Physiological vs. pharmacological effects of glucocorticoids on human monocytes.

描述（由申请人提供）：药理学糖皮质激素（GC）已有效用于治疗炎症超过半个世纪，导致GC作用的既定范例仅涉及导致抑制炎症和免疫的机制。相反，较少认识到的是，生理应激相关浓度的GC-但不是更高的药理学水平-在免疫和炎症反应的激活中发挥重要的增强作用。因此，我们提出了一项试点人体研究，以获得将支持一个新的范式的数据，一个解决机制，通过该机制，皮质醇在体内短暂增加到压力相关的水平（例如，创伤后的水平）准备在随后的激活事件发生时增强的先天免疫应答（例如，感染）。我们的研究旨在检验这一假设，即在人单核细胞和巨噬细胞中生理浓度的皮质醇诱导的制备表型和分子变化，导致与药理学GC剂量相比功能上相反的后果。我们的第二个目标，确定机制的个人特异性差异反应的“压力-皮质醇”与“药物-皮质醇”，如果成功的话，提供了一个筛选方法，具有深远的利益，几乎所有的途径的生物医学，行为和临床研究。具体而言，我们建议：1.识别区分应激-皮质醇增强与药物-皮质醇抑制人类单核细胞/巨噬细胞先天免疫激活途径的特定细胞和分子变化。我们已经发现，应激-皮质醇预处理可重复地增强LPS诱导的IL-6的产生，由人单核细胞衍生的巨噬细胞从一些受试者（响应者），但不是其他人（无响应者）。个体间差异和应激与药物皮质醇的差异效应将用于通过流式细胞术、基因谱分析和ELISA测定来区分导致应激皮质醇增强细胞因子产生的最相关分子事件。我们将特别询问应激-皮质醇增强LPS诱导的MAP激酶磷酸化增强人巨噬细胞先天免疫激活的机制。2.识别，在分子水平上，具体的单核细胞反应，差异调节应激皮质醇和pharm-cortisol在体内。每例受试者将作为他/她自己的对照，并将接受3次体内治疗;生理盐水、应激-皮质醇（40 mg/70 kg/6小时）或药物-皮质醇（400 mg/70 kg/6小时），治疗间间隔将测量值恢复至基线。将使用流式细胞术、Taqman真实的时间PCR和ELISA测定来确定应激-皮质醇与药物-皮质醇诱导的先天免疫炎症的关键细胞调节物的变化的体内发作、幅度和持久性。我们计划确定特定的分子事件，预测不同的体内应激皮质醇与药物皮质醇的影响，或个体受试者之间的先天免疫激活的差异。我们的研究结果将指导未来的研究，以确定GC的抗炎和促炎调节作用之间的重要临床区别。 公共卫生相关性：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超过半个世纪，但其在不同人群中的有效性存在无法解释的差异，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，以促进治疗急性全身性炎症的新治疗干预措施。所提出的研究是新颖的，据我们所知，我们将是第一个研究机制，生理应激相关水平的氢化可的松增强免疫和炎症途径内的人单核细胞白色血细胞在体内。

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.