Zhong Wenxian, Yin Hongmei, Xie Lixin
State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Qingdao, China.
Mol Vis. 2009 Jul 4;15:1303-11.
The aim of this study was to investigate the expression and regulation of the four major inflammatory cytokines in fungal keratitis (FK) with the goal of further understanding its pathogenesis in order to develop more effective therapeutic approaches.
Aspergillus fumigatus and Candida albicans were the corneal pathogens selected for this study to establish murine FK using epikeratophakia with the aid of corneal epithelium erasion. One, three, five, and seven days post-infection, the corneal lesions and inflammatory responses were observed by slit-lamp and histopathology, and the expressions of the four inflammatory cytokines, macrophage inflammatory protein-2 (MIP-2), cytokine-induced neutrophil chemoattractant (KC), interleukin-1beta (IL-1beta), and interleukin-6 (IL-6), in the infected corneas were determined using reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). For the intervention experiment with neutralizing antibodies, the experimental mice were then injected subconjunctivally with 5 microl (2 ng/microl) MIP-2 or IL-1beta polyclonal antibody 1 h before and 24 h after surgery. Reestablishment of the FK murine model was performed following injection. Effects of MIP-2 or IL-1beta polyclonal antibody on the corneal diseases were observed by slit-lamp microscopy, histopathology, and ELISA.
Expression of MIP-2, KC, IL-1beta, and IL-6 was upregulated significantly in the infected group one, three, five, and seven days after surgery. Following treatment with an MIP-2 polyclonal antibody, the corneal clinical scores and inflammatory responses decreased, the MIP-2 protein levels were downregulated significantly (p<0.01), and the KC protein levels decreased slightly (p>0.05). Upon administration of IL-1beta polyclonal antibodies, the decrease in clinical scores, inflammatory responses, and protein levels of MIP-2 and KC was apparent at 1 and 3 days after infection (p<0.01).
A persistent, high level expression of MIP-2 and IL-1beta is an important and even major factor in the corneal pathogenesis of FK. Specific polyclonal neutralizing antibodies may be administered to inhibit the major chemokines and cytokines responsible for corneal damage thus effectively relieving the injury caused by FK.
本研究旨在调查真菌性角膜炎（FK）中四种主要炎性细胞因子的表达及调控情况，以期进一步了解其发病机制，从而开发更有效的治疗方法。
本研究选用烟曲霉和白色念珠菌作为角膜病原体，借助角膜上皮刮除术，采用角膜表层镜片术建立小鼠FK模型。感染后1天、3天、5天和7天，通过裂隙灯和组织病理学观察角膜病变及炎症反应，并使用逆转录聚合酶链反应（RT-PCR）和酶联免疫吸附测定（ELISA）法测定感染角膜中四种炎性细胞因子，即巨噬细胞炎性蛋白-2（MIP-2）、细胞因子诱导的中性粒细胞趋化因子（KC）、白细胞介素-1β（IL-1β）和白细胞介素-6（IL-6）的表达。对于中和抗体干预实验，在手术前1小时和手术后24小时，给实验小鼠结膜下注射5微升（2纳克/微升）MIP-2或IL-1β多克隆抗体。注射后重建FK小鼠模型。通过裂隙灯显微镜检查、组织病理学和ELISA观察MIP-2或IL-1β多克隆抗体对角膜疾病的影响。
感染组术后1天、3天、5天和7天，MIP-2、KC、IL-1β和IL-6的表达显著上调。用MIP-2多克隆抗体治疗后，角膜临床评分和炎症反应降低，MIP-2蛋白水平显著下调（p<0.01），KC蛋白水平略有下降（p>0.05）。给予IL-1β多克隆抗体后，感染后1天和3天，临床评分、炎症反应以及MIP-2和KC蛋白水平的降低均很明显（p<0.01）。
MIP-2和IL-1β持续高水平表达是FK角膜发病机制中的一个重要甚至主要因素。可给予特异性多克隆中和抗体以抑制导致角膜损伤的主要趋化因子和细胞因子，从而有效减轻FK所致损伤。
