PROCESSING OF ENDOTOXINS BY LIVER MACROPHAGES

由于革兰氏阴性菌引起的感染是主要的临床问题， 循环中的细菌内毒素会导致危及生命的全身性 反应.内毒素也参与了许多肿瘤的病理学过程。 肝脏疾病包括肝硬化。肝脏是主要的部位 肠源性内毒素的代谢。细胞负责 从门静脉血中清除内毒素的是肝脏巨噬细胞 （枯否细胞），并与肝细胞合作解毒。 由于持续长期暴露于低水平的内毒素， 枯否细胞对内毒素的反应与其他巨噬细胞不同， 单核细胞和内毒素摄取和反应的分子细节， 枯否细胞不清楚。为了研究这一点，我们将：1）表征 内毒素与枯否细胞结合所必需的结构 细胞我们已经鉴定了两种新的内毒素结合蛋白31和 大鼠枯否细胞上的34 kD，是内部蛋白质， 参与细胞因子应答信号的转导。 设计实验以纯化这些蛋白质并测定其氨基 酸性序列我们将生产针对这31种病毒的单克隆抗体， 使用多抗原肽（MAP）的34 kD结合蛋白 技术.这些抗体将用于研究功能性 这些结合蛋白的意义和分布。2)我们将 鉴定、克隆和测序编码31和34 kD的基因 使用肽序列合成内毒素结合蛋白 特异性寡核苷酸探针。序列数据将用于 创建结合蛋白的潜在结构域模型， 它们与其他蛋白质的结构关系。克隆的基因将 也可用于转染到真核细胞中， 模型来研究这些新的LPS结合蛋白的生物学作用。 3)我们将确定31和34 kD结合蛋白在 与LPS与枯否细胞相互作用相关的信号传导事件， Kupffer细胞对LPS的反应分泌细胞因子。到 检查Mn/++和其他过渡金属的保护作用 抗内毒素和它们对LPS结合31和34的抑制 kD蛋白。这些研究将增加我们对 内毒素的生物学效应，并导致新的治疗方法， 中毒性休克

Infection due to gram negative bacteria is a major clinical problem and bacterial endotoxins in the circulation cause life threatening systemic reactions. Endotoxin is also involved in the pathology of a number of liver diseases including cirrhosis. The liver is the major site of metabolism for gut derived endotoxins. The cells responsible for endotoxin clearance from the portal blood is the liver macrophage (Kupffer cell) and co-operates with hepatocytes in detoxification. Because of continuous chronic exposure to low levels of endotoxin the Kupffer cell response to endotoxin is different to other macrophages and monocytes and the molecular details of endotoxin uptake and response in Kupffer cells in unclear. To investigate this we will: 1) Characterize the structures necessary for the binding of endotoxin to the Kupffer cells. We have identified two novel endotoxin binding proteins of 31 and 34 kD on rat Kupffer cells that are internal proteins that appear to be involved in transducing the signal for the cytokine response. Experiments are designed to purify these proteins and to determine amino acid sequences. We will produce monoclonal antibodies to both the 31 and 34 kD binding proteins using multiple antigenic peptide (MAP) technology. These antibodies will be used to study the functional significance and distribution of these binding proteins. 2) We will identify and clone and sequence the genes encoding the 31 and 34 kD endotoxin binding proteins using peptide sequences to synthesize specific oligonucleotide probes. The sequence data will be used to create a potential domain model for the binding proteins and to examine their structural relationships to other proteins. The cloned genes will also be available for transfection into eukaryotic cells and provide a model to study the biological role of these novel LPS binding proteins. 3) We will determine the role of the 31 and 34 kD binding proteins in signaling events associated with LPS interaction with Kupffer cells and in the secretion of cytokines by Kupffer cells in response to LPS. To examine the protective effects of Mn/++ and other transition metals against endotoxin and their inhibition of LPS binding to the 31 and 34 kD proteins. These studies will increase our understanding of the biological effects of endotoxins and lead to novel therapies against toxic shock.