McNAMARA N A, Van R, Tuchin O S, Fleiszig S M
Morton D. Sarver Laboratory for Cornea and Contact Lens Research, School of Optometry, University of California, Berkeley, CA, USA.
Exp Eye Res. 1999 Nov;69(5):483-90. doi: 10.1006/exer.1999.0722.
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 PAO1 algC::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.
人类皮肤、肺和气管可产生人β-防御素-2（hBD-2），这是一种可诱导、转录调控的抗菌肽，对革兰氏阴性菌具有活性，这或许可以解释这些组织对感染具有异常抵抗力的原因。由于完整的角膜上皮对感染也具有高度抵抗力，我们研究了人眼表上皮是否可能产生hBD-2。结膜上皮细胞取自人尸体眼，而角膜上皮细胞则取自尸体眼和活体人类患者的眼睛。使用逆转录-聚合酶链反应及针对hBD-2的定制引物，从人角膜和结膜上皮细胞cDNA中扩增出一段257 bp的序列，并将该DNA条带的氨基酸序列与通过GenBank（Z71389）获得的hBD-2已知基因序列进行计算机匹配。为了确定细菌副产物是否会上调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，即AK1012菌株和PAO1 algC::tet菌株，磷酸甘露糖变位酶活性均有缺陷，而该活性是合成完整多糖核心和LPS分子O侧链结构所必需的。这些突变均不影响LPS的脂质A部分。用铜绿假单胞菌野生型PAO1细菌培养上清液处理的细胞显示hBD-2 mRNA表达强烈上调，而用任一LPS突变体产生的培养上清液刺激的细胞，其hBD-2基因表达几乎没有变化或无变化。从细菌培养上清液中提取的LPS用于证明hBD-2的上调是由LPS引起的。染料木黄酮可阻断这种上调，提示蛋白酪氨酸激酶活性参与其中。因此，人角膜和结膜上皮均表达hBD-2的mRNA，且这种表达受细菌LPS上调。从LPS突变体获得的数据表明，引发哺乳动物内毒素诱导的许多病理生理表现的脂质A并非必需。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。
