Graduate Institute of Veterinary Pathobiology, College of Veterinary Medicine, National Chung Hsing University, 250, Kuo Kuang Road, Taichung, 402, Taiwan, ROC.
Vet Res. 2011 Feb 7;42(1):25. doi: 10.1186/1297-9716-42-25.
Actinobacillus pleuropneumoniae (A. pleuropneumoniae) causes fibrino-hemorrhagic necrotizing pleuropneumonia in pigs. Production of proinflammatory mediators in the lungs is an important feature of A. pleuropneumoniae infection. However, bacterial components other than lipopolysaccharide involved in this process remain unidentified. The goals of this study were to determine the role of A. pleuropneumoniae exotoxin ApxI in cytokine induction and to delineate the underlying mechanisms. Using real-time quantitative PCR analysis, we found native ApxI stimulated porcine alveolar macrophages (PAMs) to transcribe mRNAs of IL-1β, IL-8 and TNF-α in a concentration- and time-dependent manner. Heat-inactivation or pre-incubation of ApxI with a neutralizing antiserum attenuated ApxI bioactivity to induce cytokine gene expression. The secretion of IL-1β, IL-8 and TNF-α protein from PAMs stimulated with ApxI was also confirmed by quantitative ELISA. In delineating the underlying signaling pathways contributing to cytokine expression, we observed mitogen-activated protein kinases (MAPKs) p38 and cJun NH2-terminal kinase (JNK) were activated upon ApxI stimulation. Administration of an inhibitor specific to p38 or JNK resulted in varying degrees of attenuation on ApxI-induced cytokine expression, suggesting the differential regulatory roles of p38 and JNK in IL-1β, IL-8 and TNF-α production. Further, pre-incubation of PAMs with a CD18-blocking antibody prior to ApxI stimulation significantly reduced the activation of p38 and JNK, and subsequent expression of IL-1β, IL-8 or TNF-α gene, indicating a pivotal role of β2 integrins in the ApxI-mediated effect. Collectively, this study demonstrated ApxI induces gene expression of IL-1β, IL-8 and TNF-α in PAMs that involves β2 integrins and downstream MAPKs.
胸膜肺炎放线杆菌（A. pleuropneumoniae）可引起猪的纤维素性-出血性坏死性胸膜肺炎。肺部促炎介质的产生是 A. pleuropneumoniae 感染的一个重要特征。然而，参与这一过程的细菌成分除脂多糖外仍未被识别。本研究的目的是确定胸膜肺炎放线杆菌外毒素 ApxI 在细胞因子诱导中的作用，并阐明其潜在机制。通过实时定量 PCR 分析，我们发现天然 ApxI 以浓度和时间依赖的方式刺激猪肺泡巨噬细胞（PAMs）转录 IL-1β、IL-8 和 TNF-α 的 mRNA。ApxI 的热失活或与中和抗血清预孵育减弱了 ApxI 的生物活性，从而抑制细胞因子基因的表达。用 ApxI 刺激的 PAMs 分泌的 IL-1β、IL-8 和 TNF-α 蛋白也通过定量 ELISA 得到了证实。在阐明参与细胞因子表达的潜在信号通路时，我们观察到 ApxI 刺激后，丝裂原活化蛋白激酶（MAPKs）p38 和 cJun NH2-末端激酶（JNK）被激活。特异性 p38 或 JNK 抑制剂的给药导致 ApxI 诱导的细胞因子表达程度不同，表明 p38 和 JNK 在 IL-1β、IL-8 和 TNF-α 产生中的调节作用不同。此外，在 ApxI 刺激前用 CD18 阻断抗体预孵育 PAMs，可显著降低 p38 和 JNK 的激活，以及随后的 IL-1β、IL-8 或 TNF-α 基因的表达，表明β2 整合素在 ApxI 介导的效应中起着关键作用。总之，本研究表明 ApxI 诱导 PAMs 中 IL-1β、IL-8 和 TNF-α 的基因表达，涉及β2 整合素和下游 MAPKs。
