Cook Ellen B, Stahl James L, Graziano Frank M, Barney Neal P
Departments of Medicine, Visual Sciences, University of Wisconsin-Madison, School of Medicine and Public Health, Madison, Wisconsin, USA.
Invest Ophthalmol Vis Sci. 2008 Sep;49(9):3992-8. doi: 10.1167/iovs.08-1873. Epub 2008 May 16.
Previous studies demonstrated that mast cell-derived TNFalpha stimulation is critical to the upregulation of intercellular adhesion molecule (ICAM)-1 on human conjunctival epithelial cells (HCECs), which is an important feature of ocular allergic inflammation. Shedding of TNFR1 by TNFalpha-converting enzyme (TACE) is a primary mechanism for the regulation of TNFalpha-mediated events. This process has not been examined in HCECs. In this study, the authors examined the regulation of TNFR1 expression and shedding by TACE on primary HCECs and the IOBA-NHC conjunctival epithelial cell line.
Primary human conjunctival mast cells and epithelial cells were obtained from cadaveric conjunctival tissue. HCECs were incubated with and without activators (IgE-activated mast cell supernates, phorbol myristate acetate [PMA; to activate TACE], TNFalpha, and IFNgamma [to upregulate TNFR1]) for 24 hours. Pretreatment with the TACE inhibitor TAPI-2 was used to inhibit shedding of TNFR1. Supernates collected from the incubations were analyzed with ELISA for soluble TNFR1 (sTNFR1). With the use of flow cytometry, cells were harvested from these experiments for analysis of TNFR1 and ICAM-1 receptor expression.
IgE-activated conjunctival mast cell supernates upregulated the expression of TNFR1. TAPI-2 inhibited the PMA-induced release of sTNFR1 receptor and enhanced the surface expression of TNFR1 in HCECs in a dose-dependent manner. Upregulation of TNFR1 expression by priming with TAPI-2 and IFNgamma resulted in enhanced ICAM-1 expression in response to TNFalpha stimulation (significant change in the slope of the dose-response curve).
These results demonstrate that TACE promotes TNFR1 shedding in HCECs and that TNFR1 expression may be a more significant target than TNFalpha for intervention in ocular inflammation.
先前的研究表明，肥大细胞衍生的肿瘤坏死因子α（TNFα）刺激对于人结膜上皮细胞（HCECs）上细胞间黏附分子（ICAM）-1的上调至关重要，这是眼部过敏性炎症的一个重要特征。TNFα转换酶（TACE）介导的TNF受体1（TNFR1）脱落是调节TNFα介导事件的主要机制。该过程尚未在HCECs中进行研究。在本研究中，作者研究了TACE对原代HCECs和IOBA-NHC结膜上皮细胞系中TNFR1表达和脱落的调节作用。
从尸体结膜组织中获取原代人结膜肥大细胞和上皮细胞。将HCECs在有或无激活剂（IgE激活的肥大细胞上清液、佛波酯肉豆蔻酸酯乙酸盐[PMA；用于激活TACE]、TNFα和干扰素γ[用于上调TNFR1]）的情况下孵育24小时。用TACE抑制剂TAPI-2预处理以抑制TNFR1的脱落。从孵育物中收集的上清液用酶联免疫吸附测定（ELISA）分析可溶性TNFR1（sTNFR1）。通过流式细胞术，从这些实验中收获细胞以分析TNFR1和ICAM-1受体表达。
IgE激活的结膜肥大细胞上清液上调了TNFR1的表达。TAPI-2以剂量依赖性方式抑制PMA诱导的sTNFR1受体释放，并增强HCECs中TNFR1的表面表达。用TAPI-2和干扰素γ预处理上调TNFR1表达，导致在TNFα刺激下ICAM-1表达增强（剂量反应曲线斜率有显著变化）。
这些结果表明，TACE促进HCECs中TNFR1的脱落，并且TNFR1表达可能是眼部炎症干预中比TNFα更重要的靶点。
