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: 6818810
• 负责人: FRANK C. SCHMALSTIEG
• 金额: $ 12.31万
• 依托单位: UNIVERSITY OF TEXAS MED BR GALVESTON
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2009-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6818810
• 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.

本研究的总体目标是确定白介素-8在烟雾吸入和烧伤绵羊模型中发生的内源性肺部炎症的发生和持续中的作用。在特定目标1中，将确定肺循环中的隔离细胞是否最初在支气管循环中被激活，并含有 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中肺毛细血管中PMN隔离减少。激活多形核白细胞（PMN）后，将通过流式细胞术测量血液和肺淋巴PMN细胞表面CD 18和L-选择素。将使用静息PMN和受伤动物的PMN测量PMN和红细胞肺通过时间。IL-8 在肺淋巴液中测定LTB 4，并与PARP活性相关。具体目的2，通过Western印迹、酶活性和共聚焦激光扫描细胞术测量总JNK/SAPK和磷酸化JNK/SAPK、p38 Map激酶、ERK 1/2、Akt和PARP活性，寻找在这种损伤中介导中性粒细胞活化和F-肌动蛋白形成的细胞信号传导途径的使用。将探讨这些途径参与的后果 通过电泳迁移率变动分析，在气道细胞和中性粒细胞的核分离物中NF-κ B和AP-1转录因子的易位。将通过蛋白质印迹法测量IKappaB。将通过共聚焦显微镜和激光扫描细胞术在肺组织中的单个细胞中测量这些相同的分子，以允许鉴定细胞类型和定量每种分子。在具体目标3中，将检查IL-8在继发于烟雾和烧伤损伤的肺部炎症的发生和维持中的作用。本研究利用我们构建的CXCR 1和CXCR 2联合拮抗剂（AAR-IL-8）直接检测IL-8在烟雾吸入和烧伤肺损伤中的作用。AAR-IL-8将作为持续输注静脉内给予受伤绵羊48小时。将通过流式细胞术测量肺淋巴中中性粒细胞的活化状态。将采用激光扫描细胞术来表征肺组织中中性粒细胞的活化并估计阻塞 肺毛细血管中的中性粒细胞。将测量肺淋巴流量、PaO 2/FiO 2、左心房压、肺动脉和肺动脉、毛细血管楔压、心输出量、肺微血管和全身血管阻力。