Lee A, Chow D, Haus B, Tseng W, Evans D, Fleiszig S, Chandy G, Machen T
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA.
Am J Physiol. 1999 Jul;277(1):L204-17. doi: 10.1152/ajplung.1999.277.1.L204.
The role of tight junctions in the binding and cytoxicity of Pseudomonas aeruginosa to apical or basolateral membranes of lung airway epithelial cells was tested with fluorescence microscopy on living cells. Binding of noncytotoxic P. aeruginosa strain O1 was assessed with P. aeruginosa that expressed green fluorescent protein. Binding of cytotoxic P. aeruginosa strain 6206 was assessed with FITC-labeled P. aeruginosa; cytotoxicity was determined from nuclear uptake of the impermeant dye propidium iodide. The role of direct contact of P. aeruginosa to epithelial cells was tested with filters with small (0.45-micrometer) or large (2.0-micrometer) pores. High transepithelial resistance (R(t)) Calu-3 and cultured bovine tracheal monolayers (R(t) > 1,000 Omega. cm(2)) bound P. aeruginosa very infrequently (<1 P. aeruginosa/100 cells) at the apical membrane, but P. aeruginosa bound frequently to cells near "free edges" at holes, wounds, islands, and perimeters; cytotoxicity required direct interaction with basolateral membranes. Wounded high R(t) epithelia showed increased P. aeruginosa binding and cytotoxicity at the free edges because basolateral membranes were accessible to P. aeruginosa, and dead and living cells near the wound bound P. aeruginosa similarly. Compared with high R(t) epithelia, low R(t) CFT1 (R(t) = 100-200 Omega. cm(2)) and EGTA-treated Calu-3 monolayers were 25 times more susceptible to P. aeruginosa binding throughout the monolayer. Cytotoxicity to CFT1 cells (throughout the confluent monolayer, not only at the free edge) occurred after a shorter delay (0.25 vs. 2.0 h) and then five times faster than to Calu-3 cells, indicating that the time course of P. aeruginosa cytotoxicity may be limited by the rate of gaining access through tight junctions and that this occurred faster in low R(t) than in high R(t) airway epithelia. Cytotoxicity appeared to occur in a sequential process that led first to a loss of fura 2 and a later uptake of propidium iodide. P. aeruginosa bound three times more frequently to regions between cells (tight junctions?) than to cell membranes of low R(t) CFT1 cells.
通过对活细胞进行荧光显微镜观察，测试了紧密连接在铜绿假单胞菌与肺气道上皮细胞顶端或基底外侧膜的结合及细胞毒性中的作用。用表达绿色荧光蛋白的铜绿假单胞菌评估无细胞毒性的铜绿假单胞菌菌株O1的结合情况。用异硫氰酸荧光素（FITC）标记的铜绿假单胞菌评估细胞毒性铜绿假单胞菌菌株6206的结合情况；细胞毒性通过非渗透性染料碘化丙啶的核摄取来确定。用具有小孔径（0.45微米）或大孔径（2.0微米）的滤膜测试铜绿假单胞菌与上皮细胞直接接触的作用。高跨上皮电阻（R(t)）的Calu-3细胞和培养的牛气管单层细胞（R(t)>1000Ω·cm²）在顶端膜处极少结合铜绿假单胞菌（<1个铜绿假单胞菌/100个细胞），但铜绿假单胞菌在孔、伤口、岛屿和边缘处的“自由边缘”附近的细胞上频繁结合；细胞毒性需要与基底外侧膜直接相互作用。受伤的高R(t)上皮细胞在自由边缘处显示出铜绿假单胞菌结合增加和细胞毒性增强，因为铜绿假单胞菌可接触到基底外侧膜，伤口附近的死细胞和活细胞对铜绿假单胞菌的结合类似。与高R(t)上皮细胞相比，低R(t)的CFT1细胞（R(t)=100 - 200Ω·cm²）和用乙二醇双四乙酸（EGTA）处理的Calu-3单层细胞在整个单层中对铜绿假单胞菌结合的敏感性高25倍。对CFT1细胞的细胞毒性（在整个汇合单层中，不仅在自由边缘）延迟时间更短（0.25小时对2.0小时），然后比Calu-3细胞快五倍，这表明铜绿假单胞菌细胞毒性的时间进程可能受通过紧密连接进入细胞的速率限制，并且在低R(t)气道上皮细胞中比在高R(t)气道上皮细胞中发生得更快。细胞毒性似乎以一个连续过程发生，首先导致fura 2丢失，随后碘化丙啶摄取。铜绿假单胞菌与低R(t)的CFT1细胞的细胞间区域（紧密连接？）的结合频率比与细胞膜的结合频率高三倍。
