Douillet Christelle D, Robinson William P, Milano Peter M, Boucher Richard C, Rich Preston B
Division of Trauma and Critical Care, Department of Surgery, University of North Carolina at Chapel Hill, 4008 Burnett-Womack, Chapel Hill, NC 27599-7228, USA.
Am J Physiol Lung Cell Mol Physiol. 2006 Oct;291(4):L734-46. doi: 10.1152/ajplung.00389.2005. Epub 2006 Apr 21.
Extracellular nucleotides can mediate a variety of cellular functions via interactions with purinergic receptors. We previously showed that mechanical ventilation (MV) induces airway IL-6 and ATP release, modifies luminal nucleotide composition, and alters lung purinoceptor expression. Here we hypothesize that extracellular nucleotides induce secretion of IL-6 by small airway epithelial cells (SAEC). Human SAEC were stimulated with nucleotides in the presence or absence of inhibitors. Supernatants were analyzed for IL-6 and lysates for p38 MAPK activity by ELISA. RNA was analyzed by real-time RT-PCR. Rats (n=51) were randomized to groups as follows: control, small-volume MV, large-volume MV, large-volume MV-intratracheal apyrase, or small-volume MV-intratracheal adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS). After 1 h of MV, bronchoalveolar lavage fluid was analyzed for ATP and IL-6 by luminometry and ELISA. ATP and ATPgammaS increased SAEC IL-6 secretion in a time- and dose-dependent manner, an effect inhibited by apyrase. Agonists were ranked in the following order: ATPgammaS>ATP=UTP>ADP=adenosine>2-methylthio-ADP=control. SB-203580, but not U-0126 or JNK1 inhibitor, decreased nucleotide effects. Additionally, nucleotides induced p38 MAPK phosphorylation. Inhibitors of Ca2+ signaling, phospholipase C, transcription, and translation decreased IL-6 release. Furthermore, nucleotides increased IL-6 expression. In vivo, large-volume MV increased airway ATP and IL-6 concentrations. IL-6 release was decreased by apyrase and increased by ATPgammaS. Extracellular nucleotides induce P2Y2-mediated secretion of IL-6 by SAEC via Ca2+, phospholipase C, and p38 MAPK-dependent pathways. This effect is dependent on transcription and translation. Our findings were confirmed in an in vivo model, thus demonstrating a novel mechanism of nucleotide-induced IL-6 secretion by airway epithelia.
细胞外核苷酸可通过与嘌呤能受体相互作用介导多种细胞功能。我们之前的研究表明，机械通气（MV）可诱导气道白细胞介素-6（IL-6）和三磷酸腺苷（ATP）释放，改变管腔内核苷酸组成，并改变肺嘌呤受体表达。在此，我们假设细胞外核苷酸可诱导小气道上皮细胞（SAEC）分泌IL-6。在有或无抑制剂存在的情况下，用核苷酸刺激人SAEC。通过酶联免疫吸附测定（ELISA）分析上清液中的IL-6，并分析裂解物中的p38丝裂原活化蛋白激酶（MAPK）活性。通过实时逆转录聚合酶链反应（RT-PCR）分析RNA。将大鼠（n = 51）随机分为以下几组：对照组、小潮气量MV组、大潮气量MV组、大潮气量MV-气管内注入Apyrase组或小潮气量MV-气管内注入腺苷5'-O-（3-硫代三磷酸）（ATPγS）组。MV 1小时后，通过荧光测定法和ELISA分析支气管肺泡灌洗液中的ATP和IL-6。ATP和ATPγS以时间和剂量依赖性方式增加SAEC的IL-6分泌，该效应被Apyrase抑制。激动剂的排序如下：ATPγS>ATP =三磷酸尿苷（UTP）>二磷酸腺苷（ADP）=腺苷>2-甲硫基-ADP =对照组。SB-203580可降低核苷酸的作用，但U-0126或JNK1抑制剂则不能。此外，核苷酸可诱导p38 MAPK磷酸化。钙离子信号传导、磷脂酶C、转录和翻译的抑制剂可降低IL-6释放。此外，核苷酸可增加IL-6表达。在体内，大潮气量MV可增加气道ATP和IL-6浓度。Apyrase可降低IL-6释放，而ATPγS可增加IL-6释放。细胞外核苷酸通过钙离子、磷脂酶C和p38 MAPK依赖性途径诱导SAEC分泌P2Y2介导的IL-6。该效应依赖于转录和翻译。我们的研究结果在体内模型中得到证实，从而证明了核苷酸诱导气道上皮细胞分泌IL-6的新机制。
