Mechanisms of Lung Inflammation Following Discrete Hepatic Injury

越来越多的证据表明，肝脏损伤可导致或加重肺损伤。例如，当全身性炎症反应综合征（SIRS）患者肝功能障碍伴有急性肺损伤时，死亡率和发病率都很高。肝和肺中产生的细胞因子，包括肿瘤坏死因子α (tnf - α)和白细胞介素（IL- 1），与SIRS的发病机制有关，动物实验证明它们能够引发急性肺损伤。编码这些细胞因子的基因表达的增加涉及转录因子核因子κ B （nf - κ B）的激活，这种激活在几种炎症状态中得到了充分的证明。肝冷冻消融是最近引进的一种用于消除原发性和转移性肝脏肿瘤的非切除手术技术，当超过30-35%的肝脏被消融时，与急性肺损伤相关，但这种反应的机制尚不清楚。我们已经证明，在实验动物中，由冷冻消融产生的直接和选择性肝损伤导致肝脏中NF-kappaB的早期激活，随后从肝脏中释放tnf - α，然后肺中NF-kappaB的激活，其组织学结果与在其他中性粒细胞肺损伤条件下所见的相似；这些事件均不发生在肝切除术后。本项目的具体目的是：1)确定改变肝脏NF-kappaB活化是否会影响冷冻消融诱导肺损伤的应答。NF-kappaB的激活将通过在肝脏中表达诱导或抑制NF-kappaB活性的转基因来特异性调节。我们还将利用细胞可渗透的cSN50肽，该肽含有源自p50-NF-kappaB1亚基的核定位信号序列（NLS），在直接肝损伤前后抑制NF-kappaB。2)通过比较tnf - α和IL-1缺失小鼠的应答，确定近端细胞因子tnf - α和IL-1在肝脏冷冻消融应答中的作用；通过比较tnf - α、tnf - α受体1和/或IL-1受体1缺失小鼠的应答，确定近端细胞因子tnf - α和IL-1在肝脏冷冻消融应答中的作用。结果变量包括中性粒细胞肺炎症、组织学定义的肺损伤、NF- kappaB激活、血清和肺灌洗细胞因子和类二十烷酸浓度。3)通过改变肝或肺中环氧合酶（COX）的表达和/或活性，确定类二十烷酸在肝脏冷冻消融应答中的作用。在这些研究中，我们将定义COX-2在肺和肝脏中对冷冻消融反应的表达，并测量前列腺素产物，通过使用COX-2抑制剂和COX-2敲除小鼠来确定COX-2在冷冻消融反应中的作用，并通过将野生型肝细胞移植到COX-2敲除小鼠中并通过传递转基因在这些动物中表达COX-2来确定肝脏COX-2表达是否对调节冷冻消融反应至关重要。这些研究的结果应该加强我们对肝脏在直接肝损伤的繁殖反应中的作用的理解。

There is increasing evidence that injury to the liver can precipitate or exaggerate lung injury. For example, when hepatic dysfunction accompanies acute lung injury in patients with the Systemic Inflammatory Response Syndrom (SIRS), mortality and morbidity are high. Cytokines which are produced in both the liver and the lungs, including tumor necrosis factor alpha (TNF-alpha) and interleukin (IL- 1), are implicated in the pathogenesis of SIRS and animal experiments have demonstrated them capable of precipitating acute lung injury. Increased expression of the genes encoding these cytokines involves activation of the transcription factor nuclear factor kappa B (NF-kappaB) and such activation is well-documented in several inflammatory states. Hepatic cryoablation, a recently introduced non-resectional surgical technique for eliminating primary and metastatic liver tumors, is associated with acute lung injury when more than 30-35% of the liver is ablated but mechanisms of this response remain undefined. We have shown that the direct and selective liver injury produced by cryoablation in experimental animals causes an early activation of NF-kappaB in the liver, followed by TNF-alpha release from the liver and then NF-kappaB activation in the lungs with histologic findings similar to those seen in other conditions of neutrophilic lung injury; none of these events follows hepatic resection. The specific aims of this project are: 1) To determine whether altering NF-kappaB activation in the liver affects the response to cryoablation induced lung injury. NF-kappaB activation will be specifically modulated by expressing transgenes in the liver which induce or inhibit NF-kappaB activity. We will also utilize the cell permeable cSN50 peptide, which contains the nuclear localization signal sequence (NLS) derived from the p50-NF-kappaB1 subunit to inhibit NF-kappaB prior to and following direct liver injury. 2) To determine the role of the proximal cytokines, TNF-alpha and IL-1, in the response to hepatic cryoablation by comparing responses in mice deficient in TNF-alpha and IL-1, in the response to hepatic cryoablation by comparing responses in mice deficient in TNF-alpha, TNF-alpha receptor 1 and/or IL-1 receptor 1. Outcome variables will include neutrophilic lung inflammation, histologically defined lung injury, NF- kappaB activation, and serum and lung lavage cytokine and eicosanoid concentrations. 3) To determine the role of eicosanoids in the response to hepatic cryoablation by altering cyclooxygenase (COX) expression and/or activity in liver or lungs. In these studies we will define COX-2 expression in response to cryoablation in lung and liver and measure prostanoid products, determine the role of COX-2 in the cryoablation response by using COX-2 inhibitors and COX-2 knockout mice, and establish whether liver COX-2 expression is critical for modulating the cryoablation response by transplantation of wild type hepatocytes into COX-2 knockout mice and by delivering transgenes to express COX-2 in these animals. Results of these studies should enhance our understanding of the role of the liver in propagation in response to direct liver injury.