Pulmonary Disease Unit, G. Gaslini Institute, Genoa, Italy.
Lab Invest. 2012 Aug;92(8):1140-8. doi: 10.1038/labinvest.2012.67.
Epithelial barrier permeability is altered in inflammatory respiratory disorders by a variety of noxious agents through modifications of the epithelial cell structure that possibly involve tight junction (TJ) organization. To evaluate in vitro whether pro-inflammatory cytokines involved in the pathogenesis of respiratory disorders could alter TJ organization and epithelial barrier integrity, and to characterize the signal transduction pathway involved Calu-3 airway epithelial cells were exposed to TNF-a, IL-4 and IFN-g to assess changes in: (a) TJ assembly, that is, occludin and zonula occludens (ZO)-1 expression and localization, evaluated by confocal microscopy; (b) apoptotic activity, quantified using terminal transferase deoxyuridine triphosphate nick-end labeling staining; (c) epithelial barrier integrity, detected as transmembrane electrical resistance and expressed as G(T) values; (d) epidermal growth factor receptor (EGFR)-dependent mitogenactivated protein (MAP) kinase (MAPK)/extracellular signal-regulated kinases (ERK)1/2 phosphorylation, assessed by western blotting. Exposure to cytokines for 48 h induced a noticeable downregulation of the TJ transmembrane proteins. The degree ZO-1 and occludin colocalization was 62±2% in control cultures and significantly decreased in the presence of TNF-a (47±3%), IL-4 (43±1%) and INF-g (35±3%). Although no apoptosis induction was detected following exposure to cytokines, changes in the epithelial barrier integrity were observed, with a significant enhancement in paracellular conductance. G(T) values were, respectively, 1.030±0.0, 1.300±0.04, 1.260±0.020 and 2.220±0.015 (mS/cm²)1000 in control cultures and in those exposed to TNF-a, IFN-g and IL-4. The involvement of EGFR-dependent MAPK/ERK1/2 signaling pathway in cytokine-induced damage was demonstrated by a significant increase in threonine/tyrosine phosphorylation of ERK1/2, already detectable after 5 min incubation. All these cytokine-induced changes were markedly prevented when Calu-3 cells were cultured in the presence of an EGFR inhibitor (AG1478, 1 μM) or a MAP kinase inhibitor (U0126, 25 μM). In conclusion, cytokine-induced epithelial injury includes TJ disassembly and epithelial barrier permeability alteration and involves the EGFR-dependent MAPK/ERK1/2 signaling pathway.
上皮屏障通透性在炎症性呼吸系统疾病中发生改变，这是由多种有害因子引起的，这些因子通过改变上皮细胞结构来实现，其中可能涉及紧密连接 (TJ) 的组织。为了评估参与呼吸疾病发病机制的促炎细胞因子是否会改变 TJ 组织和上皮屏障完整性，本研究采用 Calu-3 气道上皮细胞，研究细胞因子是否会改变紧密连接的组装（通过共聚焦显微镜评估闭合蛋白和封闭蛋白-1 的表达和定位）、细胞凋亡活性（通过末端转移酶脱氧尿苷三磷酸末端标记染色进行量化）、上皮屏障完整性（通过跨膜电阻检测，以 G(T) 值表示）以及表皮生长因子受体 (EGFR)-依赖性有丝分裂原激活蛋白 (MAP) 激酶 (MAPK)/细胞外信号调节激酶 (ERK)1/2 磷酸化（通过 Western blot 进行评估）。结果发现，细胞因子暴露 48 h 后，TJ 跨膜蛋白明显下调。在对照培养物中，ZO-1 和闭合蛋白的共定位程度为 62±2%，而 TNF-a（47±3%）、IL-4（43±1%）和 INF-g（35±3%）存在时明显降低。虽然细胞因子暴露后没有检测到细胞凋亡诱导，但观察到上皮屏障完整性发生变化，细胞旁通透性显著增强。G(T) 值分别为 1.030±0.0、1.300±0.04、1.260±0.020 和 2.220±0.015(mS/cm²)1000，在对照培养物和暴露于 TNF-a、IFN-g 和 IL-4 的培养物中。通过 EGFR 依赖性 MAPK/ERK1/2 信号通路的参与，ERK1/2 的苏氨酸/酪氨酸磷酸化明显增加，在孵育 5 min 后即可检测到，这表明细胞因子诱导的损伤与细胞因子诱导的损伤有关。当 Calu-3 细胞在 EGFR 抑制剂 (AG1478，1 μM) 或 MAP 激酶抑制剂 (U0126，25 μM) 存在的情况下培养时，所有这些细胞因子诱导的变化均明显受到抑制。总之，细胞因子诱导的上皮损伤包括 TJ 解体和上皮屏障通透性改变，并涉及 EGFR 依赖性 MAPK/ERK1/2 信号通路。
