Mechanisms for the induction of bronchial smooth muscle hyperresponsiveness in allergic bronchial asthma

白细胞介素-13（IL-13）是哮喘患者气道高反应性发展的中心介质之一。然而，其对支气管平滑肌（BSM）的作用尚不清楚。最近的研究表明，RhoA/ rho激酶参与BSM收缩，这一途径现已被提出作为哮喘治疗的新靶点。为了明确IL-13在BSM高反应性诱导中的作用，我们研究了IL-13对BSM收缩性和RhoA表达的影响。将BALB/c雄性小鼠致敏，并用卵清蛋白抗原反复激发。在体外（人BSM细胞和小鼠BSM组织）和体内（经鼻注入小鼠气道）也测试了IL-13的作用。在反复抗原攻击小鼠中，观察到明显的气道炎症和BSM高反应性，支气管肺泡灌洗液中IL-13的上调。在培养的人BSM细胞中，IL-13引起信号转导和转录激活因子（STAT6）的激活和RhoA的上调，这两者都被STAT6抑制剂来氟米特抑制。在幼龄小鼠离体BSM组织中，IL-13的存在显著增强了BSM组织的收缩性。此外，在体内用1L-13处理气道，在幼稚小鼠中引起BSM高反应性和RhoA上调。这些发现表明IL-13/STAT6信号对于抗原诱导的BSM高反应性的发展至关重要，并且在BSM中特异性抑制该途径的药物可能为治疗哮喘提供新的策略。接下来，研究了一种新的STAT6抑制剂AS1517499对抗原诱导的BSM高反应性的影响。在培养的人BSM细胞中，IL-13（100 ng/mL）引起STAT6的磷酸化和RhoA的上调，RhoA是一种负责Ca^<2+>对平滑肌收缩增敏的单体GTPase，与as1517499 （100 nM）共孵育可抑制这两种事件。在BALB/c小鼠中，主动致敏和卵清蛋白抗原反复激发，在最后一次抗原激发后，观察到支气管肺泡灌洗液中1L-13水平升高，bsm中STAT6磷酸化。这些小鼠BSM对乙酰胆碱的收缩能力增强，同时BSM中RhoA水平上调。每次卵清蛋白暴露前1h腹腔注射AS1517499(10mg/kg)，几乎完全抑制抗原诱导的RhoA上调和BSM高反应性。也发现了部分但显著的抑制抗原诱导的IL-13的产生。这些发现表明，抗原暴露产生的M-13通过BSM细胞中STAT6的激活直接上调RhoA，导致BSM收缩增强，这是气道高反应性的原因之一。AS1517499对RhoA和IL-13上调的抑制作用可能有助于哮喘的治疗。少

Interleukin-13(IL-13) is one of the central mediators for development of airway hyperresponsiveness in asthma. However, its effect on bronchial smooth muscle(BSM) is not well known Recent studies revealed an involvement of RhoA/Rho-kinase in BSM contraction and this pathway has now been proposed as a new target for asthma therapy. To make clear the role of IL-13 on the induction of BSM hyperresponsiveness, effects of IL-13 on contractility and RhoA expression in BSMs were investigated. Male BALB/c mice were sensitized and repeatedly challenged with ovalbumin antigen. Both in vitro(human BSM cells and mouse BSM tissues) and in vivo(intranasal instillation into mouse airways) effects of IL-13 were also tested. In the repeatedly antigen-challenged mice, marked airway inflammation and BSM hyperresponsiveness with an upregulation of IL-13 in bronchoalveolar lavage fluids were observed In cultured human BSM cells, IL-13 caused an activation of signal transducer and activator of transcription … More 6(STAT6) and an upregulation of RhoA : both of them were inhibited by a STAT6 inhibitor leflunomide. In isolated BSM tissues of naive mice, the contractility was significantly enhanced by organ culture in the presence of IL-13. Moreover, in vivo treatment of airways with 1L-13 caused a BSM hyperresponsiveness with an upregulation of RhoA in naive mice. These findings suggest that IL-13/STAT6 signaling is critical for development of antigen-induced BSM hyperresponsiveness and that agents that specifically inhibit this pathway in BSM may provide a novel strategy for treatment of asthma. Next, the effect of a novel STAT6 inhibitor, AS1517499, on the development of antigen-induced BSM hyperresponsiveness was investigated. In cultured human BSM cells, IL-13(100 ng/mL) caused a phosphorylation of STAT6 and an upregulation of RhoA, a monomeric GTPase responsible for Ca^<2+> sensitization of smooth muscle contraction : both events were inhibited by co-incubation with AS 1517499(100 nM). In BALB/c mice that were actively sensitized and repeatedly challenged with ovalbumin antigen, an increased 1L-13 level in bronchoalveolar lavage fluids and a phosphorylation of STAT6 in BSMs were observed after the last antigen challenge. These mice had an augmented BSM contractility to acetylcholine together with an upregulation of RhoA in BSMs. Intraperitoneal injections of AS1517499(10mg/kg) 1h before each ovalbumin exposure inhibited both the antigen-induced upregulation of RhoA and BSM hyperresponsiveness, almost completely. A partial but significant inhibition of antigen-induced production of IL-13 was also found. These findings suggest that M-13, generated by antigen exposure, upregulates RhoA via an activation of STAT6 in BSM cells directly, resulting in an augmented BSM contraction, which is one of the causes of the airway hyperresponsiveness. The inhibitory effects of AS1517499 both on RhoA and IL-13 upregulations might be useful for asthma treatment. Less