REGULATION OF ALVEOLAR MACROPHAGE FUNCTION IN ASBESTOSIS

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 从石棉肺患者的肺中获得的肺泡巨噬细胞的一个重要特征是它们类似于单核细胞样细胞。 此外，从石棉肺患者获得的肺泡巨噬细胞自发释放细胞因子。 本方案的重点是确定引起暴露于石棉的巨噬细胞产生细胞因子所需的机制，并探讨正常肺泡巨噬细胞和从石棉肺患者中获得的单核细胞或单核细胞样细胞之间的功能差异。 初步数据表明，正常肺泡巨噬细胞，不像血液单核细胞，不产生细胞因子时，在体外刺激，石棉。 这些数据还表明，正常肺泡巨噬细胞产生更多的活性氧（ROS）接触石棉后比血液单核细胞。 研究表明，p38丝裂原活化蛋白（MAP）激酶通过调节TATA结合蛋白（TBP）的活化与巨噬细胞中细胞因子基因的表达相关，而ROS则不同程度地激活MAP激酶。 这些研究者假设，由于缺乏p38 MAP激酶激活，石棉不会引起正常肺泡巨噬细胞中细胞因子基因的表达。 因此，正常肺泡巨噬细胞和单核细胞样细胞之间的功能差异部分是由于p38 MAP激酶活性的差异。 在目标1中，他们将确定正常肺泡巨噬细胞中产生的石棉诱导的ROS，特别是羟基自由基，是否通过抑制激活p38的上游激酶和/或激活磷酸酶来抑制p38 MAP激酶。 具体而言，石棉的新贡献将是比较肺泡巨噬细胞和血液单核细胞中ROS的产生;确定石棉诱导的ROS是否下调肺泡巨噬细胞中的p38 MAP激酶;并确定石棉是否诱导磷酸酶的活性，磷酸酶抑制p38 MAP激酶。 在目标2中，他们将通过评估TBP与NF-κ B和AP-1蛋白的相互作用，确定石棉是否增加正常巨噬细胞中NF-κ B和AP-1驱动的转录。 他们还将比较肺泡巨噬细胞和血液单核细胞中TBP磷酸化和TATA盒结合。 在目标3中，他们将确定在这些条件下，p38 MAP激酶的再活化是否导致正常巨噬细胞表达细胞因子基因的能力。 他们还计划评估从石棉肺患者中获得的巨噬细胞中p38 MAP激酶活性和ROS生成。 虽然本方案中的研究与石棉肺有关，但它们是新颖的研究，也为理解巨噬细胞细胞因子基因调控以及肺泡巨噬细胞和单核细胞样细胞之间的功能差异提供了重要的基本线索。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. One important characteristic of alveolar macrophages obtained from the lungs of asbestosis patients is they resemble monocyte-like cells. In addition, alveolar macrophages obtained from patients with asbestosis spontaneously release cytokines. The focus of this protocol is to determine the mechanisms necessary to elicit cytokine production by macrophages exposed to asbestos and to explore the functional differences between normal alveolar macrophages and monocytes or monocyte-like cells obtained from patients with asbestosis. Preliminary data indicate that normal alveolar macrophages, unlike blood monocytes, do not produce cytokines when stimulated, in vitro, with asbestos. These data also show that normal alveolar macrophages produce more reactive oxygen species (ROS) after exposure to asbestos than do blood monocytes. Studies have demonstrated that the p38 mitogen-activated protein (MAP) kinase is linked to cytokine gene expression in macrophages through its modulation of TATA-binding protein (TBP) activation, and ROS differentially activate MAP kinases. These investigators hypothesize that asbestos does not cause cytokine gene expression in normal alveolar macrophages due to lack of p38 MAP kinase activation. Thus, the functional difference between normal alveolar macrophages and monocyte-like cells is, in part, due to differences in p38 MAP kinase activity. In Aim 1, they will determine if asbestos-induced ROS, especially hydroxyl radical, produced in normal alveolar macrophages inactivate the p38 MAP kinase by inhibiting the upstream kinases that activate p38 and/or by activating phosphatases. Specifically, the new contributions for asbestos will be to compare the generation of ROS in alveolar macrophages and blood monocytes; to determine if asbestos-induced ROS down-regulates the p38 MAP kinase in alveolar macrophages; and to determine if asbestos induces the activity of phosphatases that inactivate the p38 MAP kinase. In Aim 2 they will determine if asbestos increases NF-kB- and AP-1-driven transcription in normal macrophages compared to monocytes by evaluating the interaction of TBP with NF-kB and AP-1 proteins. They will also compare TBP phosphorylation and binding to the TATA box in alveolar macrophages and blood monocytes. In Aim 3 they will determine if reactivation of p38 MAP kinase results in the ability of normal macrophages, under these conditions, to express cytokine genes. They also plan to evaluate p38 MAP kinase activity and ROS generation in macrophages obtained from patients with asbestosis. Although the studies in this protocol relate to asbestosis, they are novel studies that also provide important basic clues to understand macrophage cytokine gene regulation and the functional difference between alveolar macrophages and monocyte-like cells.