Morishima Hirotaka, Kajiwara Keiichi, Akiyama Kazuo, Yanagihara Yukiyoshi
Clinical Research Center for Allergy and Rheumatology, National Hospital Organization, Sagamihara Hospital, Sagamihara, Japan.
Int Arch Allergy Immunol. 2008;145(2):163-74. doi: 10.1159/000108141. Epub 2007 Sep 11.
Viral infection causes asthma exacerbations and airway hyperreactivity. Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA) of viral or synthetic origin in a fashion different from protein kinase R (PKR). The aim of this study was to examine the expression and function of TLR3 in human airway smooth muscle (ASM) cells.
Expression of TLR3 and muscarinic receptor (MR), histamine receptor (HR), and cysteinyl leukotriene receptor (CysLTR) subtypes was analyzed by quantitative real-time PCR, flow cytometry, or Western blotting. It was assessed whether ASM cells respond to polyinosinic-polycytidylic acid (poly I:C), a synthetic analog of dsRNA, with alterations in M2R, M3R, H1R, and CysLT1R expression. The function of these subtypes was evaluated by cholinergic regulation of forskolin-stimulated cyclic AMP accumulation or by mobilization of intracellular calcium upon stimulation.
ASM cells expressed TLR3 and PKR, and intracellular TLR3 expression was demonstrated. Poly I:C caused decreased M2R and increased M3R expression, without affecting H1R and CysLT1R expression. Poly I:C-treated cells showed decreased cholinergic inhibition of forskolin-stimulated cyclic AMP accumulation and enhanced calcium flux in response to acetylcholine, but not to histamine and LTD4. These modulating effects of poly I:C were reversed by chloroquine, but not by 2-aminopurine.
The data indicate that poly I:C internalized by ASM cells differentially regulates M2R and M3R expression and function by interacting with TLR3 rather than with PKR, suggesting that these changes may contribute to airway hyperreactivity.
病毒感染会导致哮喘发作和气道高反应性。Toll样受体3（TLR3）以不同于蛋白激酶R（PKR）的方式识别病毒或合成来源的双链RNA（dsRNA）。本研究的目的是检测TLR3在人气道平滑肌（ASM）细胞中的表达及功能。
通过定量实时PCR、流式细胞术或蛋白质印迹法分析TLR3和毒蕈碱受体（MR）、组胺受体（HR）及半胱氨酰白三烯受体（CysLTR）亚型的表达。评估ASM细胞是否对dsRNA的合成类似物聚肌苷酸-聚胞苷酸（poly I:C）产生反应，导致M2R、M3R、H1R和CysLT1R表达改变。通过福司可林刺激的环磷酸腺苷积累的胆碱能调节或刺激后细胞内钙的动员来评估这些亚型的功能。
ASM细胞表达TLR3和PKR，并证实有细胞内TLR3表达。Poly I:C导致M2R表达降低和M3R表达增加，而不影响H1R和CysLT1R表达。经poly I:C处理的细胞对福司可林刺激的环磷酸腺苷积累的胆碱能抑制作用降低，对乙酰胆碱刺激的钙通量增强，但对组胺和白三烯D4无此反应。Poly I:C的这些调节作用可被氯喹逆转，但不能被2-氨基嘌呤逆转。
数据表明，ASM细胞内化的poly I:C通过与TLR3而非PKR相互作用，差异性地调节M2R和M3R的表达及功能，提示这些变化可能导致气道高反应性。
