Narayanan Srihari, Glasser Adrian, Hu Ying-Sheng, McDermott Alison M
College of Optometry, University of Houston, 505 J. Davis Armistead Building, 4901 Calhoun Road, Houston, TX 77204-2020, USA.
Exp Eye Res. 2005 Feb;80(2):175-83. doi: 10.1016/j.exer.2004.08.027.
The purpose of this study was to characterize the pattern of cytokine gene expression by human corneal epithelial cells (HCEC) in response to interleukin-1 (IL-1). Primary cultured HCEC (P-HCEC) or SV40 transformed HCEC (SV40-HCEC) were treated for 6 hr with serum-free growth-media alone or with recombinant human IL-1beta or IL-1alpha (10 ng ml(-1)). 33P labeled cDNA was generated from total RNA, then hybridized to a human cytokine expression array. An autoradiograph was generated for each experimental condition and results analysed semi-quantitatively. Reverse transcription polymerase chain reaction (RT-PCR) was performed to detect mRNA for IL-8, growth related oncogene-beta (GRO-beta), intercellular adhesion molecule (ICAM)-1 and Ephrin A5. P-HCEC and SV40-HCEC demonstrated comparable cytokine profiles. For P-HCEC (n=2) the expression of 35 genes was upregulated or only detectable following IL-1beta treatment whereas the expression of nine genes was downregulated or undetectable after IL-1beta treatment. In SV40-HCEC (n=3), the expression of 48 genes was upregulated or only detectable following IL-1beta treatment and the expression of 10 genes was downregulated or undetectable after IL-1beta treatment. Some genes that demonstrated increased expression included cadherin-5, ICAM-1, GRO-alpha, GRO-beta, GRO-gamma, Activin A (bA subunit), tumor necrosis factor-alpha, IL-6, and IL-8. Genes that showed decreased expression included the chemokine receptor-CXCR-4, ciliary neurotrophic factor (CNTF), c-kit ligand, Ephrin A5, G-protein coupled receptor RDC-1 and FGF family FGFR2. Bayesian analysis of the SV40-HCEC data (n=3) revealed the expression of 15 genes that were significantly (p<0.05) differentially regulated. Within these 15 genes, the expression of chemokines (GRO-alpha, GRO-beta, IL-8), fibroblast growth factor 13 and the cytokine IL-6 were the most upregulated, while ephrin A5 and chemokine receptor-4 were the most downregulated. IL-1alpha treatment (n=1 P-HCEC; n=1 SV40-HCEC) produced results very similar to IL-1beta treatment. RT-PCR revealed differential regulation of IL-8, GRO-beta, ICAM-1 and ephrin A5 in accordance with gene array data. In conclusion, the data demonstrate that IL-1 treatment of HCEC differentially regulates the expression of other cytokine and related genes, thus adding to the body of evidence that IL-1 is a major mediator of ocular surface inflammatory reactions. Since the expression of a large number of genes can be studied simultaneously, gene array studies such as these offers the advantage of understanding global changes in response to a specific stimulus. Thus our study provides insight in to the ocular surface response in conditions of inflammation and corneal wound healing where the levels of IL-1 are known to be increased.
本研究的目的是描述人角膜上皮细胞（HCEC）对白细胞介素-1（IL-1）应答时细胞因子基因表达的模式。原代培养的HCEC（P-HCEC）或SV40转化的HCEC（SV40-HCEC）分别用无血清生长培养基单独处理，或用重组人IL-1β或IL-1α（10 ng/ml）处理6小时。从总RNA生成33P标记的cDNA，然后与人类细胞因子表达阵列杂交。针对每个实验条件生成放射自显影片，并对结果进行半定量分析。进行逆转录聚合酶链反应（RT-PCR）以检测IL-8、生长相关癌基因-β（GRO-β）、细胞间黏附分子（ICAM）-1和Ephrin A5的mRNA。P-HCEC和SV40-HCEC表现出相当的细胞因子谱。对于P-HCEC（n = 2），35个基因的表达在IL-1β处理后上调或仅可检测到，而9个基因的表达在IL-1β处理后下调或不可检测。在SV40-HCEC（n = 3）中，48个基因的表达在IL-1β处理后上调或仅可检测到，10个基因的表达在IL-1β处理后下调或不可检测。一些表达增加的基因包括钙黏蛋白-5、ICAM-1、GRO-α、GRO-β、GRO-γ、激活素A（βA亚基）、肿瘤坏死因子-α、IL-6和IL-8。表达降低的基因包括趋化因子受体-CXCR-4、睫状神经营养因子（CNTF）、c-kit配体、Ephrin A5、G蛋白偶联受体RDC-1和FGF家族FGFR2。对SV40-HCEC数据（n = 3）的贝叶斯分析显示15个基因的表达有显著差异（p<0.05）。在这15个基因中，趋化因子（GRO-α、GRO-β、IL-8）、成纤维细胞生长因子13和细胞因子IL-6的表达上调最为明显，而Ephrin A5和趋化因子受体-4的表达下调最为明显。IL-1α处理（n = 1 P-HCEC；n = 1 SV40-HCEC）产生的结果与IL-1β处理非常相似。RT-PCR显示IL-8、GRO-β、ICAM-1和Ephrin A5的差异调节与基因阵列数据一致。总之，数据表明IL-1处理HCEC可差异调节其他细胞因子及相关基因的表达，从而进一步证明IL-1是眼表炎症反应的主要介质。由于可以同时研究大量基因的表达，此类基因阵列研究具有了解对特定刺激的整体反应变化的优势。因此，我们的研究为了解炎症和角膜伤口愈合情况下眼表反应提供了见解，已知在这些情况下IL-1水平会升高。
