Toll Like Receptors on Conjunctival Epithelium

描述(申请人提供)：我们的目标是研究人结膜上皮细胞上Toll样受体(TLR)的表达，并确定微生物配体激活TLR在眼部过敏性炎症中的潜在作用。将结膜上皮细胞作为免疫调节剂的研究主要集中在促炎细胞因子的作用上，而微生物制品的直接作用尚未被研究。初步研究表明，结膜上皮细胞表达一种模式识别受体TLR2，并对金黄色葡萄球菌多肽聚糖(SA-PGN)产生反应，释放介质，上调表面受体。通过TLRs和肥大细胞介质同时激活结膜上皮细胞可能是一种常见的现象，这可能是慢性眼部变态反应性疾病的发病原因之一。第一个具体目标将集中于确定结膜上皮细胞表达的TLR(主要是TLR1，2，4，6，9和辅助受体CD14)，以及表达的TLR是否可以被细胞因子和/或TLR配体(TLR1，2和6的SA-PGN和酵母酵母多糖；TLR4的铜绿假单胞菌脂多糖，TLR9的CpG DNA)诱导。这将包括通过印迹细胞学对从人类受试者(正常和各种疾病状态)的眼表获得的细胞进行活体检查。第二个特定目标将研究结膜上皮细胞与TLR配体的激活，微生物产物与其各自TLRs的特异性结合以及信号转导(辅助蛋白MyD88、MD2和TIRAP)。激活将被定义为上调ICAM-1和/或人类白细胞抗原-DR的表达和/或促进肿瘤坏死因子α、IL-6、IL-8和/或RANTES的释放。第三个特定目标将检测TLR配体和IgE激活的纯化结膜肥大细胞上清液同时和顺序激活结膜上皮细胞的效果。联合激活的结果将与单独使用TLR配体或IgE激活的肥大细胞上清液的激活进行比较。结膜上皮细胞和肥大细胞将从身体结膜组织中获得。评估蛋白质表达的技术包括流式细胞术、蛋白质印迹、免疫-聚合酶链式反应(印迹细胞学细胞)和酶联免疫吸附试验。评估信使核糖核酸表达的技术包括Northern印迹和RT-PCR(印迹细胞学细胞)。检查结合特异性和信号复合体的技术将包括阻断抗体、免疫共沉淀和凝胶移位分析。

DESCRIPTION (provided by applicant): Our goal is to investigate expression of Toll-like receptors (TLR) on human conjunctival epithelium and determine the potential contributions of TLR activation by microbial ligands to ocular allergic inflammation. Studies examining conjunctival epithelial cells as immunomodulators have been focused on the effects of pro-inflammatory cytokines, while the direct effects of microbial products have not been examined. Preliminary studies suggest that conjunctival epithelial cells express a pattern recognition receptor, TLR2, and respond to Staphylococcus aureus peptidoglycan (SA-PGN) with mediator release and surface receptor upregulation. It is likely that simultaneous activation of conjunctival epithelial cells via TLRs and mast cell mediators is a frequent occurrence, which may contribute to the development of chronic ocular allergic disease. The first specific aim will focus on determining the repertoire of TLRs (focusing on TLR1,2,4,6,9 and accessory receptor, CD14) expressed by conjunctival epithelial cells and whether the TLRs expressed are inducible by cytokines and/or TLR ligands (SA-PGN and yeast zymosan for TLRs 1,2, and 6; Pseudomonas aeruginosa LPS for TLR4, CpG DNA for TLR9). This will include in vivo examination of cells obtained from the ocular surface of human subjects (normal vs various disease states) via impression cytology. The second specific aim will investigate activation of conjunctival epithelial cells with TLR ligands, specific binding of microbial products to their respective TLRs and signaling (accessory proteins MyD88, MD2, and TIRAP). Activation will be defined as upregulation of ICAM-1 and/or HLA-DR expression and/or enhanced release of TNFalpha, IL-6, IL-8, and/or RANTES. The third specific aim will examine the effect of simultaneous and sequential activation of conjunctival epithelial cells with TLR ligands and supernates from IgE-activated purified conjunctival mast cells. The results of combined activation will be compared to activation with either TLR ligands or IgE-activated mast cell supernates alone. Conjunctival epithelial and mast cells will be obtained from cadaveric conjunctival tissues. Techniques to evaluate protein expression will include flow cytometry, Western blot, Immuno-PCR (in impression cytology cells), and ELISA. Techniques to evaluate mRNA expression will include Northern blot and RT-PCR (in impression cytology cells). Techniques to examine binding specificity and signaling complexes will include blocking antibodies, co-immunoprecipitation and gel shift assays.