Toll Like Receptors on Conjunctival Epithelium

描述（由申请方提供）：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对眼部过敏性炎症的潜在贡献。检查结膜上皮细胞作为免疫调节剂的研究集中在促炎细胞因子的作用上，而尚未检查微生物产品的直接作用。初步研究表明，结膜上皮细胞表达模式识别受体TLR 2，并响应金黄色葡萄球菌肽聚糖（SA-PGN）与介质释放和表面受体上调。很可能是结膜上皮细胞通过TLR和肥大细胞介质的同时激活是经常发生的，这可能有助于慢性眼部过敏性疾病的发展。第一个具体目标将集中于确定由结膜上皮细胞表达的TLR的库（集中于TLR 1、2、4、6、9和辅助受体CD 14）以及所表达的TLR是否可由细胞因子和/或TLR配体诱导（SA-PGN和酵母聚糖用于TLR 1、2和6;铜绿假单胞菌LPS用于TLR 4，CpG DNA用于TLR 9）。这将包括通过印迹细胞学对从人类受试者（正常与各种疾病状态）的眼表面获得的细胞进行体内检查。第二个具体目标将研究结膜上皮细胞与TLR配体的活化，微生物产物与其各自TLR的特异性结合和信号传导（辅助蛋白MyD 88，MD 2和TIRAP）。活化将被定义为ICAM-1和/或HLA-DR表达的上调和/或TNF α、IL-6、IL-8和/或RANTES的释放增强。第三个具体目标将检查用TLR配体和来自IgE活化的纯化结膜肥大细胞的上清液同时和顺序活化结膜上皮细胞的效果。将组合活化的结果与单独用TLR配体或IgE活化的肥大细胞上清液的活化进行比较。结膜上皮细胞和肥大细胞将从尸体结膜组织中获得。评估蛋白质表达的技术将包括流式细胞术、蛋白质印迹、免疫-PCR（在印迹细胞学细胞中）和ELISA。评估mRNA表达的技术将包括北方印迹和RT-PCR（在印迹细胞学细胞中）。检查结合特异性和信号复合物的技术将包括阻断抗体、免疫共沉淀和凝胶位移测定。

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.