Department of Visual Sciences, Division of Ophthalmology, Nihon University School of Medicine, Tokyo, Japan.
Invest Ophthalmol Vis Sci. 2014 Apr 15;55(4):2419-23. doi: 10.1167/iovs.13-13265.
We evaluated an anti-inflammatory effect of TNF receptor 1 (TNFR1) ectodomain shedding in ocular surface.
Human corneal epithelial cell (HCEC) was first pretreated by TNF-α. Ectodomain shedding was stimulated by uridine triphosphate (UTP) or peptidoglycan (PGN), with or without shedding inhibition using TNF-α processing inhibitor (TAPI). The phosphorylation of the NF-κB inhibitory protein, IκB, was assessed by Western blotting and concentrations of soluble TNFR1 (sTNFR1) in culture medium were analyzed by ELISA. Tear fluid from patients with Sjögren syndrome and graft-versus-host disease (GVHD) was collected and analyzed by ELISA for sTNFR1 concentration. Five dry eye patients underwent topical treatment using diquafosol sodium eye drops, a purinergic P2Y2 receptor agonist, and the tear fluid of the patients was sampled before and 4 weeks after the treatment for sTNFR1 ELISA.
Phosphorylation of IκB was diminished by adding UTP or PGN, and this down-regulation of IκB phosphorylation was reversed by adding TAPI. In HCEC medium, sTNFR1 release was increased significantly by adding UTP or PGN, and inhibited significantly by adding TAPI. In the tears of the patients with Sjögren syndrome and GVHD, sTNFR1 expression was upregulated. In the tears of the patients with short breakup time (BUT) dry eye, sTNFR1 concentrations (ng/mL) in the tears were 1.30 ± 0.58 ng/mL for the pretreatment baseline, and 1.64 ± 0.70 after treatment, statistically significantly higher than those for the pretreatment (P < 0.01).
Ectodomain shedding of sTNFR1 blocked TNF-α-induced intracellular signaling in corneal epithelium. The upregulation of sTNFR1 in inflamed ocular surfaces suggests an anti-inflammatory role of sTNFR1 ectodomain shedding at the ocular surface.
我们评估了肿瘤坏死因子受体 1（TNFR1）胞外结构域脱落对眼表的抗炎作用。
首先用 TNF-α 预处理人角膜上皮细胞（HCEC）。用尿苷三磷酸（UTP）或肽聚糖（PGN）刺激胞外结构域脱落，用 TNF-α 处理抑制剂（TAPI）抑制脱落。用 Western blot 检测 NF-κB 抑制蛋白 IκB 的磷酸化，用 ELISA 分析培养上清液中可溶性 TNFR1（sTNFR1）的浓度。收集干燥综合征和移植物抗宿主病（GVHD）患者的泪液，用 ELISA 分析 sTNFR1 浓度。5 例干眼症患者使用嘌呤能 P2Y2 受体激动剂二氯酚酸钠滴眼剂进行局部治疗，在治疗前和治疗后 4 周采集患者的泪液，用于 sTNFR1 ELISA。
加入 UTP 或 PGN 可使 IκB 磷酸化减少，而加入 TAPI 可逆转 IκB 磷酸化的下调。在 HCEC 培养基中，加入 UTP 或 PGN 可显著增加 sTNFR1 的释放，并显著抑制 TAPI 的释放。在干燥综合征和 GVHD 患者的泪液中，sTNFR1 表达上调。在短泪膜破裂时间（BUT）干眼症患者的泪液中，sTNFR1 浓度（ng/mL）在治疗前基础值为 1.30±0.58ng/mL，治疗后为 1.64±0.70ng/mL，与治疗前相比显著升高（P<0.01）。
sTNFR1 胞外结构域脱落阻断了角膜上皮细胞中 TNF-α 诱导的细胞内信号转导。炎性眼表 sTNFR1 的上调提示 sTNFR1 胞外结构域脱落在眼表具有抗炎作用。
