MD-2在人角膜上皮细胞应答LPS刺激中的作用研究
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摘要
细菌性角膜炎(bacterial keratitis)是指由细菌感染引起的角膜化脓性炎症,至今仍是世界性的常见致盲眼病。在许多国家和地区,铜绿假单胞菌是首位的细菌性角膜炎致病菌,铜绿假单胞菌属于革兰氏阴性的条件致病菌,该菌可以产生多种致病因子,脂多糖(lipopolysaccharide, LPS)是其最主要的致病因子,LPS通过诱导角膜细胞产生TNF-α、IL-1、IL-6、IL-8和IL-12等,发挥关键的致炎作用。哺乳动物细胞对LPS刺激产生应答,需要通过一系列蛋白辅助,其中产生应答的起始为LPS受体复合物。目前认为LPS受体复合物至少包括四个组成部分：LPS结合蛋白(LPS binding protein, LBP)、CD14、髓样分化蛋白2(myeloid differentiation-2, MD-2)以及Toll样受体4(Toll-like receptor 4, TLR4). MD-2分子的发现是近些年来LPS受体复合物研究方面最重要的进展之一,MD-2关联LPS与TLR4,为LPS/TLR4信号途径提供必需的桥接。然而,人角膜上皮细胞对LPS刺激是否发生应答,可能与人角膜上皮细胞MD-2的表达与否,以及TLR4的分布位置有关。
研究目的：本课题以人角膜上皮细胞SDHCEC1(spontaneously derived human corneal epithelial cell linel, SDHCEC1)作为实验对象,体外探讨人角膜上皮细胞对LPS的刺激应答机制。
研究方法：传代培养来源于中山大学眼科中心王智崇教授团队连续培养成功的人角膜上皮细胞SDHCEC1,通过光学显微镜观察细胞形态,免疫组化法鉴定角膜上皮细胞的特异蛋白CK3/CK12的表达。不同浓度的LPS刺激SDHCEC1细胞不同时间后,Real-time PCR检测TLR4、CD14、IL-8和MD-2 mRNA; ELISA检测细胞培养基IL-8; Western Blot检测IκB-α和p-IκB-α的活化；流式细胞术检测SDHCEC1细胞膜表面和细胞内TLR4、CD14的表达。构建MD-2克隆质粒并鉴定,构建重组MD-2表达质粒并鉴定。重组MD-2表达质粒转染293T细胞,Western Blot检测重组MD-2表达。将293T细胞分泌的重组MD-2蛋白加入SDHCEC1细胞培养基中,Real-time PCR检测TLR4、CD14和IL-8；流式细胞术检测TLR4、CD14; ELISA检测细胞培养基IL-8; Western Blot检测IκB-α和p-IκB-α。
结果：光学显微镜下,SDHCEC1细胞呈典型角膜上皮细胞形态；SDHCEC1细胞内CK3/CK12阳性表达。不同浓度的LPS刺激SDHCEC1细胞不同时间长度后,Real-time PCR结果显示TLR4、CD 14弱阳性表达,统计学无显著差异,IL-8和MD-2则未见表达；ELISA检测培养基无IL-8的分泌；Western Blot检测未见活化的p-IκB-α;流式细胞术发现SDHCEC1细胞膜表面未见TLR4、CD 14表达,而细胞质中TLR4、CD 14弱阳性表达。MD-2克隆质粒和重组MD-2表达质粒构建成功。MD-2表达质粒转染293T细胞后,Western Blot检测重组MD-2蛋白表达。与正常细胞相比较,添加重组MD-2蛋白培养的SDHCEC1细胞对LPS产生应答,表现为：Real-time PCR检测TLR4、CD 14和IL-8 mRNA表达上调；流式细胞术检测TLR4、CD 14在细胞表面和细胞内均阳性表达；ELISA检测细胞培养基IL-8含量增高；Western Blot检测到NF-κB的活化。
结论：人角膜上皮细胞SDHCEC1 MD-2表达缺失,细胞膜表面缺乏功能性的TLR4表达,LPS/TLR4/MD-2信号途径中,NF-κB不活化,细胞未见分泌促炎细胞因子IL-8,这可能是在正常情况下,人角膜上皮细胞通过调控MD-2与细胞膜表面的TLR4表达,抑制对LPS刺激的先天免疫反应,避免炎症细胞在角膜中聚集和角膜结构被炎症破坏,维持角膜局部的免疫自稳状态。但当出现外源性或内源性MD-2的时候,角膜上皮细胞可能在MD-2的诱导下,TLR4重新分布在细胞膜表面,并且募集下游分子,NF-κB活化,分泌促炎细胞因子,激活LPS/TLR4信号途径,参与炎症的发生。
Bacterial keratitis is a kind of suppurative keratitis caused by bacterial infection and can often lead to blindness. In many countries and areas,pseudomonas aeruginosa is the main pathogen which belongs to gram-negative opportunistic bacteria group.This strain can generate many pathogenic factors,among which, lipopolysaccharide(LPS)is the most important one.LPS plays a critical role in inflammation by producing TNF-α, IL-1, IL-6, IL-8 and IL-12 from corneal epithelial cells.The mammal's cells need some accessory proteins to response to LPS. It is currently known that LPS receptor complex is composed of 4 parts at least, which are LPS binding protein(LBP), CD 14, myeloid differentiation-2(MD-2)and toll-like receptor 4(TLR4). MD-2 is one of the most important progresses in this field until now and it acts as a necessary bridge in the signaling pathway associated with LPS and TLR4.But now it is still unclear whether the human corneal epithelial cells can response to LPS, if yes, are expression of MD-2 and location of TLR4 involved in the signaling pathway?
