Role of IL-8 Inflammation in smoke inhalation and burn therapy/Project 3

IL-8 炎症在烟雾吸入和烧伤治疗中的作用/项目 3

• 批准号: 7271413
• 负责人: FRANK C. SCHMALSTIEG
• 金额: $ 13.06万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7271413
• 关键词: arginine; biological signal transduction; burns; chemokine; chemokine receptor; confocal scanning microscopy; flow cytometry; inflammation; interleukin 8; lung injury; lymphatic circulation; neutrophil; proline; protein transport; pulmonary circulation; respiratory system; sheep; smoke inhalation; transcription factor; western blottings

The overall goal of this research is to establish the role of intedeukin-8 in the genesis and persistence of endogenous pulmonary inflammation that occurs in this ovine model of smoke inhalation and bum injury. In Specific Aim 1, it will be determined if sequestered cells in the pulmonary circulation are initially activated in the bronchial circulation and contain F-actin. The bronchial circulation will be ablated and the pulmonary circulation examined for decreased sequestration of PMNs in pulmonary capillaries by confocal microscopy. Activation of polymorphonuclear leukocytes (PMNs) will be followed by flow cytometric measurement of blood and lung lymph PMN cell surface CD18 and L-selectin. PMN and red blood cell pulmonary transit times will be measured using both resting PMNs and PMNs from the injured animals. IL-8 and LTB4 will be determined in lung lymph and correlated with PARP activity. Specific Aim 2, usage of cell signaling pathways mediating activation of neutrophils and formation of F-actin in this injury are sought through measurement of total and phosphorylated JNK/SAPK, p38 Map kinase, ERK 1/2, Akt, and PARP activity by Western blotting, enzyme activity, and confocal laser scanning cytometry. Consequences of involvement of these pathways will be explored through translocation of NF-KappaB and AP-1 transcription factors in nuclear isolates of airway cells and neutrophils by electrophoretic mobility shift assays. IKappaB will be measured by Western blotting. These same molecules will be measured in individual cells in lung tissue by confocal microscopy and laser scanning cytometry to allow for identification of cell type and quantification of each molecule. In Specific Aim 3, the role of IL-8 in the genesis and maintenance of pulmonary inflammation secondary to smoke and bum injuries will be examined. Advantage will be taken of our construction of a combined CXCR1 and CXCR2 antagonist (AAR-IL-8) to directly test the role of IL-8 in pulmonary injury in smoke inhalation and bum trauma. AAR-IL-8 will be given intravenously as a constant infusion in injured sheep through 48 h. The activation status of neutrophils in lung lymph will be measured by flow cytometry. Laser scanning cytometry will be employed to characterize activation of neutrophils in lung tissue and to estimate obstructing neutrophils in pulmonary capillaries. Lung lymph flow, PaO2/FiO2, left atdal pressure, pulmonary artery and pulmonary, capillary wedge pressure, cardiac output, pulmonary microvascular and systemic vascular resistance will be measured.

这项研究的总体目标是确定INTDEKIN-8在烟雾吸入和烧伤绵羊模型中内源性肺部炎症的发生和持续中的作用。在具体目标1中，将确定肺循环中的隔离细胞最初是否在支气管循环中被激活并包含 F-肌动蛋白。将消融支气管循环，并通过共聚焦显微镜检查肺循环以减少肺毛细血管内中性粒细胞的隔离。在中性粒细胞(PMN)激活之后，将通过流式细胞仪检测血液和肺淋巴液中PMN细胞表面CD18和L-选择素的水平。PMN和红细胞肺通过时间将使用静息PMN和来自受伤动物的PMN进行测量。IL-8 肺淋巴组织中LTB4含量与PARP活性呈正相关。具体目的2，通过Western blotting、酶活性和激光共聚焦扫描细胞术检测总的和磷酸化的JNK/SAPK、p38Map激酶、ERK1/2、Akt和PARP活性，寻找细胞信号通路在中性粒细胞活化和F-肌动蛋白形成中的作用。我们将探索这些途径参与的后果 通过电泳迁移率改变分析，核因子-kappaB和AP-1转录因子在呼吸道细胞和中性粒细胞核分离中的移位。用免疫印迹法检测IKappaB。这些相同的分子将通过共聚焦显微镜和激光扫描细胞仪在肺组织的单个细胞中进行测量，以便于识别细胞类型和量化每个分子。在具体目标3中，将研究IL-8在吸烟和烧伤继发肺部炎症的发生和维持中的作用。我们将利用我们构建的CXCR1和CXCR2联合拮抗剂(AAR-IL-8)来直接测试IL-8在烟雾吸入和烧伤所致肺损伤中的作用。伤后48h持续静脉滴注AAR-IL-8，用流式细胞仪检测肺淋巴中性粒细胞的活化状态。激光扫描细胞术将被用来表征肺组织中性粒细胞的激活并估计阻塞 肺毛细血管内的中性粒细胞。测量肺淋巴流量、PaO2/FiO2、左心房压、肺动脉和肺组织、毛细血管楔压、心输出量、肺微血管和体循环阻力。