Regulation of Pseudomonas aeruginosa internalization after contact lens wear in vivo and in serum-free culture by ocular surface cells.
Regulation of Pseudomonas aeruginosa internalization after contact lens wear in vivo and in serum-free culture by ocular surface cells.
复制标题
DOI：
10.1167/iovs.05-1332
复制
发表时间：
2006-08
影响因子：
4.4
通讯作者：
Naoka Yamamoto;Nobutaka Yamamoto;M. Petroll;J. Jester;H. Cavanagh
中科院分区：
文献类型：
--
作者：
Naoka Yamamoto;Nobutaka Yamamoto;M. Petroll;J. Jester;H. Cavanagh
目的探讨角膜接触镜（CL）和铜绿假单胞菌（PA）感染对角膜上皮细胞囊性纤维化跨膜传导调节因子（CFTR）定位的影响，以及CFTR介导PA结合和内化过程中脂筏形成与CFTR的关系。方法在PA暴露前后和无血清人角膜上皮细胞培养物（hTCEpi）中，在兔角膜结膜上皮（有/无CL磨损）中评价CFTR免疫定位。用Alexa 555缀合的霍乱毒素β-亚基观察脂筏形成。局部用菲律宾肽预处理后，通过庆大霉素存活试验在体内测定脂筏参与PA内化。通过用CFTR肽或LPS阻断来评估CFTR在PA结合和内化中的参与。结果CL配戴后抗CFTR染色增强，但CFTR定位与眼表细胞结合PA无关。结膜上皮细胞CFTR染色，但不结合或内化PA。角膜上皮细胞在体内没有染色CFTR，除非受到接触镜诱导的缺氧的挑战。LPS能显著抑制hTCEpi对PA的内化（P < 0.01），而CFTR肽对hTCEpi的内化无明显影响。值得注意的是，正常结膜上皮细胞显示脂筏形成和CFTR染色，但不结合PA。在CL磨损后，通过菲律宾肽抑制筏形成阻断了PA在体内的内化。结论CFTR不是眼表PA感染的主要受体，缺氧CL刺激后，无论是脂筏还是CFTR定位都不能单独预测PA结合;然而，脂筏对CL介导的PA内化至关重要。
PURPOSE To determine the effects of contact lenses (CLs) and Pseudomonas aeruginosa (PA) infection on localization of cystic fibrosis transmembrane conductance regulator (CFTR) on corneal surface epithelial cells and the association between lipid raft formation and CFTR in mediating PA binding and internalization in ocular surface epithelium. METHODS CFTR immunolocalization was evaluated in vivo in rabbit corneal-conjunctival epithelium (with/without CL wear) before and after PA exposure and in serum-free human corneal epithelial cell culture (hTCEpi). Lipid raft formation was visualized with Alexa555-conjugated cholera toxin beta-subunit. Lipid raft involvement in PA internalization was assayed in vivo by gentamicin survival assays after topical filipin pretreatment. Involvement of CFTR in PA binding and internalization was evaluated by blockade with CFTR peptides or LPS. RESULTS CL wear in vivo enhanced anti-CFTR staining, but CFTR localization did not correlate with the PA binding by ocular surface cells. Conjunctival epithelial cells stained for CFTR but did not bind or internalize PA. Corneal epithelial cells in vivo did not stain for CFTR unless challenged by contact lens-induced hypoxia. PA internalization by hTCEpi was significantly inhibited by LPS (P < 0.01), but not by CFTR peptides. Remarkably, normal conjunctival epithelial cells showed lipid raft formation and CFTR staining but did not bind PA. Inhibition of raft formation by filipin blocked PA internalization in vivo after CL wear. CONCLUSIONS CFTR is not the predominant receptor for ocular surface PA infection, and after hypoxic CL challenge, neither lipid rafts nor CFTR localization alone predicts PA binding; however, lipid rafts are critical to CL-mediated PA internalization.