Geisler Fabian, Algül Hana, Riemann Marc, Schmid Roland M
Department of Internal Medicine II, Klinikum Rechts der Isar, Technical University of Munich, Munich, Germany.
J Immunol. 2005 May 15;174(10):6431-9. doi: 10.4049/jimmunol.174.10.6431.
The systemic inflammatory response syndrome is responsible for pancreatitis-associated mortality. Recent in vitro and in vivo studies have suggested that pancreatic elastase is one missing link between the localized inflammatory process in the pancreas and distant organ dysfunction and failure. It has been shown that pancreatic elastase activates transcription factors, including NF-kappaB, and induces TNF-alpha secretion in myeloid cells via TLRs. In this study we demonstrate that a highly purified low endotoxin pancreatic elastase preparation (El-UP) failed both to activate NF-kappaB and to induce TNF-alpha release in RAW 264.7 cells and bone marrow-derived macrophages. In contrast, a less purified elastase preparation (El-IV) caused activation of NF-kappaB and was able to induce TNF-alpha release at very low concentrations. These effects were sensitive to pretreatment of the cells with polymyxin B and were resistant to heat inactivation. Endotoxin activity as determined by the Limulus amebocyte lysate assay was >3 orders of magnitude lower in the low endotoxin elastase preparation (El-UP) compared with less purified elastase preparations (El-IV). In contrast to contaminated elastase or LPS, elastase free of contamination (El-UP) failed to induce elevated serum TNF-alpha levels or pulmonary neutrophil infiltration after i.p. application in mice and did not induce lethality when coinjected with d-galactosamine. Failure of low endotoxin elastase (El-UP) to induce proinflammatory effects in vivo and in vitro was not due to functional inactivity of the elastase preparation, as determined by elastase activity assay. These results question current concepts of direct proinflammatory effects attributed to pancreatic elastase.
全身炎症反应综合征是胰腺炎相关死亡的原因。最近的体外和体内研究表明,胰腺弹性蛋白酶是胰腺局部炎症过程与远处器官功能障碍及衰竭之间缺失的一个环节。研究表明,胰腺弹性蛋白酶可激活包括核因子κB在内的转录因子,并通过Toll样受体(TLR)诱导髓样细胞分泌肿瘤坏死因子-α(TNF-α)。在本研究中,我们证明一种高度纯化的低内毒素胰腺弹性蛋白酶制剂(El-UP)在RAW 264.7细胞和骨髓来源的巨噬细胞中既不能激活核因子κB,也不能诱导TNF-α释放。相比之下,一种纯化程度较低的弹性蛋白酶制剂(El-IV)可引起核因子κB的激活,并能在极低浓度下诱导TNF-α释放。这些效应对用多粘菌素B预处理细胞敏感,且对热失活有抗性。通过鲎试剂法测定,低内毒素弹性蛋白酶制剂(El-UP)中的内毒素活性比纯化程度较低的弹性蛋白酶制剂(El-IV)低3个数量级以上。与受污染的弹性蛋白酶或脂多糖(LPS)不同,无污染的弹性蛋白酶(El-UP)经腹腔注射给小鼠后,不会诱导血清TNF-α水平升高或肺部中性粒细胞浸润,与d-半乳糖胺共同注射时也不会诱导致死性。通过弹性蛋白酶活性测定确定,低内毒素弹性蛋白酶(El-UP)在体内和体外未能诱导促炎效应并非由于弹性蛋白酶制剂功能失活。这些结果对目前归因于胰腺弹性蛋白酶的直接促炎作用的概念提出了质疑。