REGULATION OF ALVEOLAR MACROPHAGE FUNCTION IN ASBESTOSIS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 从石棉肺患者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。此外，石棉肺患者的肺泡巨噬细胞会自发释放细胞因子。该方案的重点是确定暴露于石棉的巨噬细胞产生细胞因子的必要机制，并探索正常肺泡巨噬细胞与石棉肺患者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，与单核细胞不同，正常的肺泡巨噬细胞在体外受到石棉刺激时不会产生细胞因子。这些数据还表明，接触石棉后，正常肺泡巨噬细胞比单核细胞产生更多的活性氧(ROS)。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)的激活与巨噬细胞中细胞因子基因的表达有关，而ROS对MAP激酶的激活具有差异性。这些研究人员假设，石棉不会导致正常肺泡巨噬细胞中细胞因子基因的表达，这是因为缺乏p38 MAP激酶的激活。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p38MAPK活性的差异。在目标1中，他们将确定石棉诱导的ROS，特别是正常肺泡巨噬细胞产生的羟基自由基是否通过抑制激活p38的上游激酶和/或激活磷酸酶来灭活p38蛋白激酶。具体地说，石棉的新贡献将是比较肺泡巨噬细胞和血单核细胞中ROS的生成；确定石棉诱导的ROS是否下调肺泡巨噬细胞中的p38蛋白激酶；以及确定石棉是否诱导使p38蛋白激酶失活的磷酸酶的活性。在目标2中，他们将通过评估TBP与NF-kB和AP-1蛋白的相互作用，确定石棉是否与单核细胞相比，增加了正常巨噬细胞中由NF-kB和AP-1驱动的转录。他们还将比较TBP在肺泡巨噬细胞和血单核细胞中的磷酸化和与TATA盒的结合。在目标3中，他们将确定p38蛋白激酶的重新激活是否导致正常巨噬细胞在这些条件下表达细胞因子基因的能力。他们还计划评估从石棉肺患者获得的巨噬细胞中p38蛋白激酶的活性和ROS的产生。虽然该方案中的研究与石棉肺有关，但它们也是为了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础线索。

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.