TGF-BETA, CHLORIDE CHANNELS AND MIGRATION OF EOSINOPHILS

描述(由申请人提供)：在慢性哮喘中，嗜酸性粒细胞在反复接触变应原后在肺中的迁移与呼吸道重塑有关。嗜酸性粒细胞在哮喘患者呼吸道中募集和激活的机制尚不清楚。我们发现，鼻腔注射ClC3 siRNA可以预防过敏性哮喘小鼠肺对乙酰胆碱、BALF嗜酸性粒细胞增多和呼吸道炎症的高反应性。在体外条件下，转化生长因子-21可增加嗜酸性粒细胞ClC3的mRNA转录，诱导嗜酸性粒细胞趋化、形态改变和跨内皮细胞迁移，这一作用可被氯通道阻滞剂抑制。利用全细胞膜片钳技术，我们还证实了转化生长因子-21引起的人血嗜酸细胞氯电流的显著增加，而用siRNA抑制ClC3和用rotlerin处理细胞都取消了这一作用。我们的中心假设是，在哮喘中，转化生长因子-2的释放、嗜酸性粒细胞氯离子通道和细胞体积之间的关系是细胞跨内皮细胞迁移和气道侵袭的关键决定因素。在目标1中，我们将确定参与选择性激活电压门控氯电流和转化生长因子-2调节人血嗜酸细胞ClC3水平的胞浆激酶。假设转化生长因子-2激活蛋白激酶C-4(PKC-4)，增加电压门控性氯电流，并诱导p38/JNK蛋白激酶和蛋白激酶C的磷酸化，增加ClC-3的表达，从而增强ClC3电流的激活。这反过来会诱导嗜酸性粒细胞的趋化和跨内皮细胞迁移。在目标2中，我们将分析ClC3启动子中的转录起始点(S)，并寻找参与转化生长因子-2选择性调节人血嗜酸细胞中ClC-3水平的转录因子。我们还将检测体内抑制AP-1转录对抗原致敏和攻击小鼠肺嗜酸细胞ClC-3表达和活性的影响。假设转化生长因子-2激活了激酶的磷酸化，进而使AP-1家族转录因子和Smads磷酸化，从而调节ClC3的转录。在目标3中，我们将研究转化生长因子-2对人血嗜酸粒细胞和变态反应性鼻炎和变态反应性哮喘患者鼻腔冲洗液中嗜酸粒细胞的氯电流、形态变化和迁移的影响，以及其潜在的细胞和分子途径。假设过敏性哮喘患者嗜酸性粒细胞的氯离子电流和迁移增加，无论是在未刺激的细胞和/或经转化生长因子-2刺激后，取决于哮喘的慢性化程度。长期目标是确定转化生长因子-2对嗜酸性粒细胞电压门控氯电流的影响，并检测免疫调节剂的作用。这样的研究将为慢性哮喘的病理生理过程以及预防或逆转这种疾病的方法提供独特的见解。 哮喘肺中的嗜酸性粒细胞是参与慢性哮喘发病机制的主要炎性细胞之一。在这个项目中，我们计划通过实验在细胞和分子水平上研究嗜酸性粒细胞迁移到肺组织的精确机制。从这项研究中获得的信息应该为制定更好的慢性哮喘治疗方法提供机会。

DESCRIPTION (provided by applicant): Migration of eosinophils in the lung in response to repeated allergen exposure is associated with airway remodeling in chronic asthma. Mechanism involved in eosinophil recruitment and activation in the airways of asthmatic individuals are still unclear. We have discovered that intranasal administration of ClC3 siRNA prevented airway hyperresponsiveness to methacholine, BALF eosinophilia and airway inflammation in the lungs of allergic asthmatic mice. Under in vitro conditions, TGF-21 increased mRNA transcripts of ClC3 in eosinophils, and induced eosinophil chemotaxis, shape change, and transendothelial migration, which was inhibited by chloride channel blockers. Using whole cell patch-clamp, we also demonstrated a significant increase in Cl- current in human blood eosinophils in response to TGF-21 and that both ClC3 knock-down with siRNA and treatment of the cells with rottlerin abolished this effect. Our central hypothesis is that the relationships between the release of TGF-2 in the airways, eosinophil Cl- channels, and cell volume are critical determinants of cell transendothelial migration and airway invasion in bronchial asthma. In Aim 1, we will identify cytosolic kinases involved in selective activation of voltage-gated chloride currents and regulation of ClC3 levels by TGF-2 in human blood eosinophils. The hypothesis is that TGF-2 activates protein kinase C-4 (PKC-4) to increase voltage-gated chloride current and induces phosphorylation of p38/JNK MAP kinase and protein kinase C to increase ClC-3 expression leading to heightened activation of ClC3 current. This, in turn, induces chemotaxis and transendothelial migration of eosinophils. In Aim 2, we will analyze the transcription initiation site(s) in the ClC3 promoter and identify transcription factors involved in selective regulation of ClC- 3 levels by TGF-2 in human blood eosinophils. We will also examine the effect of the in vivo inhibition of AP-1 transcription on the expression and activity of ClC-3 in lung eosinophils of antigen-sensitized and challenged mice. The hypothesis is that TGF-2 activates phosphorylation of kinases, which in turn phosphorylate AP-1 family transcription factors and Smads, to regulate ClC3 transcription. In Aim 3, we will examine the effect of TGF-2 on Cl- currents, shape change and migration, and the underlying cellular and molecular pathway in human blood eosinophils and eosinophils isolated from nasal washings of allergic rhinitis and allergic asthmatic patients. The hypothesis is that Cl- currents in and migration of eosinophils of allergic asthmatics will be heightened, either in unstimulated cells and/or after stimulation with TGF-2, depending upon the chronicity of asthma. The long term goal is to ascertain the effects that TGF-2 have on voltage-gated chloride currents in eosinophils and to examine the effect of immunomodulators. Such investigations would provide unique insights to the pathophysiologic process of chronic asthma and the means to prevent or reverse the disease.Project Narrative Eosinophils in the asthmatic lungs are one of the major inflammatory cells that have been implicated in the pathogenesis of chronic asthma. In this project experiments are proposed to examine the precise mechanisms of eosinophil migration into the lung tissues at the cellular and molecular level. The information obtained from this study should provide an opportunity to formulate superior therapeutic approaches in chronic asthma.