McClellan Sharon A, Huang Xi, Barrett Ronald P, Lighvani Shahrzad, Zhang Yunfan, Richiert Dawn, Hazlett Linda D
Department of Anatomy/Cell Biology, Wayne State University School of Medicine, 540 East Canfield Avenue, Detroit, Michigan 48201, USA.
Invest Ophthalmol Vis Sci. 2006 Jan;47(1):256-64. doi: 10.1167/iovs.05-1050.
The purpose of this study was to determine the role of matrix metalloproteinases (MMP) in Pseudomonas aeruginosa keratitis.
Gene array and selective real-time PCR examined MMP expression in the cornea of susceptible (C57BL/6, B6) versus resistant (BALB/c) mice before and after infection; zymography tested enzyme activity for MMP-2 and -9. Clinical score, Langerhans cell (LC), and Neutrophil (PMN) quantitation were done in recombinant (r) MMP-9, antibody neutralized, and MMP-9(-/-) mice. The chemotactic potential of MMP-9 was tested in a Boyden chamber assay; light and transmission microscopy and immunostaining for collagen IV and MMP-9 were used to examine the effects and the source of MMP-9 after infection. ELISA was used to assess IL-1beta and MIP-2 levels.
Gene array (confirmed by PCR) revealed sixfold more MMP-9, and zymography showed greater enzyme activity in the infected cornea of B6 over BALB/c mice. rMMP-9 injection of BALB/c mice enhanced, whereas MMP-9 antibody neutralization in B6 mice and its absence in MMP-9(-/-) mice decreased corneal disease. MMP-9(-/-) and antibody neutralized mice had fewer LCs in cornea; rMMP-9-treated mice had more. A myeloperoxidase (MPO) assay showed a similar pattern for PMN. MMP-9 was not chemotactic for LC or PMN. The basement membrane was more intact in MMP-9(-/-) over wild-type infected mice and correlated with staining for collagen IV; PMN was a source of MMP-9. IL-1beta and MIP-2 were increased in rMMP-9 but decreased in MMP-9 antibody neutralized and MMP-9(-/-) over control groups.
MMP-9 regulates immune function in cornea by proteolysis, potentiating P. aeruginosa keratitis by degrading collagen IV and upregulating chemotactic cytokines/chemokines IL-1beta and MIP-2.
本研究旨在确定基质金属蛋白酶（MMP）在铜绿假单胞菌角膜炎中的作用。
基因芯片和选择性实时聚合酶链反应检测易感（C57BL/6，B6）和抗性（BALB/c）小鼠感染前后角膜中MMP的表达；酶谱法检测MMP-2和-9的酶活性。对重组（r）MMP-9、抗体中和及MMP-9基因敲除（-/-）小鼠进行临床评分、朗格汉斯细胞（LC）和中性粒细胞（PMN）定量分析。采用博伊登小室试验检测MMP-9的趋化潜力；利用光学显微镜和透射显微镜以及IV型胶原和MMP-9免疫染色来检测感染后MMP-9的作用及来源。采用酶联免疫吸附测定法评估白细胞介素-1β（IL-1β）和巨噬细胞炎性蛋白-2（MIP-2）水平。
基因芯片（经聚合酶链反应证实）显示，B6小鼠感染角膜中的MMP-9比BALB/c小鼠多6倍，酶谱法显示其酶活性更高。给BALB/c小鼠注射rMMP-9会加重角膜疾病，而在B6小鼠中中和MMP-9抗体以及在MMP-9（-/-）小鼠中缺乏MMP-9则会减轻角膜疾病。MMP-9（-/-）小鼠和抗体中和小鼠角膜中的LC较少；rMMP-9处理的小鼠则较多。髓过氧化物酶（MPO）检测显示PMN呈现类似模式。MMP-9对LC或PMN无趋化作用。与野生型感染小鼠相比，MMP-9（-/-）小鼠的基底膜更完整，且与IV型胶原染色相关；PMN是MMP-9的来源之一。与对照组相比，rMMP-9组中IL-1β和MIP-2升高，而MMP-9抗体中和组和MMP-9（-/-）组中则降低。
MMP-9通过蛋白水解调节角膜免疫功能，通过降解IV型胶原并上调趋化细胞因子/趋化因子IL-1β和MIP-2来加重铜绿假单胞菌角膜炎。
