TNF-α 介导致支气管屏障破坏及 src 家族激酶激活的调控。

TNF-α-mediated bronchial barrier disruption and regulation by src-family kinase activation.

机构信息

Academic Unit of Clinical and Experimental Sciences, Sir Henry Wellcome Laboratories, University of Southampton Faculty of Medicine, University Hospital Southampton, Southampton, United Kingdom.

Academic Unit of Clinical and Experimental Sciences, Sir Henry Wellcome Laboratories, University of Southampton Faculty of Medicine, University Hospital Southampton, Southampton, United Kingdom; Southampton NIHR Respiratory Biomedical Research Unit, Sir Henry Wellcome Laboratories, University of Southampton Faculty of Medicine, University Hospital Southampton, Southampton, United Kingdom.

出版信息

J Allergy Clin Immunol. 2013 Sep;132(3):665-675.e8. doi: 10.1016/j.jaci.2013.03.005. Epub 2013 Apr 28.

DOI:10.1016/j.jaci.2013.03.005

PMID:23632299

Abstract

BACKGROUND

Because TNF-α is increased in severe asthma, we hypothesized that TNF-α contributes to barrier dysfunction and cell activation in bronchial epithelial cells. We further hypothesized that src-family kinase inhibition would improve barrier function in healthy cells in the presence of TNF-α and directly in cultures of severe asthmatic cells where the barrier is disrupted.

OBJECTIVES

We assessed the effect of TNF-α, with or without src-family kinase inhibitor SU6656, on barrier properties and cytokine release in differentiated human bronchial epithelial cultures. Further, we tested the effect of SU6656 on differentiated primary cultures from severe asthma.

METHODS

Barrier properties of differentiated human bronchial epithelial air-liquid interface cultures from healthy subjects and subjects with severe asthma were assessed with transepithelial electrical resistance and fluorescent dextran passage. Proteins were detected by immunostaining or Western blot analysis and cytokines by immunoassay. Mechanisms were investigated with src kinase and other inhibitors.

RESULTS

TNF-α lowered transepithelial electrical resistance and increased fluorescent dextran permeability, caused loss of occludin and claudins from tight junctions with redistribution of p120 catenin and E-cadherin from adherens junctions, and also increased endogenous TNF-α, IL-6, IL-1β, IL-8, thymic stromal lymphoprotein, and pro-matrix metalloprotease 9 release. SU6656 reduced TNF-α-mediated paracellular permeability changes, restored occludin, p120, and E-cadherin and lowered autocrine TNF-α release. Importantly, SU6656 improved the barrier properties of severe asthmatic air-liquid interface cultures. Redistribution of E-cadherin and p120 was observed in bronchial biopsies from severe asthmatic airways.

CONCLUSIONS

Inhibiting TNF-α or src kinases may be a therapeutic option to normalize barrier integrity and cytokine release in airway diseases associated with barrier dysfunction.

摘要

背景

由于 TNF-α 在严重哮喘中增加，我们假设 TNF-α 有助于支气管上皮细胞的屏障功能障碍和细胞激活。我们进一步假设，在 TNF-α 存在的情况下，Src 家族激酶抑制剂 SU6656 会改善健康细胞的屏障功能，并直接改善屏障功能受损的严重哮喘细胞培养物的屏障功能。

目的

我们评估了 TNF-α，无论是否存在 Src 家族激酶抑制剂 SU6656，对分化的人支气管上皮细胞培养物的屏障特性和细胞因子释放的影响。此外，我们测试了 SU6656 对严重哮喘原代培养物的影响。

方法

使用跨上皮电阻和荧光葡聚糖通过评估来自健康受试者和严重哮喘患者的分化的人支气管上皮细胞气液界面培养物的屏障特性。通过免疫染色或 Western blot 分析检测蛋白质，通过免疫测定法检测细胞因子。通过Src 激酶和其他抑制剂研究机制。

结果

TNF-α 降低了跨上皮电阻并增加了荧光葡聚糖的通透性，导致紧密连接中的 occludin 和 claudins 丢失，p120 连环蛋白和 E-钙粘蛋白从粘着连接重新分布，还增加了内源性 TNF-α、IL-6、IL-1β、IL-8、胸腺基质淋巴生成素和促基质金属蛋白酶 9 的释放。SU6656 减少了 TNF-α 介导的旁细胞通透性变化，恢复了 occludin、p120 和 E-钙粘蛋白，并降低了自分泌 TNF-α 的释放。重要的是，SU6656 改善了严重哮喘气液界面培养物的屏障特性。在严重哮喘气道的支气管活检中观察到 E-钙粘蛋白和 p120 的重新分布。