Narayanan Srihari, Miller William L, McDermott Alison M
College of Optometry, University of Houston, Houston, Texas 77204, USA.
Invest Ophthalmol Vis Sci. 2003 Sep;44(9):3795-801. doi: 10.1167/iovs.02-1301.
The goals of this study were to investigate whether beta-defensins are differentially expressed in the conjunctival epithelium of patients with moderate dry eye when compared with normal subjects and whether proinflammatory cytokines or bacteria can modulate the expression of human beta-defensins (hBDs)-1, -2, and -3 by conjunctival epithelial cells.
RNA extracted from conjunctival impression cytology specimens of eight normal subjects and nine patients with moderate dry eye was used in RT-PCR to detect mRNA for hBDs-1, -2, and -3. Two conjunctival epithelial cell lines and primary cultured conjunctival epithelial cells were treated with proinflammatory cytokines or heat-killed Pseudomonas aeruginosa. RT-PCR and immunoblot analysis were used to detect mRNA for hBD-1, -2, and -3 and protein secretion of hBD-2, respectively.
hBD-2 message was detected in RNA samples of eight of nine patients with dry eye, but not in any of the normal subjects' samples, whereas hBD-1 and -3 were detected in all subjects tested. RT-PCR revealed an upregulation of hBD-2 but no difference in expression of hBD-1 and -3 in cultured conjunctival cells after a 24-hour treatment with 10 ng/mL interleukin (IL)-1beta, IL-1beta and tumor necrosis factor-alpha (10 ng/mL) or heat-killed Pseudomonas aeruginosa (1 million colony-forming units; n = 3). hBD-2 expression was upregulated from 4 hours of treatment with IL-1beta (at 10 ng/mL; (n = 2-3) and at a concentration of 0.1 ng/mL IL-1beta (24-hour treatment; n = 2-3). Immunoblots demonstrated protein secretion results corresponding to the RT-PCR data.
hBD-2 was expressed only in the conjunctival epithelium of patients with moderate dry eye. Because cytokines such as IL-1beta and TNF-alpha induced the expression of hBD-2 by conjunctival epithelial cells and because increased proinflammatory cytokine activity is a feature of dry eye disease, it can be speculated that the hBD-2 upregulation observed in subjects with moderate dry eye is mediated by proinflammatory cytokine activity.
本研究的目的是调查与正常受试者相比，β-防御素在中度干眼症患者结膜上皮中是否存在差异表达，以及促炎细胞因子或细菌是否能调节结膜上皮细胞中人β-防御素（hBDs）-1、-2和-3的表达。
从8名正常受试者和9名中度干眼症患者的结膜印迹细胞学标本中提取RNA，用于逆转录聚合酶链反应（RT-PCR）检测hBDs-1、-2和-3的信使核糖核酸（mRNA）。用促炎细胞因子或热灭活的铜绿假单胞菌处理两种结膜上皮细胞系和原代培养的结膜上皮细胞。分别用RT-PCR和免疫印迹分析检测hBD-1、-2和-3的mRNA以及hBD-2的蛋白分泌。
9名干眼症患者中有8名的RNA样本中检测到hBD-2信使，但正常受试者的样本中均未检测到，而在所有检测的受试者中均检测到hBD-1和-3。RT-PCR显示，在用10纳克/毫升白细胞介素（IL）-1β、IL-1β和肿瘤坏死因子-α（10纳克/毫升）或热灭活的铜绿假单胞菌（100万个菌落形成单位；n = 3）处理24小时后，培养的结膜细胞中hBD-2上调，但hBD-1和-3的表达无差异。在用IL-1β（10纳克/毫升；n = 2 - 3）和0.1纳克/毫升IL-1β（24小时处理；n = 2 - 3）处理4小时后，hBD-2表达上调。免疫印迹显示蛋白分泌结果与RT-PCR数据一致。
hBD-2仅在中度干眼症患者的结膜上皮中表达。由于IL-1β和肿瘤坏死因子-α等细胞因子可诱导结膜上皮细胞表达hBD-2，且促炎细胞因子活性增加是干眼症的一个特征，因此可以推测，在中度干眼症患者中观察到的hBD-2上调是由促炎细胞因子活性介导的。
