Defensin expression by the cornea:: Multiple signalling pathways mediate IL-1β stimulation of hBD-2 expression by human corneal epithelial cells
Defensin expression by the cornea:: Multiple signalling pathways mediate IL-1β stimulation of hBD-2 expression by human corneal epithelial cells
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DOI：
10.1167/iovs.02-0787
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发表时间：
2003-05-01
影响因子：
4.4
通讯作者：
Proske, RJ
中科院分区：
文献类型：
--
作者：
McDermott, AM;Redfern, RL;Proske, RJ
目的。目的：研究人β -防御素（hBD）在人角膜上皮细胞中的表达，探讨促炎因子对培养的人角膜上皮细胞中β -防御素(hBD)-2表达的影响。从尸体角膜刮取的角膜上皮细胞和培养的HCECs中提取RNA， RT-PCR检测hBD-1、-2和-3 mRNA。为了研究促炎细胞因子对防御素表达的影响，培养HCECs，然后在浓度范围（0.01-100 ng/mL）下暴露于白细胞介素(IL)-1 β或肿瘤坏死因子(TNF)- α长达36小时。在一些实验中，在加入IL-10之前，用各种细胞信号通路抑制剂对细胞进行预处理。孵育结束后，收集细胞进行RT-PCR，收集培养基用于免疫印迹法检测分泌的防御素肽。所有尸体角膜上皮组织均表达hBD-1 mRNA。8名供体角膜中有2名检测到hBD-2，而5名检测到hBD-3。所有HCECs原代培养均表达hBD-1和-3。在8个培养物中的3个中检测到微弱的hBD-2条带。猴病毒（SV）40转化的HCECs培养物在对照条件下始终表达hBD-1和-3，而不表达hBD-2。IL-10和TNFalpha分别刺激HCECs中hBD-2的表达，并且联合使用比单独使用更有效。IL-10对hBD-2 mRNA表达和蛋白分泌的影响呈浓度依赖性（10 ng/mL时最大）和时间依赖性（分别在12 h和24 h时最大）。在去除IL-10后，hBD-2 mRNA的上调持续至少24小时。NFkappaB抑制剂吡咯烷二硫代氨基甲酸酯（PDTC, 100 muM）、咖啡酸苯乙酯（CAPE, 90 muM）和MG-132 （25 muM）可阻断il -1 β刺激的hBD-2表达。p38丝裂原活化蛋白（MAP）激酶抑制剂SB203580 （5 muM）和c-Jun nh2末端激酶（JNK）抑制剂SP600125 （25 muM）部分阻断IL-10的作用（分别阻断47%和59%）。然而，PD98059，一种ERK抑制剂，没有效果。染料木黄酮（50毫微克）和地塞米松（1毫微克）也部分阻断il -10的作用（分别阻断26%和28%）。人角膜上皮表达hBD-1和-3。hBD-2通常不存在，但它的表达可以通过促炎细胞因子如il -1 β刺激，通过丝裂原活化蛋白（MAP）激酶和核因子(NF)-kappaB途径起作用。由于已知IL-1在损伤后眼表升高，目前的观察结果提供了一种机制来解释先前的发现，即hBD-2在角膜上皮再生中上调。细胞因子刺激hBD-2表达很可能提供了额外的抗感染保护，并提高了这种防御素本身可能参与伤口愈合反应的可能性。
PURPOSE. To investigate the expression of human beta-defensins (hBDs) by human corneal epithelium and determine the effects of proinflammatory cytokines on expression of human beta-defensin (hBD)-2 by human corneal epithelial cells (HCECs) in culture.METHODS. RNA was extracted from corneal epithelial cells scraped from cadaveric corneas and from cultured HCECs, and RT-PCR was performed to detect hBD-1, -2, and -3 mRNA. To study the effects of proinflammatory cytokines on expression of defensin, HCECs were cultured and then exposed to interleukin (IL)-1beta or tumor necrosis factor (TNF)-alpha for up to 36 hours,with a range of concentrations (0.01-100 ng/mL). In some experiments, cells were pretreated with various cell signaling pathway inhibitors before the addition of IL-10. At the end of the incubations, the cells were harvested for RT-PCR and the culture media collected for the detection by immunoblot analysis of secreted defensin peptide.RESULTS. All epithelial tissue collected from cadaveric corneas expressed mRNA for hBD-1. hBD-2 was detectable in two of eight donors corneas, whereas hBD-3 was detected in five. All primary cultures of HCECs expressed hBD-1 and -3. A faint band for hBD-2 was detectable in three of eight cultures. Cultures of simian virus (SV)40-transformed HCECs always expressed hBD-1 and -3, but did not express hBD-2 under control conditions. IL-10 and TNFalpha each stimulated the expression of hBD-2 in HCECs and were more effective in combination than alone. The effects of IL-10 were concentration- (maximal at 10 ng/mL) and time-dependent (maximal at 12 hours and 24 hours for hBD-2 mRNA expression and protein secretion, respectively). The upregulation of hBD-2 mRNA persisted for at least 24 hours after removal of IL-10. The NFkappaB inhibitors pyrrolidinedithiocarbamate (PDTC; 100 muM), caffeic acid phenethyl ester (CAPE; 90 muM), and MG-132 (25 muM), blocked IL-1beta-stimulated expression of hBD-2. The p38 mitogen-activated protein (MAP) kinase inhibitor SB203580 (5 muM) and the c-Jun NH2-terminal kinase (JNK) inhibitor SP600125 (25 muM) partially blocked (by 47% and 59%, respectively) the effect of IL-10. However, PD98059, an ERK inhibitor, had no effect. Genistein (50 muM) and dexamethasone (1 muM) also partially blocked (by 26% and 28%, respectively) the effect of IL-10.CONCLUSIONS. Human corneal epithelium expresses hBD-1 and -3. hBD-2 is not typically present, but its expression can be stimulated by proinflammatory cytokines such as IL-1beta, acting through mitogen-activated protein (MAP) kinase and nuclear factor (NF)-kappaB pathways. Because IL-1 is known to be increased at the ocular surface after injury, the current observations provide a mechanism to explain the previous finding that hBD-2 is upregulated in regenerating corneal epithelium. Cytokine stimulation of hBD-2 expression most likely provides additional protection against infection and raises the possibility that this defensin in particular may be involved in the wound-healing response, per se.