Objects:To study the LPS responding mechanisms of human corneal epithelial cells in vitro.
Methods:Spontaneously derived human corneal epithelial cell linel(SDHCEC1), donated by professor Wang Zhichong from Zhongshan Ophthalmic Center, were subcultured and specific protein CK3/CK12 were identified by immunohistochemistry. TLR4, CD 14, IL-8 and MD-2 expression in SDHCEC1 were analyzed by real-time PCR after incubating with different concentrations of LPS at different times. IL-8 in the supernatant was tested by ELISA. The activation of NF-κB was analyzed by western blot. The expression of TLR4 and CD 14 were tested by flow cytometry. MD-2 recombinant cloning and expression plasmids were constructed and identified. 293T cells were transfected with MD-2 recombinant expression plasmids.Western blot was used to test the MD-2 expression. SDHCEC1 was co-cultured with supernatant secreted by 293T cells transfected with MD-2 recombinant plasmids. TLR4, CD 14, IL-8, and NF-κB in SDHCEC1 were tested as the previous.
Results:SDHCECl showed typical characters of corneal epithelial cells with the positive staining of CK3/CK12. SDHCEC1 cultured with different concentrations of LPS at different times only came up with weak expression of TLR4 and CD 14 (P＜0.05). IL-8 and MD-2 expression were negative. No IL-8 secretion was seen in the supernatant. Activation of NF-κB did not show up. TLR4 and CD 14 were not expressed at the cell surface with only weak expression within cytoplasm. MD-2 cloning plasmids and recombinant plasmids were successfully constructed and expressed in 293T cells. TLR4, CD 14 and IL-8 mRNA were up-regulated in the SDHCEC1 cells co-cultured with supernatant secreted by 293T cells transfected with MD-2 recombinant plasmids. TLR4 and CD 14 were also positive at the cell surface and within cell cytoplasm. IL-8 concentration was also up-regulated with the activation of NF-κB at the same time.
Conclusions:MD-2 expression is absent in SDHCEC1, which may lead to the deficiency of TLR4 at the cell surface, inactivation of NF-κB and no secretion of IL-8 in LPS/TLR4/MD-2 signaling pathway. According to this, we speculate that under normal conditions, in order to maintain corneal immunosilence, corneal epithelial cells may use this way to inhibit the innate immune response induced by LPS and avoid corneal epithelial cells'destruction caused by invasive inflammatory cells. But with endogenous or exogenous MD-2, TLR4 may express at the cell surface with the activation of NF-κB and secretion of IL-8, which activates the whole signaling pathway and plays an important role in sequent inflammation.
