Cell lines of pulmonary and non-pulmonary origin as tools to study the effects of house dust mite proteinases on the regulation of epithelial permeability.
Cell lines of pulmonary and non-pulmonary origin as tools to study the effects of house dust mite proteinases on the regulation of epithelial permeability.
复制标题
肺部和非肺部来源的细胞系作为研究屋尘螨蛋白酶对上皮通透性调节的影响的工具。
DOI：
10.1046/j.1365-2222.1998.00354.x
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Robinson,C
中科院分区：
文献类型：
--
作者：
Winton,HL;Wan,H;Cannell,MB;Gruenert,DC;Thompson,PJ;Garrod,DR;Stewart,GA;Robinson,C
屋尘螨(HDM)产生的过敏性和非过敏性蛋白水解酶(HDM)可导致呼吸道上皮细胞之间的黏附丧失，从而导致细胞间的功能凝聚力丧失，从而有助于过敏原的呈递。目的观察人呼吸道上皮细胞系CALU-3、16HBE140、NCI-H292和A549，以及狗肾上皮细胞株−，并建立其在HDM蛋白酶作用研究中的应用。翼状旁腺上皮通透性。建立一种改进的方法来测量HDM蛋白酶和其他刺激物诱导的上皮细胞通透性的变化。方法利用一种允许数学估计和减少非细胞扩散限制的技术，通过测量清除来计算单层上皮细胞对甘露醇的通透性。在控制条件下和用HDM蛋白水解酶(分为丝氨酸蛋白酶和半胱氨酸蛋白酶)或细胞外钙离子螯合单层后，研究其通透性。用荧光抗体染色观察细胞是否表达紧密连接(ZO-1染色)、桥粒(桥粒染色)和小带黏附(E-钙粘附素染色)。结果CALU-3细胞系表达功能性紧密连接、桥粒和小带黏附。CALU-3单层对甘露醇的清除率和渗透率较低，与广泛鉴定的MDCK细胞株相似。HDM蛋白水解酶部分或钙离子螯合处理均可显著提高膜的清除率和通透性。在对照条件下，16HBE14o−细胞对甘露醇的通透性也很低，并且表达来自主要细胞间连接的类似的功能蛋白。相比之下，NCI-H292和A549细胞株在紧密连接方面存在功能缺陷，尽管它们确实在更大程度上表达桥粒和小带粘附性。结论CALU-3和16HBE140−细胞系是研究HDM蛋白对呼吸道上皮细胞功能影响机制的有用工具。半胱氨酸和丝氨酸类的HDM蛋白被发现扰乱上皮细胞的黏附和功能。
BackgroundAllergenic and non‐allergenic proteinases from house dust mites (HDM) cause loss of adhesion between airway epithelial cells that may result in a loss of functional cohesion between the cells and thus assist in allergen presentation. Improved cellular assay systems are needed to ascertain the mechanisms involved.ObjectivesTo survey a series of epithelial cell lines (Calu‐3, 16HBE14o−, NCI‐H292 and A549 from human airways, and MDCK from dog kidney) and establish their utility for studies of the effects of HDM proteinases fromD. pteronyssinuson epithelial permeability. To develop an improved method for measuring changes in epithelial permeability induced by HDM proteinases and other provocants.MethodsThe permeability of epithelial monolayer cultures to mannitol was calculated from measurements of clearance using a technique that permits mathematical estimation and reduction of non‐cellular diffusional constraints. Permeability was studied under control conditions and after perturbation of monolayers with HDM proteinases (separated into serine‐ and cysteine‐proteinase classes) or chelation of extracellular Ca2+. Fluorescent antibody staining was used to investigate whether the cells expressed tight junctions (staining of ZO‐1), desmosomes (staining of desmoplakin) and zonulae adherentes (staining of E‐cadherin).ResultsThe Calu‐3 line was identified as an airway cell line that expressed functional tight junctions, desmosomes and zonulae adherentes. Calu‐3 monolayers exhibited a low clearance and permeability to mannitol, similar to that seen in the extensively characterized MDCK cell line. Clearance and permeability were significantly increased by treatment with either HDM proteinase fraction or by calcium chelation. 16HBE14o−cells also had a low permeability to mannitol under control conditions and expressed a similar repertoire of functional proteins from major intercellular junctions. In contrast, NCI‐H292 and A549 cell lines were functionally deficient in tight junctions, although they did express desmosomes and zonulae adherentes to a greater extent. Epithelial permeability was found to be a more appropriate and sensitive index of epithelial perturbation than was tracer clearance.ConclusionThese results suggest that the Calu‐3 and 16HBE14o−cell lines are useful tools in studying the mechanism of HDM proteinases on airway epithelial cell function. HDM proteinases of both cysteine and serine mechanistic classes were found to perturb epithelial adhesion and function.