Brito Beatriz E, Zamora David O, Bonnah Robert A, Pan Yuzhen, Planck Stephen R, Rosenbaum James T
Cellular and Molecular Pathology Laboratory, Experimental Medicine Center, Venezuelan Institute for Scientific Research (IVIC), Apdo 21827, Caracas 1020-A, Venezuela.
Exp Eye Res. 2004 Aug;79(2):203-8. doi: 10.1016/j.exer.2004.03.012.
We investigated the expression of the functional endotoxin receptor proteins Toll-like receptor-4 and CD14 in human eyes. Toll-like receptor-4 and CD14 proteins were detected by immunohistochemical analysis of sections of whole human eyes embedded in paraffin with monoclonal antibodies against human toll-like receptor-4 (HTA-125), human CD14 (RPA-M1), or as a control, an irrelevant mouse IgG1k (MOPC-21). Incubation of explants with a neutralizing anti-toll-like receptor-4 monoclonal antibody was used to determine if lipopolysaccharide stimulation of tumor necrosis factor or interleukin-6 secretion was dependent on Toll-like receptor-4 activity. Reverse transcription-polymerase chain reaction was used to detect mRNAs for toll-like receptor-4, tumor necrosis factor-alpha, interleukin-1beta, interleukin-6 and interleukin-8, 3 hr after stimulation of cultured iris microvascular endothelial cells. By immunohistochemistry, human ciliary body non-pigmented epithelial cells showed strong expression of the endotoxin receptor proteins, toll-like receptor-4 and CD14. Toll-like receptor-4 antibodies significantly inhibited lipopolysaccharide-stimulated tumor necrosis factor secretion by the ciliary body. Toll-like receptor-4 mRNA was constitutively expressed in iris endothelial cells and slightly down-regulated by endotoxin. mRNA levels for tumor necrosis factor-alpha, interleukin-1beta, interleukin-6 and interleukin-8 were all increased by endotoxin treatment. This is the first report that shows intraocular (ciliary body and iris) expression of toll-like receptor-4, other than in cornea. Our results show that the ciliary body also expresses CD14, which is anatomically colocalized with toll-like receptor-4. This suggests a potential interaction between both molecules during endotoxin activation of ciliary body cells. The juxtaposition of toll-like receptor-4 and CD14 in the anterior uveal tract helps to explain the sensitivity of the iris/ciliary body to bacterial endotoxin as seen in the standard animal model of endotoxin-induced uveitis.
我们研究了功能性内毒素受体蛋白Toll样受体4（Toll-like receptor-4，TLR-4）和CD14在人眼中的表达情况。采用抗人Toll样受体4（HTA-125）、人CD14（RPA-M1）的单克隆抗体，或作为对照的无关小鼠IgG1k（MOPC-21），对石蜡包埋的整个人眼切片进行免疫组织化学分析，以检测Toll样受体4和CD14蛋白。用中和性抗Toll样受体4单克隆抗体孵育外植体，以确定脂多糖刺激肿瘤坏死因子或白细胞介素-6分泌是否依赖于Toll样受体4的活性。在培养的虹膜微血管内皮细胞受到刺激3小时后，采用逆转录聚合酶链反应检测Toll样受体4、肿瘤坏死因子-α、白细胞介素-1β、白细胞介素-6和白细胞介素-8的mRNA。通过免疫组织化学方法发现，人睫状体无色素上皮细胞中内毒素受体蛋白Toll样受体4和CD14呈强表达。Toll样受体4抗体可显著抑制脂多糖刺激的睫状体肿瘤坏死因子分泌。Toll样受体4 mRNA在虹膜内皮细胞中组成性表达，并在内毒素作用下略有下调。内毒素处理可使肿瘤坏死因子-α、白细胞介素-1β、白细胞介素-6和白细胞介素-8的mRNA水平均升高。这是首次报道除角膜外，眼内（睫状体和虹膜）存在Toll样受体4的表达。我们的结果表明，睫状体也表达CD14，其在解剖学上与Toll样受体4共定位。这提示在睫状体细胞的内毒素激活过程中，这两种分子之间可能存在相互作用。Toll样受体4和CD14在前葡萄膜中的并置有助于解释在内毒素诱导的葡萄膜炎标准动物模型中所观察到的虹膜/睫状体对细菌内毒素的敏感性。
