NEUTROPHIL LOCALIZATION TO PULMONARY CAPILLARIES IN ARDS

ARDS 中中性粒细胞定位于肺毛细血管

• 批准号: 6242000
• 负责人: G S WORTHEN
• 金额: $ 18.45万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-12-01 至 1997-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6242000
• 关键词: G protein; adult respiratory distress syndrome; capillary; cell adhesion; gene expression; genetic promoter element; human tissue; interleukin 1; interleukin 8; lipopolysaccharides; lung injury; mitogen activated protein kinase; neutrophil; northern blottings; protein kinase C; protein tyrosine kinase; pulmonary circulation; receptor; transcription factor

Neutrophil localization to the lung to the pulmonary capillary and migration in the lung parenchyma are central events in the genesis of the adult respiratory distress syndrome (ARDS). In the previous grant period, we have promulgated the hypothesis that the viscoelastic properties of neutrophils in response to lipopolysaccharide (LPS) and other stimuli circulating in patients with ARDS accounts, at least in part, for the neutrophil localization in the pulmonary capillary. We now hypothesize that LPS and LPS-binding protein (LBP) interact with CD14, a glycosyl-phosphatidyl inositol linked surface glycoprotein results in activation of a src-family cytoplasmic tyrosine kinase, which results in the activation of MAP kinase and cytoskeletal assembly through involvement of the rho family of small molecular weigh GTP-binding proteins. In the endothelial cell, we postulate a similar sequence of events (although the transduction) mechanisms more clearly involve protein kinase C as an important pathway) which leads to the induction of IL-8 gene expression through the involvement of the NF-kB class of transcriptional regulators. These hypotheses will be tested in human neutrophils and human endothelial cells in vitro. The tyrosine kinases involved in neutrophil activation will be sought through a co- immunoprecipitation approach as will the identity of a putative transmembrane transducer which is hypothesized to interact with src-type tyrosine kinases. The potential role of other phosphorylated products will be linked to activation of low molecular weight G proteins by expression of rho A and other members of this family in Sf9 cells and their addition or microinjection into neutrophils and neutrophil-like cell lines. The transduction mechanisms involving activation both tyrosine kinases and PKC will be characterized in endothelial cells as will the presence of the putative transmembrane transducer. The IL-8 promoter region will be cloned and reporter gene studies carried out. Finally, DNAse I hypersensitivity will be used to confirm the precise sites of protein-DNA interaction in induction of IL-8 gene expression. These studies will elucidate mechanisms by which LPS induces cytoskeletal assembly in neutrophils and new gene expression in endothelial cells, which we suggest contribute to lung injury induced by sepsis in humans.

神经元定位于肺至肺毛细血管， 在肺实质中的迁移是肺动脉高压发生的中心事件。 成人呼吸窘迫综合征（ARDS）。 在上一次赠款中， 期间，我们公布了粘弹剂的假设 中性粒细胞对脂多糖（LPS）的反应特性， 在ARDS患者中循环的其他刺激因素，至少在 肺毛细血管内中性粒细胞定位。 我们现在 假设LPS和LPS结合蛋白（LBP）与CD 14相互作用， 糖基-磷脂酰肌醇连接的表面糖蛋白导致 src家族细胞质酪氨酸激酶的激活，这导致 通过MAP激酶的激活和细胞骨架组装 小分子量GTP结合的rho家族的参与 proteins. 在内皮细胞中，我们假设类似的序列， 事件（虽然转导）机制更清楚地涉及 蛋白激酶C作为一个重要的途径），导致诱导 IL-8基因的表达，通过参与NF-κ B类， 转录调节因子。 这些假设将在人类中进行测试。 中性粒细胞和人内皮细胞。 酪氨酸激酶 参与中性粒细胞活化的将通过共同的， 免疫沉淀方法，将身份的一个假定的 跨膜转导物，其被假设为与SRC型相互作用， 酪氨酸激酶 其他磷酸化产物的潜在作用 将与低分子量G蛋白的激活有关， rho A和该家族的其他成员在Sf 9细胞中的表达， 将其加入或微量注射到中性粒细胞和嗜中性粒细胞样细胞中， 细胞系 涉及激活的转导机制， 酪氨酸激酶和PKC将在内皮细胞中表征为 是否存在假定的跨膜转换器。 的IL-8 将克隆启动子区并进行报告基因研究。 最后，DNA酶I超敏反应将用于确认精确的 在诱导IL-8基因表达中蛋白质-DNA相互作用的位点。 这些研究将阐明LPS诱导细胞骨架的机制， 中性粒细胞中的组装和内皮细胞中的新基因表达， 我们认为这可能与人类脓毒症引起的肺损伤有关。