Ocular surface epithelia express mRNA for human beta defensin-2
Ocular surface epithelia express mRNA for human beta defensin-2
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DOI：
10.1006/exer.1999.0722
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发表时间：
1999-11-01
影响因子：
3.4
通讯作者：
Fleiszig, SMJ
中科院分区：
文献类型：
--
作者：
McNamara, NA;Van, R;Fleiszig, SMJ
人类皮肤、肺和气管产生人β防御素-2 (hBD-2)，这是一种可诱导的、转录调节的抗生素肽，具有抗革兰氏阴性菌的活性，这可能解释了这些组织对感染的异常抵抗力。由于完整的角膜上皮对感染也具有高度抵抗力，因此我们检查了人眼表面上皮是否可能产生 hBD-2。结膜上皮细胞取自人类尸体眼睛，而角膜上皮细胞则取自尸体眼睛和活体人类患者的眼睛。使用逆转录聚合酶链式反应和 hBD-2 定制引物，从人角膜和结膜上皮细胞 cDNA 中扩增出 257 bp 序列，并且该 DNA 带的氨基酸序列与 GenBank 中提供的已知 hBD-2 基因序列 (Z71389) 进行计算机匹配。为了确定细菌副产物是否上调 hBD-2 mRNA 表达，我们用由野生型铜绿假单胞菌菌株 PAO1 或 PAO1 的两种不同脂多糖 (LPS) 突变体制备的细菌培养上清液刺激汇合的 SV 40 永生化人角膜上皮细胞。这两种突变体菌株 AK1012 和 PAO1algC::tet 均缺乏磷酸甘露糖变位酶活性，而磷酸甘露糖变位酶活性是合成完整多糖核心和 LPS 分子 O 侧链结构所必需的。这些突变都不影响 LPS 的脂质 A 部分。用铜绿假单胞菌野生型PAO1细菌培养物上清液处理的细胞表现出hBD-2 mRNA表达的强烈上调，而用由任一LPS突变体产生的培养物上清液刺激的细胞显示hBD-2基因表达很少或没有变化。从细菌培养物上清液中提取的LPS用于证明hBD-2的上调是由LPS引起的。金雀异黄素阻断了这种上调，表明蛋白酪氨酸激酶活性参与其中。因此，人角膜和结膜上皮都表达 hBD-2 的 mRNA，并且这种表达会被细菌 LPS 上调。从 LPS 突变体获得的数据表明，脂质 A 不是必需的，脂质 A 负责引发哺乳动物中内毒素诱导的许多病理生理表现。通过 LPS 的活性部分刺激内源性 hBD-2 的产生可能具有治疗潜力。 (C) 1999 年学术出版社。
Human skin, lung and trachea produce human beta defensin-2 (hBD-2), an inducible, transcriptionally regulated antibiotic peptide with activity against gram negative bacteria which may explain the unusual resistance of these tissues to infection. Since an intact corneal epithelium is also highly resistant to infection, we examined whether human ocular surface epithelia might produce hBD-2. Conjunctival epithelial cells were obtained from a human cadaver eye, while corneal epithelial cells were obtained from both a cadaver eye and the eye of a living human patient. Using reverse transcription-polymerase chain reaction and custom primers for hBD-2, a 257 bp sequence was amplified from both human corneal and conjunctival epithelial cell cDNA, and the amino acid sequence of this DNA band was computer-matched with the known gene sequence of hBD-2 available through GenBank (Z71389). To determine whether bacterial by-products upregulate hBD-2 mRNA expression, we stimulated confluent SV 40-immortalized human corneal epithelial cells with bacterial culture supernatant prepared from either wild-type P. aeruginosa strain PAO1 or two different lipopolysaccharide (LPS) mutants of PAO1. Both of these mutants, strains AK1012 and PAO1algC::tet, are deficient in phosphomannomutase activity which is required for the synthesis of both a complete polysaccharide core and the O side chain structures of the LPS molecule. Neither of these mutations affects the lipid A portion of LPS. Cells treated with P. aeruginosa wild-type PAO1 bacterial culture supernatant demonstrated strong upregulation of hBD-2 mRNA expression, whereas cells stimulated with culture supernatant produced by either of the LPS mutants showed little or no change in hBD-2 gene expression. LPS extracted from the bacterial culture supernatant was used to demonstrate that upregulation of hBD-2 is caused by LPS. Genistein blocked this upregulation suggesting that protein tyrosine kinase activity is involved. Thus, both human corneal and conjunctival epithelium express mRNA for hBD-2, and this expression is upregulated by bacterial LPS. Data obtained from LPS mutants suggest that lipid A, which is responsible for initiating a number of the pathophysiological manifestations induced by endotoxin in mammals, is not required. Stimulation of endogenous hBD-2 production via the active portion of LPS might have therapeutic potential. (C) 1999 Academic Press.