研究目的：本课题以人角膜上皮细胞SDHCEC1(spontaneously derived human corneal epithelial cell linel, SDHCEC1)作为实验对象,体外探讨人角膜上皮细胞对LPS的刺激应答机制。
研究方法：传代培养来源于中山大学眼科中心王智崇教授团队连续培养成功的人角膜上皮细胞SDHCEC1,通过光学显微镜观察细胞形态,免疫组化法鉴定角膜上皮细胞的特异蛋白CK3/CK12的表达。不同浓度的LPS刺激SDHCEC1细胞不同时间后,Real-time PCR检测TLR4、CD14、IL-8和MD-2 mRNA; ELISA检测细胞培养基IL-8; Western Blot检测IκB-α和p-IκB-α的活化；流式细胞术检测SDHCEC1细胞膜表面和细胞内TLR4、CD14的表达。构建MD-2克隆质粒并鉴定,构建重组MD-2表达质粒并鉴定。重组MD-2表达质粒转染293T细胞,Western Blot检测重组MD-2表达。将293T细胞分泌的重组MD-2蛋白加入SDHCEC1细胞培养基中,Real-time PCR检测TLR4、CD14和IL-8；流式细胞术检测TLR4、CD14; ELISA检测细胞培养基IL-8; Western Blot检测IκB-α和p-IκB-α。
结果：光学显微镜下,SDHCEC1细胞呈典型角膜上皮细胞形态；SDHCEC1细胞内CK3/CK12阳性表达。不同浓度的LPS刺激SDHCEC1细胞不同时间长度后,Real-time PCR结果显示TLR4、CD 14弱阳性表达,统计学无显著差异,IL-8和MD-2则未见表达；ELISA检测培养基无IL-8的分泌；Western Blot检测未见活化的p-IκB-α;流式细胞术发现SDHCEC1细胞膜表面未见TLR4、CD 14表达,而细胞质中TLR4、CD 14弱阳性表达。MD-2克隆质粒和重组MD-2表达质粒构建成功。MD-2表达质粒转染293T细胞后,Western Blot检测重组MD-2蛋白表达。与正常细胞相比较,添加重组MD-2蛋白培养的SDHCEC1细胞对LPS产生应答,表现为：Real-time PCR检测TLR4、CD 14和IL-8 mRNA表达上调；流式细胞术检测TLR4、CD 14在细胞表面和细胞内均阳性表达；ELISA检测细胞培养基IL-8含量增高；Western Blot检测到NF-κB的活化。
结论：人角膜上皮细胞SDHCEC1 MD-2表达缺失,细胞膜表面缺乏功能性的TLR4表达,LPS/TLR4/MD-2信号途径中,NF-κB不活化,细胞未见分泌促炎细胞因子IL-8,这可能是在正常情况下,人角膜上皮细胞通过调控MD-2与细胞膜表面的TLR4表达,抑制对LPS刺激的先天免疫反应,避免炎症细胞在角膜中聚集和角膜结构被炎症破坏,维持角膜局部的免疫自稳状态。但当出现外源性或内源性MD-2的时候,角膜上皮细胞可能在MD-2的诱导下,TLR4重新分布在细胞膜表面,并且募集下游分子,NF-κB活化,分泌促炎细胞因子,激活LPS/TLR4信号途径,参与炎症的发生。
Bacterial keratitis is a kind of suppurative keratitis caused by bacterial infection and can often lead to blindness. In many countries and areas,pseudomonas aeruginosa is the main pathogen which belongs to gram-negative opportunistic bacteria group.This strain can generate many pathogenic factors,among which, lipopolysaccharide(LPS)is the most important one.LPS plays a critical role in inflammation by producing TNF-α, IL-1, IL-6, IL-8 and IL-12 from corneal epithelial cells.The mammal's cells need some accessory proteins to response to LPS. It is currently known that LPS receptor complex is composed of 4 parts at least, which are LPS binding protein(LBP), CD 14, myeloid differentiation-2(MD-2)and toll-like receptor 4(TLR4). MD-2 is one of the most important progresses in this field until now and it acts as a necessary bridge in the signaling pathway associated with LPS and TLR4.But now it is still unclear whether the human corneal epithelial cells can response to LPS, if yes, are expression of MD-2 and location of TLR4 involved in the signaling pathway?
Objects:To study the LPS responding mechanisms of human corneal epithelial cells in vitro.
Methods:Spontaneously derived human corneal epithelial cell linel(SDHCEC1), donated by professor Wang Zhichong from Zhongshan Ophthalmic Center, were subcultured and specific protein CK3/CK12 were identified by immunohistochemistry. TLR4, CD 14, IL-8 and MD-2 expression in SDHCEC1 were analyzed by real-time PCR after incubating with different concentrations of LPS at different times. IL-8 in the supernatant was tested by ELISA. The activation of NF-κB was analyzed by western blot. The expression of TLR4 and CD 14 were tested by flow cytometry. MD-2 recombinant cloning and expression plasmids were constructed and identified. 293T cells were transfected with MD-2 recombinant expression plasmids.Western blot was used to test the MD-2 expression. SDHCEC1 was co-cultured with supernatant secreted by 293T cells transfected with MD-2 recombinant plasmids. TLR4, CD 14, IL-8, and NF-κB in SDHCEC1 were tested as the previous.
