Glucocorticoid/cytokine mechanisms in endotoxemia.

描述(由申请人提供)：本提案中要检验的假设是，糖皮质激素(GC)对脂多糖(LPS)诱导的单核/巨噬细胞(MOMAC)激活程序的关键成分的控制具有双相效应。PI和Co-I提出了一个模型，以协调中等皮质醇水平的刺激(“允许”)作用和高皮质醇水平的抑制作用。该模型预测皮质醇对炎症介质的影响呈钟形曲线，达到或接近皮质醇昼夜变化的最高水平。由于GC的允许作用比抑制作用了解得更少，它们提供了我们建议的关于Momac激活的介体和调节因子的研究重点。其具体目的是：(1)验证GC对细胞因子的产生和Momac表型的双相影响的假说，以及(2)阐明GC对Momac激活程序的允许和抑制的分子机制。我们建议使用体外和人体体内系统来解决这些问题。最初，我们将使用淋洗出的MO的培养，这些培养将在皮质醇保持在预计低于允许、允许或抑制的水平的条件下暴露于脂多糖。我们将使用ELISA和流式细胞仪分析来确定皮质醇和内毒素对MOMAC激活计划的两个关键成分--IL-6和CD163--的交互作用。在首次被确定为最大限度地增强或抑制CD163和IL-6的剂量和时间内，我们将使用脂多糖和皮质醇，我们将检测已证明由脂多糖诱导或抑制的其他细胞因子、信号分子和受体。我们还将使用受控良好的人类实验性内毒素血症范例来阐明在体内对内毒素的反应中，GC抑制或增强可能的促炎和抗炎分子的产生和作用的生理和分子机制。我们相信，这些研究将对GC增强和抑制免疫和炎症功能的机制产生重要的见解，导致更有效的方法将它们用于临床炎症环境。

DESCRIPTION (provided by applicant): The hypothesis to be tested in this proposal is that glucocorticoids (GC) exert a biphasic effect on the control of key components of the lipopolysaccharride (LPS)-induced monocyte/ macrophage (MOMac) activation program. A model was proposed by the PI and Co-I to reconcile the stimulatory ("permissive") actions of moderate cortisol levels with the suppressive actions of high cortisol levels. This model predicts a bell-shaped curve for the effects of cortisol on inflammatory mediators that peaks at or near the upper levels of diurnal cortisol variation. As the permissive effects of GC are less well understood than the inhibitory actions, they provide the focus of our proposed studies on mediators and regulators of MOMac activation. The Specific Aims are: (1) To test the hypothesis that GC exert a biphasic influence on cytokine production and MOMac phenotype in response to LPS, and (2) To elucidate the molecular mechanisms of permissive and suppressive effects of GC on the MOMac activation program. We propose to address these questions employing both in vitro and human in vivo systems. Initially, we will use cultures of elutriated MO that will be exposed to LPS under conditions in which cortisol is maintained at levels predicted to be sub-permissive, permissive or suppressive. We will use ELISA and flow cytometric analyses to determine the interactive effects of cortisol and LPS on two key components of the MOMac activation program--IL-6 and CD163. Using LPS and cortisol at dosages and times that are first determined to result in maximal enhancement or inhibition of CD 163 and IL-6, we will examine additional cytokines, signaling molecules and receptors that have been shown to be induced or suppressed by LPS. We will also use a well-controlled paradigm of human experimental endotoxemia to elucidate the physiologic and molecular mechanisms that lead GC to either suppress or enhance the production and actions of putative pro- and anti-inflammatory molecules during the response to endotoxin in vivo. We believe that these studies will yield important insights into the mechanisms by which GC can both enhance and suppress immune and inflammatory functions, leading to more effective approaches to their use in clinical settings of inflammation.