School of Optometry, Indiana University, Bloomington, Indiana 47405, USA.
Invest Ophthalmol Vis Sci. 2010 Mar;51(3):1575-82. doi: 10.1167/iovs.09-4343. Epub 2009 Sep 24.
TNF-alpha is elevated in the cornea and aqueous humor during allograft rejection and anterior uveitis. The authors investigated the involvement of p38 MAP kinase in the TNF-alpha-induced loss of barrier integrity in monolayers of cultured bovine corneal endothelial cells.
Transendothelial electrical resistance (TER), a measure of barrier integrity, was determined by electrical cell-substrate impedance sensing. Barrier integrity was further assessed in terms of permeability to FITC dextran. Reorganization of the apical junctional complex (AJC) in response to TNF-alpha was visualized by immunofluorescence. The expression of TNF-alpha receptors was confirmed by RT-PCR. Activation of p38 MAP kinase in response to TNF-alpha was determined by Western blot analysis.
Exposure to TNF-alpha induced a continuous decline in TER that persisted for more than 20 hours. It also led to a significant increase in permeability to FITC dextran. At the AJC, the cytokine caused disassembly of microtubules, disruption of perijunctional actomyosin ring (PAMR), and dislocation of ZO-1 and cadherins. Western blot analysis showed that TNF-alpha also led to the activation of p38 MAP kinase. All these responses to the cytokine were opposed by treatment with SB-203580, a selective p38 MAP kinase inhibitor. TNFR1, but not TNFR2, was expressed in untreated cells with no change in the expression pattern on treatment with the cytokine.
TNF-alpha breaks down the barrier integrity of corneal endothelium, concomitant with the disruption of PAMR, remodeling of AJC, and disassembly of microtubules. These effects are mediated by transient activation of p38 MAP kinase. Thus, the TNF-alpha-induced barrier dysfunction in the corneal endothelium can be suppressed by inhibitors of p38 MAP kinase and agents downstream of the kinase that affect the cytoskeleton.
在同种异体移植物排斥和前葡萄膜炎中，TNF-α在角膜和房水中升高。作者研究了 p38MAP 激酶在 TNF-α诱导的培养牛角膜内皮细胞单层中屏障完整性丧失中的作用。
通过电细胞-底物阻抗感应测定，跨内皮电阻（TER）作为屏障完整性的测量。用 FITC 葡聚糖的通透性进一步评估屏障完整性。通过免疫荧光观察 TNF-α 对顶端连接复合体（AJC）的重排。通过 RT-PCR 证实 TNF-α受体的表达。通过 Western blot 分析确定 p38MAP 激酶对 TNF-α的激活。
暴露于 TNF-α导致 TER 持续下降超过 20 小时，持续下降。它还导致 FITC 葡聚糖通透性显著增加。在 AJC，细胞因子引起微管解体、周缘肌动球蛋白环（PAMR）破坏以及 ZO-1 和钙粘蛋白的脱位。Western blot 分析表明，TNF-α还导致 p38MAP 激酶的激活。用选择性 p38MAP 激酶抑制剂 SB-203580 处理可拮抗细胞因子的所有这些反应。在未处理的细胞中表达 TNFR1，但不表达 TNFR2，在用细胞因子处理后，其表达模式没有变化。
TNF-α破坏角膜内皮的屏障完整性，同时破坏 PAMR、AJC 重塑和微管解体。这些作用是通过 p38MAP 激酶的短暂激活介导的。因此，TNF-α诱导的角膜内皮屏障功能障碍可通过 p38MAP 激酶抑制剂和影响细胞骨架的激酶下游的药物来抑制。
