TGF-BETA, CHLORIDE CHANNELS AND MIGRATION OF EOSINOPHILS

描述（由申请人提供）：慢性哮喘患者反复接触过敏原后，肺部嗜酸性粒细胞的迁移与气道重塑有关。哮喘个体气道中嗜酸性粒细胞募集和激活的机制尚不清楚。我们发现，经鼻给药ClC3 siRNA可防止过敏性哮喘小鼠气道对甲胆碱的高反应性、BALF嗜酸性粒细胞增多和气道炎症。在体外条件下，TGF-21增加嗜酸性粒细胞ClC3的mRNA转录，诱导嗜酸性粒细胞趋化、形状改变和跨内皮迁移，这些被氯离子通道阻滞剂抑制。使用全细胞膜片钳，我们还证明了TGF-21对人血液嗜酸性粒细胞Cl-电流的显著增加，并且用siRNA敲除ClC3和用rotlerin处理细胞都可以消除这种影响。我们的中心假设是，气道中TGF-2的释放、嗜酸性粒细胞Cl-通道和细胞体积之间的关系是支气管哮喘中细胞跨内皮迁移和气道侵袭的关键决定因素。在Aim 1中，我们将确定参与选择性激活电压门控氯离子电流和调节人嗜酸性粒细胞中TGF-2 ClC3水平的胞质激酶。假设TGF-2激活蛋白激酶C-4 （PKC-4）增加电压门控氯电流，诱导p38/JNK MAP激酶和蛋白激酶C磷酸化，增加ClC-3表达，导致ClC3电流激活增强。这反过来又诱导嗜酸性粒细胞趋化性和跨内皮迁移。在Aim 2中，我们将分析cl3启动子中的转录起始位点，并确定参与人血液嗜酸性粒细胞中TGF-2选择性调节ClC- 3水平的转录因子。我们还将研究体内抑制AP-1转录对抗原致敏和激发小鼠肺嗜酸性粒细胞中ClC-3的表达和活性的影响。假设是TGF-2激活激酶的磷酸化，进而磷酸化AP-1家族转录因子和Smads，从而调节ClC3的转录。在Aim 3中，我们将研究TGF-2对人类血液嗜酸性粒细胞Cl-电流、形状变化和迁移的影响，以及从变应性鼻炎和过敏性哮喘患者的洗鼻液中分离的嗜酸性粒细胞的潜在细胞和分子途径。假设过敏性哮喘患者嗜酸性粒细胞的Cl-电流和迁移会增加，无论是在未刺激的细胞中还是在TGF-2刺激后，这取决于哮喘的慢性性。长期目标是确定TGF-2对嗜酸性粒细胞中电压门控氯离子电流的影响，并检查免疫调节剂的作用。这些研究将为慢性哮喘的病理生理过程和预防或逆转疾病的手段提供独特的见解。项目的叙述

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.