TGF-BETA, CHLORIDE CHANNELS AND MIGRATION OF EOSINOPHILS

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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.