Immunomodulatory mechanism of volatile anesthetics

说明书（由申请人提供）：挥发性麻醉药是围手术期全身麻醉的主要药物。虽然主要目的是提供镇痛，催眠和静止，但它们被认为具有免疫调节作用。挥发性麻醉药对免疫功能的改变具有重要的临床意义，特别是对危重病人。候选人的长期目标是1)阐明各种临床使用的麻醉药的分子机制和免疫调节作用的程度，从而为临床医生在患者潜在疾病的背景下合理使用一种麻醉剂而不是其他麻醉剂提供依据；2)重新设计不含免疫调节的新麻醉药。我们将重点放在两个粘附分子L2和M2上，它们只在白细胞上表达，在白细胞对内皮的阻滞、吞噬、免疫突触的形成、自然杀伤细胞的细胞毒性等方面发挥作用。在我们的初步实验中，我们发现异氟醚对L2和M2具有功能性抑制作用。此外，七氟醚抑制L2，而非M2。我们假设挥发性麻醉剂与这些整合素结合并改变其功能，从而调节免疫功能。在这个提议中，我们将研究挥发性麻醉剂如何在结构上与这些分子相互作用。我们提出的研究包括三个目标，并将在免疫疾病研究所（波士顿儿童医院细胞和分子医学项目）进行。在Aim1和Aim 2中，我们将确定临床使用的麻醉药与L2和M2及其结合位点的相互作用。挥发性麻醉剂对配体结合的影响将在无细胞和基于细胞的实验中使用流式细胞术和v底实验进行评估。结合参数用等温滴定量热法测定。将结合X射线晶体学和光激活麻醉剂评估麻醉结合部位。在Aim3中，我们将研究挥发性麻醉剂对这些粘附分子功能的损害所产生的生物学效应。为此，我们将使用自然杀伤细胞细胞毒性试验、Fc受体介导的中性粒细胞细胞毒性试验和吞噬试验。随后，我们的实验结果将提供深入了解挥发性麻醉药诱导免疫调节的潜在机制。

DESCRIPTION (provided by applicant): Volatile anesthetics are major drugs to provide general anesthesia during perioperative period. While the primary purpose is to provide analgesia, hypnosis and immobility, they are suggested to possess immunomodulatory effects. Alternation of immune function by volatile anesthetics can be clinically significant particularly fr critical-ill patients. Candidate's long-term goal is 1) to elucidate the molecular mechanism and degree of immunomodulatory effects by various, clinically-used anesthetics, thereby providing clinicians the rational to use one anesthetic over others in the context of patient's underlying diseases, and 2) to redesign new anesthetics without immunomodulation. We focused our target on two adhesion molecules L2 and M2 that are expressed exclusively on leukocytes, playing a role in leukocyte arrest on the endothelium, phagocytosis, immunological synapse formation, natural killer cells cytotoxicity, and so on. In our preliminary experiments, we showed that L2 and M2 are functionally inhibited by isoflurane. Also L2, not M2 was inhibited by sevoflurane. We hypothesize that volatile anesthetics bind to these integrins and modify their functions, thereby modulating immune function. In this proposal, we will investigate how volatile anesthetics interact with these molecules structurally. Our proposed research consists of three Aims, and will be performed in Immune Disease Institute (Program in Cellular and Molecular Medicine in Children's Hospital Boston). In Aim1 and Aim 2, we will determine the interaction of clinically used anesthetics with L2 and M2 and their binding site(s). The effects of volatile anesthetics on ligand binding will be assessed in cell-free and cell-based assays using flow cytometery and V-bottom assay. The binding parameters will be measured with isothermal titration calorimetry. Anesthetic binding site(s) will be assessed in the combination of X ray crystallography and photoactivatable anesthetics. In Aim3, we will examine biological effects by volatile anesthetics resulting from the impairment of these adhesion molecules' function. We will use natural killer cells cytotoxicity assay, Fc receptor mediated neutrophil cytotoxicity and phagocytosis assay for this purpose. Subsequently, our experimental results will provide insight into the underlying mechanism of volatile anesthetics-induced immunomodulation.