQ的mRNA。关于N/OFQ从免疫细胞中的释放知之甚少，在单细胞水平上也一无所知。我们有一个中国人卵巢（CHO）细胞克隆，表达嵌合Galpha-i/q蛋白，使Gi偶联受体能够增加细胞内钙;这可以用荧光法测量。该细胞系与NOP共转染;有效地允许受体NOP活化作为钙信号进行监测; N/OFQ的生物传感器。如果我们用多形核白细胞覆盖Galpha-i/q细胞并用fMLP刺激它们去角质，我们已经能够首次在单细胞水平上测量N/OFQ释放。这种释放对NOP拮抗剂敏感，但对嘌呤能拮抗剂不敏感。我们的应用有两个假设; 1-白细胞差异释放N/OFQ，我们将开发一种新的基于细胞的Galpha-i/q生物传感器系统，以在单细胞水平上测量这一点; 2-通过N/OFQ激活NOP将在细胞生理水平上调节白细胞功能，我们将使用细胞内信号传导途径和迁移作为读数。宗旨和目标：我们将开发和开发基于Galpha-i/q嵌合体的生物传感器测定，用于在共聚焦显微镜中表达重组人NOP的细胞。将志愿者血液分离为单个免疫细胞群（单核细胞、B和T淋巴细胞以及嗜碱性粒细胞、中性粒细胞和嗜酸性粒细胞），并评估每个免疫细胞群的释放能力。基于初步数据显示差异ppNoc表达，我们预测，粒细胞群体，嗜酸性粒细胞将是N/OFQ的来源。此外，我们预测单核细胞和淋巴细胞也将释放N/OFQ，我们可以从单个细胞中测量。我们将评估拮抗剂敏感性以确认NOP为靶点，探索释放过程的钙依赖性，并通过免疫荧光确认每种细胞免疫细胞类型。N/OFQ肽的细胞含量也将通过用特异性N/OFQ抗体的免疫荧光来测量。值得注意的是，没有NOP可靠的抗体可用于Western印迹。我们将通过使用Western印迹测量ERK 1/2磷酸化和JNK来验证肽和非肽激动剂对免疫细胞NOP活化的影响。ERK 1/2的激活与细胞凋亡有关，因此我们还将测量细胞色素c和切割的半胱天冬酶3，就像我们以前在神经元细胞中所做的那样。由于Rho GTPases与细胞迁移相关，我们将对此进行评估，我们将使用Active RHO pull down和检测试剂盒检查NOP激活对Rho GTPases的影响。我们还将使用Luminex xMAP技术和人25-TNF板测量在不存在和存在N/OFQ的情况下响应于脱粒刺激的细胞群的细胞因子释放谱。我们将使用transwell迁移分析来评估自分泌和旁分泌对迁移的控制以及NOP激活的影响。应用/益处：本项目的数据将提供：（1）测量单细胞释放的新技术，（2）N/OFQ从免疫细胞的第一个详细释放曲线，目前缺乏的文献和（3）在生物化学和功能水平的释放的后果的详细信息。此外，我们相信这些实验技术和数据将适用于其他细胞和系统（例如神经递质释放）。

Context: Classical opioid receptors are classified as MOP(mu), DOP(delta) and KOP(kappa). In addition to these three receptor types there is a fourth non-classical type, the receptor for nociceptin/orphaninFQ (N/OFQ) or NOP. The peptide N/OFQ is produced from a precursor pre-pro-N/OFQ (ppNoc). Opioids cause immune suppression but the mechanisms are poorly understood; in particular for the N/OFQ-NOP system. In this regard all immune cells we have tested to date express mRNA for NOP but variably express mRNA for N/OFQ. Little is known about the release of N/OFQ from immune cells and nothing at the single cell level. We have a Chinese Hamster Ovary (CHO) cell clone that expresses a chimeric Galpha-i/q protein enabling Gi coupled receptors to increases in intracellular Calcium; this can be measured fluorimetrically. This cell line is co-transfected with NOP; effectively allowing receptor NOP activation to be monitored as a Calcium signal; a biosensor for N/OFQ. If we overlay Galpha-i/q cells with polymorphonuclear leucocytes and stimulate them to degranulate with fMLP we have been able to measure N/OFQ release at the single cell level for the first time. This release was sensitive to NOP antagonists but not purinergic antagonists. There are two hypotheses to our application; 1-Leucocytes differentially release N/OFQ and we will develop a novel cell based Galpha-i/q biosensor system to measure this at the single cell level; 2-NOP activation by N/OFQ will modulate leucocyte function at the cellular physiological level and we will use the intracellular signalling pathways and migration as readouts. Aims and objectives: We will develop and characterise a Galpha-i/q chimera based biosensor assay for use with cells expressing recombinant human NOP in confocal microscopy. Volunteer human blood will be separated into individual immune cell populations (monocytes, B and T lymphoctyes and basophil, neutrophil and eosinophil granulocytes) with each assessed for release capability. Based on preliminary data showing differential ppNoc expression we predict that of the granulocyte population, eosinophils will be the source of N/OFQ. In addition we predict that monocytes and lymphocytes will also release N/OFQ and that we can measure this from single cells. We will assess antagonist sensitivity to confirm NOP as the target, explore Calcium dependence of the release process and confirm each cell immune cell type by immunofluoresence. Cellular content of N/OFQ peptide will also be measured by immunofluoresence with specific N/OFQ antibodies. Of note, there are no NOP reliable antibodies for use in Western blotting.We will characterise the effects of immune cell NOP activation with peptide and non-peptide agonists by measuring ERK1/2 phosphorylation and JNK using Western blot. Activation of ERK1/2 has been linked to apoptosis so we will also measure cytochrome c and cleaved caspase 3 as we have done previously in neuronal cells. As Rho GTPases are linked with cell migration and we will be assessing this we will examine the effects of NOP activation on Rho GTPase using the Active RHO pull down and detection kit. We will also measure the cytokine release profile from cell populations in response to degranulation stimuli in the absence and presence of N/OFQ using Luminex xMAP technology and the Human 25-Plex panel. We will use transwell migration assays to assess autocrine and paracrine control of migration and the effects of NOP activation.Applications/Benefits: Data from this project will provide: (1) a novel technique for measuring single-cell release, (2) the first detailed release profile of N/OFQ from immune cells, currently lacking in the literature and (3) detailed information of the consequences of that release at a biochemical and functional level. Furthermore, we believe that these experimental techniques and data will have applicability to other cells and systems (e.g. neuronal transmitter release).