Sharif N A, Wiernas T K, Howe W E, Griffin B W, Offord E A, Pfeifer A M
Alcon Laboratories, Inc., Fort Worth, Texas 76134-2099, USA.
Invest Ophthalmol Vis Sci. 1998 Dec;39(13):2562-71.
To investigate the epithelial nature of primary and SV40 virus-immortalized human corneal epithelial (CEPI) cells and to study a variety of functional responses to some key inflammatory agents (bradykinin [BK], histamine, and platelet-activating factor [PAF]) and their antagonists in these cells.
Primary CEPI (P-CEPI) and clone 4 of the SV40 virus-immortalized (CEPI-17-CL4) cells were analyzed for their interaction with several monoclonal antibodies selective for various cytokeratins to define their immunocytochemical characteristics and phenotypic traits. Both cell types were tested for their ability to respond to BK, histamine, and PAF and their antagonists, using the production of [3H]inositol phosphates ([3H]IPs) as an index of receptor activation. The ability of BK, PAF, and histamine to stimulate cytokine release and the induction of mRNA for matrix metalloproteinase-1 (MMP-1) were also studied using enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction techniques, respectively.
P-CEPI and CEPI-17-CL4 cells were both shown to possess the epithelial cell cytokeratins labeled with AE1 and AE3 antibodies. The potencies (EC50s) of BK, histamine, and PAF were similar for stimulating [3H]IPs production in P-CEPI and CEPI-17-CL4 cells: BK = 2.27 to 2.99 nM, PAF = 17.1 to 18.26 nM, and histamine = 1.65 to 5.74 microM (all n = 3 to 6). Both cell types also responded similarly to receptor-selective antagonists for BK, PAF, and histamine (Hoe-140: Ki = 10.1 to 11.9 nM; PCA-4248: Ki = 315 to 421 nM; triprolidine: Ki = 0.8 to 4.76 nM; all n = 5 to 10). Histamine (100 microM) and interleukin-1alpha (IL-1alpha, 10 ng/ml) significantly stimulated IL-6 and granulocyte macrophage colony-stimulating factor release, and histamine, BK, and PAF stimulated the mRNA for MMP-1 in these cells.
These studies have shown that the primary and immortalized human corneal epithelial cells express functional BK (a B2 subtype), histamine (an H1 subtype), and PAF receptors and exhibit very similar immunocytochemical, signal transduction, and pharmacological properties. Therefore, the CEPI-17-CL4 cells (currently at passage 220) appear to provide a useful representative in vitro model system to study the physiological and pathologic aspects of the human corneal epithelium.
研究原代及SV40病毒永生化人角膜上皮（CEPI）细胞的上皮性质，并研究这些细胞对一些关键炎症介质（缓激肽[BK]、组胺和血小板活化因子[PAF]）及其拮抗剂的多种功能反应。
分析原代CEPI（P-CEPI）细胞和SV40病毒永生化（CEPI-17-CL4）细胞的克隆4与几种针对不同细胞角蛋白的单克隆抗体的相互作用，以确定其免疫细胞化学特征和表型特征。使用[3H]肌醇磷酸酯（[3H]IPs）的产生作为受体激活的指标，检测这两种细胞类型对BK、组胺和PAF及其拮抗剂的反应能力。还分别使用酶联免疫吸附测定和逆转录-聚合酶链反应技术研究了BK、PAF和组胺刺激细胞因子释放以及诱导基质金属蛋白酶-1（MMP-1）mRNA的能力。
P-CEPI细胞和CEPI-17-CL4细胞均显示表达用AE1和AE3抗体标记的上皮细胞角蛋白。BK、组胺和PAF在P-CEPI细胞和CEPI-17-CL4细胞中刺激[3H]IPs产生的效力（EC50）相似：BK = 2.27至2.99 nM，PAF = 17.1至18.26 nM，组胺 = 1.65至5.74 microM（所有n = 3至6）。这两种细胞类型对BK、PAF和组胺的受体选择性拮抗剂也有相似的反应（Hoe-140：Ki = 10.1至11.9 nM；PCA-4248：Ki = 315至421 nM；曲普利啶：Ki = 0.8至4.76 nM；所有n = 5至10）。组胺（100 microM）和白细胞介素-1α（IL-1α，10 ng/ml）显著刺激IL-6和粒细胞巨噬细胞集落刺激因子释放，组胺、BK和PAF刺激这些细胞中MMP-1的mRNA。
这些研究表明，原代及永生化人角膜上皮细胞表达功能性BK（B2亚型）、组胺（H1亚型）和PAF受体，并表现出非常相似的免疫细胞化学、信号转导和药理学特性。因此，CEPI-17-CL4细胞（目前传代至220代）似乎为研究人角膜上皮的生理和病理方面提供了一个有用的体外模型系统。
