Zhang Jing, Wu Xin-Yi, Yu Fu-Shin X
Department of Cellular Biology and Anatomy, Medical College of Georgia, Augusta, Georgia 30912, USA.
Curr Eye Res. 2005 Jul;30(7):527-34. doi: 10.1080/02713680590968150.
We hypothesized that corneal epithelium plays a role in the innate immune response by sensing the presence of pathogens and providing signals that activate the corneal defense system. We sought to determine the mechanisms involved in the activation of the signaling pathways and subsequent production of proinflammatory cytokines in human corneal epithelial cells (HCECs) in response to Pseudomonas aeruginosa infection.
Epithelial monolayers of a telomerase-immortalized HCEC line, HUCL, and primary cultures of HCECs were exposed to P. aeruginosa (PA01 strain) with or without the presence of the NF-kappaB inhibitor kamebakaurin, the p38 inhibitor SB203580, or the JNK inhibitor SP600125. IkappaB-alpha phosphorylation and degradation and p38 and JNK phosphorylation were assessed at different time points by Western blot analysis. Interleukin (IL)-6, IL-8, and TNF-alpha levels were determined by reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA).
Exposure of HUCL cells and primary HCECs to P. aeruginosa resulted in rapid activation of NF-kappaB as indicated by an increase in IkappaB-alpha phosphorylation observed within 15 min and by IkappaB-alpha degradation, which peaked in 1 hr. Two stress-activated mitogen-activated protein kinases, p38 and JNK, were also activated as their phosphorylation was induced by P. aeruginosa infection. Concomitant with the activation of these Toll-like receptor-mediated signaling pathways, transcriptional expression and subsequent secretion of IL-6 and IL-8 in HUCL cells were also induced by P. aeruginosa. Presence of the NF-kappaB inhibitor kamebakaurin in culture medium blocked P. aeruginosa-induced NF-kappaB activation and inhibited IL-6, IL-8, and TNF-alpha expression and secretion. Inhibition of p38 or JNK also resulted in a decrease in bacteria-induced expression and secretion of these cytokines.
P. aeruginosa triggers an innate immune response in HCECs, and NF-kappaB and, to a lesser extent, the p38/JNK signal pathways are responsible for P. aeruginosa-induced proinflammatory cytokine production in these cells.
我们推测角膜上皮通过感知病原体的存在并提供激活角膜防御系统的信号，在先天性免疫反应中发挥作用。我们试图确定在铜绿假单胞菌感染后，人角膜上皮细胞（HCECs）中信号通路激活及随后促炎细胞因子产生所涉及的机制。
将端粒酶永生化的HCEC系HUCL的上皮单层细胞和原代培养的HCECs暴露于铜绿假单胞菌（PA01菌株），同时存在或不存在NF-κB抑制剂甘茶素、p38抑制剂SB203580或JNK抑制剂SP600125。通过蛋白质印迹分析在不同时间点评估IκB-α磷酸化和降解以及p38和JNK磷酸化情况。通过逆转录-聚合酶链反应（RT-PCR）和酶联免疫吸附测定（ELISA）测定白细胞介素（IL）-6、IL-8和肿瘤坏死因子-α水平。
HUCL细胞和原代HCECs暴露于铜绿假单胞菌后，NF-κB迅速激活，表现为15分钟内IκB-α磷酸化增加以及IκB-α降解，1小时达到峰值。两种应激激活的丝裂原活化蛋白激酶p38和JNK也被激活，因为它们的磷酸化是由铜绿假单胞菌感染诱导的。与这些Toll样受体介导的信号通路激活同时，铜绿假单胞菌也诱导了HUCL细胞中IL-6和IL-8的转录表达及随后的分泌。培养基中存在NF-κB抑制剂甘茶素可阻断铜绿假单胞菌诱导的NF-κB激活，并抑制IL-6、IL-8和肿瘤坏死因子-α的表达和分泌。抑制p38或JNK也导致细菌诱导的这些细胞因子的表达和分泌减少。
铜绿假单胞菌在HCECs中引发先天性免疫反应，NF-κB以及在较小程度上p38/JNK信号通路负责铜绿假单胞菌诱导的这些细胞中促炎细胞因子的产生。
