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

背景：经典的阿片受体分为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，即痛觉肽/孤儿蛋白q （N/OFQ）或NOP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，但机制尚不清楚；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 mRNA，但表达N/OFQ mRNA的差异很大。我们对免疫细胞释放N/OFQ知之甚少，对单细胞水平的释放也一无所知。我们有一个中国仓鼠卵巢（CHO）细胞克隆，表达嵌合的α - 1 /q蛋白，使Gi偶联受体增加细胞内钙；这可以用荧光法测量。该细胞系共转染NOP；有效地将受体NOP激活作为钙信号进行监测；用于N/OFQ的生物传感器。如果我们用多形核白细胞覆盖α - 1 /q细胞，并用fMLP刺激它们脱粒，我们就能够首次在单细胞水平上测量N/OFQ释放。该释放物对NOP拮抗剂敏感，而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α - 1 /q生物传感器系统，在单细胞水平上测量N/OFQ；通过N/OFQ激活2-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通路和迁移作为读数。目的和目的：我们将开发和表征一种基于α - 1 /q嵌合体的生物传感器检测方法，用于共聚焦显微镜下表达重组人NOP的细胞。志愿者的血液将被分离成单独的免疫细胞群（单核细胞、B淋巴细胞和T淋巴细胞以及嗜碱性细胞、中性细胞和嗜酸性粒细胞），并评估每种免疫细胞的释放能力。根据初步数据显示不同的ppNoc表达，我们预测在粒细胞群体中，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会释放N/OFQ，我们可以从单个细胞中测量到这一点。我们将评估拮抗剂敏感性以确认NOP为靶点，探索释放过程中的钙依赖性，并通过免疫荧光确认每种细胞的免疫细胞类型。N/OFQ肽的细胞含量也将用特异性N/OFQ抗体免疫荧光法测定。值得注意的是，在Western blotting中没有NOP可靠的抗体。我们将通过使用Western blot检测ERK1/2磷酸化和JNK，来描述肽和非肽激动剂对免疫细胞NOP激活的影响。ERK1/2的激活与细胞凋亡有关，因此我们也将测量细胞色素c和裂解半胱天蛋白酶3，正如我们之前在神经元细胞中所做的那样。由于Rho GTPase与细胞迁移有关，我们将对此进行评估，我们将使用Active Rho下拉和检测试剂盒检查NOP激活对Rho GTPase的影响。我们还将使用Luminex xMAP技术和Human 25-Plex面板，测量细胞群在N/OFQ缺失和存在的情况下对脱颗粒刺激的细胞因子释放谱。我们将使用井外迁移试验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：该项目的数据将提供：(1)测量单细胞释放的新技术，(2)免疫细胞中N/OFQ的第一个详细释放剖面，目前文献中缺乏，(3)在生化和功能水平上释放后果的详细信息。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（例如神经元递质释放）。

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).