Physiological vs. pharmacological effects of glucocorticoids on human monocytes.

描述(申请人提供)：半个多世纪以来，药理糖皮质激素(GC)一直被有效地用于治疗炎症，导致了GC作用的既定范例，仅针对导致抑制炎症和免疫的机制。相比之下，较少被认识到的事实是，与生理应激相关的GC浓度--但不是更高的药理水平--在免疫和炎症反应的激活中发挥着重要的增强作用。因此，我们建议进行一项试验性的人体研究，以获得支持一种新范式的数据，该范式解决了体内皮质醇瞬时增加到应激相关水平(例如，创伤后的水平)为随后的激活事件发生(例如，感染)时增强的先天免疫反应做准备的机制。我们的研究旨在检验这样一个假设，即生理浓度的皮质醇在人单核细胞和巨噬细胞中诱导的制备性表型和分子变化，导致与药物GC剂量相反的功能后果。我们的次要目标是确定个体对“压力-皮质醇”和“药用皮质醇”的不同反应机制，如果成功，将为生物医学、行为和临床研究的几乎所有途径提供一种具有深远好处的筛查方法。具体地说，我们建议：1.确定区分应激-皮质醇增强和药物-皮质醇抑制人单核/巨噬细胞天然免疫激活途径的特定细胞和分子变化。我们发现，应激-皮质醇预处理可重复地增强来自某些受试者(应答者)的人单核细胞来源的巨噬细胞产生的IL-6，但不能增加其他受试者(无应答者)的IL-6的产生。应激与药物皮质醇的个体差异和差异效应将被用来通过流式细胞术、基因图谱分析和ELISA法分析导致应激-皮质醇增加细胞因子产生的最相关的分子事件来区分。我们将具体询问应激-皮质醇增强脂多糖诱导的MAP激酶磷酸化增强人巨噬细胞天然免疫激活的机制。2.在分子水平上鉴定体内受应激-皮质醇和药用皮质醇差异调节的特异性单核细胞反应。每个受试者将作为他/她自己的对照，并将接受3种活体治疗：生理盐水、应激-皮质醇(40 mg/70 kg/6小时)或药用皮质醇(400 mg/70 kg/6小时)，治疗间隔时间将测量结果恢复到基线。流式细胞术、Taqman实时聚合酶链式反应和酶联免疫吸附试验将被用来确定应激皮质醇和药用皮质醇诱导的先天性免疫炎症的关键细胞调节因子在体内的起始、幅度和持久性。我们计划确定特定的分子事件，以预测应激皮质醇与药用皮质醇在体内的不同影响，或个体之间先天免疫激活的差异。我们的结果将指导未来的研究，以确定GCs抗炎和促炎调节作用之间的重要临床差异。 与公共卫生相关：半个多世纪以来，与天然肾上腺激素氢化可的松相关的类固醇已被用于有效治疗炎症和自身免疫，但其在不同人身上的有效性存在不明原因的差异，这是一个令人烦恼的临床挑战。需要更好地了解这些类固醇调节免疫和炎症介质的机制，以促进治疗急性系统性炎症的新的治疗干预措施。提出的研究是新颖的，因为据我们所知，我们将第一次检查生理应激相关水平的氢化可的松在体内增强人类单核细胞内的免疫和炎症途径的机制。

DESCRIPTION (provided by applicant): Pharmacological glucocorticoids (GCs) have been effectively used to treat inflammation for over a half century, resulting in the established paradigm of GC action that addresses only mechanisms that lead to suppression of inflammation and immunity. In contrast, and less appreciated, is the fact that physiological stress-associated concentrations of GCs-but not higher pharmacological levels-play an essential enhancing role in the activation of immune and inflammatory responses. We therefore propose a pilot human study to obtain data that will support a new paradigm, one addressing the mechanisms by which a transient in vivo increase in cortisol to stress-associated levels (e.g., levels that follow trauma) prepares for an enhanced innate immune response when a subsequent activation event occurs (e.g., an infection). Our study is designed to test the hypothesis that preparative phenotypic and molecular changes induced by physiological concentrations of cortisol in human monocytes and macrophages, lead to functionally opposing consequences compared with pharmacological GC doses. Our secondary aim, identifying mechanisms for individual-specific differential responses to 'stress- cortisol' versus 'pharm-cortisol' would, if successful, provide a screening approach with far reaching benefits for nearly all avenues of biomedical, behavioral, and clinical research. Specifically, we propose to: 1. Identify specific cellular and molecular changes that distinguish stress-cortisol enhancement from pharm- cortisol suppression of human monocyte/macrophage innate immune activation pathways. We have found that stress-cortisol pretreatment reproducibly enhances LPS-induced IL-6 production by human monocyte-derived macrophages from some subjects (responders) but not others (non-responders). Inter-individual differences and differential effects of stress- vs. pharm-cortisol will be used to discriminate by flow cytometry, gene profiling analysis and ELISA assays the most relevant molecular events that lead to stress-cortisol enhanced cytokine production. We will specifically interrogate mechanisms by which stress-cortisol enhancement of LPS- induced MAP kinase phosphorylation augments innate immune activation in human macrophages. 2. Identify, at the molecular level, specific monocyte responses that are differentially regulated by stress- cortisol and pharm-cortisol in vivo. Each subject will serve as his/her own control, and will receive 3 in vivo treatments; saline, stress-cortisol (40mg/70kg/6hours), or pharm-cortisol (400mg/70kg/6hours) with an inter- treatment interval that returns measurements to baseline. Flow cytometry, Taqman real time PCR and ELISA assays will be used to determine the in vivo onset, magnitude and durability of stress-cortisol versus pharm- cortisol-induced changes in key cellular regulators of innate immune inflammation. We plan to identify specific molecular events that predict differential in vivo effects of stress-cortisol versus pharm-cortisol, or differences in innate immune activation among individual subjects. Our results will direct future studies to determine important clinical distinctions between anti-inflammatory and pro-inflammatory regulatory effects of GCs. PUBLIC HEALTH RELEVANCE: Steroids related to the natural adrenal hormone hydrocortisone have been used to effectively treat inflammation and autoimmunity for over a half century, but unexplained differences in their effectiveness in different people presents a vexing clinical challenge. Improved understanding of the mechanisms by which these steroids regulate mediators of immunity and inflammation is needed to foster new therapeutic interventions for the treatment of acute systemic inflammation. The studies proposed are novel in that, to the best of our knowledge, we will be the first to examine mechanisms by which physiological stress-associated levels of hydrocortisone enhance immune and inflammatory pathways within human monocytic white blood cells in vivo.