Results:SDHCECl showed typical characters of corneal epithelial cells with the positive staining of CK3/CK12. SDHCEC1 cultured with different concentrations of LPS at different times only came up with weak expression of TLR4 and CD 14 (P＜0.05). IL-8 and MD-2 expression were negative. No IL-8 secretion was seen in the supernatant. Activation of NF-κB did not show up. TLR4 and CD 14 were not expressed at the cell surface with only weak expression within cytoplasm. MD-2 cloning plasmids and recombinant plasmids were successfully constructed and expressed in 293T cells. TLR4, CD 14 and IL-8 mRNA were up-regulated in the SDHCEC1 cells co-cultured with supernatant secreted by 293T cells transfected with MD-2 recombinant plasmids. TLR4 and CD 14 were also positive at the cell surface and within cell cytoplasm. IL-8 concentration was also up-regulated with the activation of NF-κB at the same time.
Conclusions:MD-2 expression is absent in SDHCEC1, which may lead to the deficiency of TLR4 at the cell surface, inactivation of NF-κB and no secretion of IL-8 in LPS/TLR4/MD-2 signaling pathway. According to this, we speculate that under normal conditions, in order to maintain corneal immunosilence, corneal epithelial cells may use this way to inhibit the innate immune response induced by LPS and avoid corneal epithelial cells'destruction caused by invasive inflammatory cells. But with endogenous or exogenous MD-2, TLR4 may express at the cell surface with the activation of NF-κB and secretion of IL-8, which activates the whole signaling pathway and plays an important role in sequent inflammation.
引文
[1]钟文贤,孙士营,赵靖,等.1054例化脓性角膜炎的回顾性分析.中华眼科杂志,2007.43(3)：245-250.
[2]梁艳闯,王智群,李然,等.细菌性角膜炎病原学及耐药性分析.眼科研究,2007.25(3)：306-309.
[3]Houang E, Lam D, Fan D, et al. Microbial keratitis in Hong Kong:relationship to climate, environment and contact-lens disinfection. Transactions of the Royal Society of Tropical Medicine and Hygiene,2001.95(4):361-367.
[4]Fong CF, Hu FR, Tseng CH, et al. Antibiotic susceptibility of bacterial isolates from bacterial keratitis cases in a university hospital in Taiwan. American Journal of Ophthalmology, 2007.144(5):682-689.
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[7]Pachigolla G, Blomquist PH, Cavanagh HD. Microbial keratitis pathogens and antibiotic susceptibilities:a 5-year review of cases at an urban county hospital in north Texas. Eye Contact Lens,2007.33(1):45-49.
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[1]钟文贤,孙士营,赵靖,等.1054例化脓性角膜炎的回顾性分析.中华眼科杂志,2007.43(3)：245-250.
[2]梁艳闯,王智群,李然,等,细菌性角膜炎病原学及耐药性分析.眼科研究,2007.25(3)：306-309.
[3]Houang E, Lam D, Fan D, et al. Microbial keratitis in Hong Kong:relationship to climate, environment and contact-lens disinfection. Transactions of the Royal Society of Tropical Medicine and Hygiene,2001.95(4):361-367.
[4]Fong CF, Hu FR, Tseng CH, et al. Antibiotic susceptibility of bacterial isolates from bacterial keratitis cases in a university hospital in Taiwan. American Journal of Ophthalmology,2007.144(5):682-689.
[5]Tan DT, Lee CP, Lim AS.Corneal ulcers in two institutions in Singapore:analysis of causative factors, organisms and antibiotic resistance. Ann Acad Med Singapore,1995.24(6):823-829.
[6]Norina TJ, Raihan S, Bakiah S, et al. Microbial keratitis:aetiological diagnosis and clinical features in patients admitted to Hospital Universiti Sains Malaysia. Singapore Med J,2008.49(l): 67-71.
[7]Pachigolla G, Blomquist PH, Cavanagh HD. Microbial keratitis pathogens and antibiotic susceptibilities:a 5-year review of cases at an urban county hospital in north Texas. Eye Contact Lens,2007.33(1):45-49.
[8]Green M, Apel A, Stapleton F. Risk factors and causative organisms in microbial keratitis. Cornea,2008.27(1):22-27.
[9]Kipnis E, Sawa T, Wiener-Kronish J. Targeting mechanisms of Pseudomonas aeruginosa pathogenesis. Med Mal Infect,2006.36(2):78-91.
