Baumgarten G, Knuefermann P, Wrigge H, Putensen C, Stapel H, Fink K, Meyer R, Hoeft A, Grohé C
Universitätsklinikum Bonn, Department of Anesthesiology and Intensive Care Medicine, Bonn, Germany.
Eur J Anaesthesiol. 2006 Dec;23(12):1041-8. doi: 10.1017/S0265021506001098. Epub 2006 Jul 12.
Proinflammatory cytokines as well as nitric oxide (NO) play a major role in mediating the response to lipopolysaccharide (LPS). The present study tested the hypothesis that LPS induces proinflammatory cytokines in the lung via the Toll-like receptor 4 (TLR4)/CD14 signalling cascade.
Control mice and TLR4-deficient (TLR4-D) mice were used to test TLR4-mediated effects of LPS. Both strains received either Escherichia coli LPS (20 mg kg-1 intraperitoneal) or saline and their lungs were collected at different time points. Pulmonary nuclear factor kappaB (NFkappaB) activation was investigated with electromobility shift assay. mRNA expression of inflammatory mediators and their corresponding receptors were detected with Ribonuclease Protection Assay. Protein expression was detected by ELISA and western blotting. Inducible NO synthase (iNOS) expression was monitored by RT-PCR and iNOS activity by conversion of l-arginine to citrulline. Immune cells were sampled by bronchoalveolar lavage (BAL) and classified.
LPS application induced CD14-, but not TLR4 protein expression in control mice. Activation of pulmonary NFkappaB was observed within 60 min in control, but not in TLR4-D mice. Six hours of LPS administration induced a significant increase in pulmonary tumour necrosis factor alpha-, interleukin-1beta- and interleukin-6 mRNA and protein expression in control mice compared to TLR4-D mice. Furthermore, LPS induced a significantly higher increase of the iNOS expression and catalytic activity in control mice than in TLR4-D mice. BAL revealed an increase in total cell count in all LPS treated mice.
Our findings suggest that TLR4 plays a key role for regulating the expression of relevant cytokines within the lung during endotoxic shock.
促炎细胞因子以及一氧化氮（NO）在介导对脂多糖（LPS）的反应中起主要作用。本研究检验了LPS通过Toll样受体4（TLR4）/CD14信号级联在肺中诱导促炎细胞因子的假说。
使用对照小鼠和TLR4缺陷（TLR4-D）小鼠来测试LPS的TLR4介导效应。两种品系的小鼠均接受大肠杆菌LPS（20 mg/kg腹腔注射）或生理盐水，并在不同时间点采集它们的肺。用凝胶迁移率变动分析研究肺细胞核因子κB（NFκB）的激活。用核糖核酸酶保护分析检测炎症介质及其相应受体的mRNA表达。通过酶联免疫吸附测定和蛋白质印迹法检测蛋白质表达。通过逆转录-聚合酶链反应监测诱导型一氧化氮合酶（iNOS）的表达，通过将L-精氨酸转化为瓜氨酸来监测iNOS活性。通过支气管肺泡灌洗（BAL）对免疫细胞进行采样并分类。
在对照小鼠中，LPS诱导了CD14蛋白表达，但未诱导TLR4蛋白表达。在对照小鼠中，60分钟内观察到肺NFκB激活，而TLR4-D小鼠中未观察到。与TLR4-D小鼠相比，给予LPS 6小时后，对照小鼠肺中肿瘤坏死因子α、白细胞介素-1β和白细胞介素-6的mRNA和蛋白质表达显著增加。此外，与TLR4-D小鼠相比，LPS在对照小鼠中诱导的iNOS表达和催化活性增加明显更高。BAL显示所有接受LPS治疗的小鼠的总细胞数增加。
我们的研究结果表明，TLR4在内毒素休克期间对调节肺内相关细胞因子的表达起关键作用。