[10]Pier GB. Pseudomonas aeruginosa lipopolysaccharide:a major virulence factor, initiator of inflammation and target for effective immunity. Int J Med Microbiol,2007.297(5):277-295.
[11]Lu YC, Yeh WC, Ohashi PS. LPS/TLR4 signal transduction pathway. Cytokine,2008.42(2): 145-151.
[12]Raoust E, Balloy V, Garcia-Verdugo I, et al. Pseudomonas aeruginosa LPS or Flagellin Are Sufficient to Activate TLR-Dependent Signaling in Murine Alveolar Macrophages and Airway Epithelial Cells. Plos One,2009.4(10):e7259.
[13]Miyake K. Innate recognition of lipopolysaccharide by CD14 and toll-like receptor 4-MD-2: unique roles for MD-2. International Immunopharmacology,2003.3(1):119-128.
[14]Visintin A, Mazzoni A, Spitzer JH, et al. Secreted MD-2 is a large polymeric protein that efficiently confers lipopolysaccharide sensitivity to Toll-like receptor 4. Proc Natl Acad Sci U S A, 2001.98(21):12156-12161.
[15]Shimazu R, Akashi S, Ogata H, et al. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med,1999.189(11):1777-1782.
[16]Ueta M, Nochi T, Jang MH, et al. Intracellularly expressed TLR2s and TLR4s contribution to an immunosilent environment at the ocular mucosal epithelium. Journal of Immunology,2004.173(5):3337-3347.
[17]Song PI, Abraham TA, Park Y, et al. The expression of functional LPS receptor proteins CD14 and toll-like receptor 4 in human corneal cells. Investigative Ophthalmology & Visual Science,2001.42(12):2867-2877.
[18.]Zhang J, Kumar A, Wheater M, et al. Lack of MD-2 expression in human corneal epithelial cells is an underlying mechanism of lipopolysaccharide (LPS) unresponsiveness. Immunol Cell Biol,2009.87(2):141-148.
[19]Lyczak JB, Cannon CL, Pier GB. Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect,2000.2(9):1051-1060.
[20]Kurpakus-Wheater M, Kernacki KA, Hazlett LD. Maintaining corneal integrity how the "window" stays clear-Introduction. Progress in Histochemistry and Cytochemistry,2001.6(3): 185-259.
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[23]Baldwin AS Jr. The NF-kappa B and I kappa B proteins:new discoveries and insights. Annu Rev Immunol,1996.14:649-683.
[24]Driscoll KE, Hassenbein DQ Carter JM,et al. Cloning, expression, and functional characterization of rat MIP-2:a neutrophil chemoattractant and epithelial cell mitogen. J Leukoc Biol,1995.58(3):359-364.
[25]Roebuck KA, Rahman A, Lakshminarayanan V, et al. H2O2 and tumor necrosis factor-alpha activate intercellular adhesion molecule 1 (ICAM-1) gene transcription through distinct cis-regulatory elements within the ICAM-1 promoter. J Biol Chem,1995.270(32):18966-18974.
[26]Liang Y, Zhou Y, Shen P. NF-kappaB and its regulation on the immune system. Cell Mol Immunol,2004.1(5):343-350.
[27]Hazlett LD. Corneal response to Pseudomonas aeruginosa infection. Prog Retin Eye Res, 2004.23(1):1-30.
[28]Carlson EC, Drazba J, Yang X, et al. Visualization and characterization of inflammatory cell recruitment and migration through the corneal stroma in endotoxin-induced keratitis. Invest Ophthalmol Vis Sci,2006.47(l):241-248.
[29]Yoshimura T, Matsushima K, Oppenheim JJ, et al. Neutrophil chemotactic factor produced by lipopolysaccharide (LPS)-stimulated human blood mononuclear leukocytes:partial characterization and separation from interleukin 1 (IL 1). J Immunol,1987.139(3):788-93.
[30]Baggiolini M. Chemokines in pathology and medicine. J Intern Med,2001.250(2):91-104.
[31]Cario E, Rosenberg IM, Brandwein SL, et al. Lipopolysaccharide activates distinct signaling pathways in intestinal epithelial cell lines expressing Toll-like receptors. J Immunol,2000.164(2): 966-972.
[32]Becker MN, Diamond G, Verghese MW, et al. CD14-dependent lipopolysaccharide-induced beta-defensin-2 expression in human tracheobronchial epithelium. J Biol Chem,2000.275(38): 29731-29736.
[33]Wolfs TG, Buurman WA, van Schadewijk A, et al. In vivo expression of Toll-like receptor 2 and 4 by renal epithelial cells:IFN-gamma and TNF-alpha mediated up-regulation during inflammation. J Immunol,2002.168(3):1286-1293.
[34]Schilling JD, Mulvey MA, Vincent CD, et al. Bacterial invasion augments epithelial cytokine responses to Escherichia coli through a lipopolysaccharide-dependent mechanism. J Immunol,2001.166(2):1148-1155.
[35]Uehara A, Sugawara S, Takada H. Priming of human oral epithelial cells by interferon-gamma to secrete cytokines in response to lipopolysaccharides, lipoteichoic acids and peptidoglycans. J Med Microbiol,2002.51(8):626-634.
[36]Talreja J, Dileepan K, Puri S, et al. Human conjunctival epithelial cells lack lipopolysaccharide responsiveness due to deficient expression of MD2 but respond after interferon-gamma priming or soluble MD2 supplementation. Inflammation,2005.29(4-6): 170-181.
[37]Ohnishi T, Muroi M, Tanamoto K. MD-2 is necessary for the toll-like receptor 4 protein to undergo glycosylation essential for its translocation to the cell surface. Clin Diagn Lab Immunol,2003.10(3):405-410.
[1]Talreja J, Dileepan K, Puri S, et al. Human conjunctival epithelial cells lack lipopolysaccharide responsiveness due to deficient expression of MD2 but respond after interferon-gamma priming or soluble MD2 supplementation. Inflammation,2005.29(4-6): 170-181.
[2]Shimazu R, Akashi S, Ogata H, et al. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med,1999.189(11):1777-1782.
[3]Nagai Y, Shimazu R, Ogata H, et al. Requirement for MD-1 in cell surface expression of RP105/CD180 and B-cell responsiveness to lipopolysaccharide.Blood,2002.99(5):1699-1705.
[4]Inohara N, Nunez G. ML — a conserved domain involved in innate immunity and lipid metabolism. Trends Biochem Sci,2002.27(5):219-221.
[5]Visintin A, Mazzoni A, Spitzer JA, et al. Secreted MD-2 is a large polymeric protein that efficiently confers lipopolysaccharide sensitivity to Toll-like receptor 4. Proc Natl Acad Sci U S A,2001.98(21):12156-12161.
[6]Nagai Y, Akashi S, Nagafuku M, et al. Essential role of MD-2 in LPS responsiveness and TLR4 distribution. Nat Immunol,2002.3(7):667-672.
[7]Ohnishi T, Muroi M, Tanamoto K. MD-2 is necessary for the toll-like receptor 4 protein to undergo glycosylation essential for its translocation to the cell surface. Clin Diagn Lab Immunol, 2003.10(3):405-410.
[8]刘亚伟,刘靖华.用FRET技术研究TLR4和MD-2的相互作用.南方医科大学学报,2006.26(8)：1101-1105.
[9]Hyakushima N, Mitsuzawa H, Nishitani C, et al. Interaction of soluble form of recombinant extracellular TLR4 domain with MD-2 enables lipopolysaccharide binding and attenuates TLR4-mediated signaling. Journal of Immunology,2004.173(11):6949-6954.
[10]Mitsuzawa H, Nishitani C, Hyakushima N, et al. Recombinant soluble forms of extracellular TLR4 domain and MD-2 inhibit lipopolysaccharide binding on cell surface and dampen lipopolysaccharide-induced pulmonary inflammation in mice. J Immunol,2006.177(11): 8133-8139.
[11]Latz E, Visintin A, Lien E, et al. Lipopolysaccharide rapidly traffics to and from the Golgi apparatus with the toll-like receptor 4-MD-2-CD14 complex in a process that is distinct from the initiation of signal transduction. J Biol Chem,2002.277(49):47834-47843.
[12]Zhang J, Kumar A, Wheater M, et al. Lack of MD-2 expression in human corneal epithelial cells is an underlying mechanism of lipopolysaccharide (LPS) unresponsiveness. Immunol Cell Biol,2009.87(2):141-148.
[13]Wright SD, Ramos RA, Tobias PS, et al. CD14, a receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein. Science,1990.249(4975):1431-1433.
[14]Nau GJ, Schlesinger A, Richmond JF, et al. Cumulative Toll-like receptor activation in human macrophages treated with whole bacteria. J Immunol,2003.170(10):5203-5209.
[15]Lyczak JB, Cannon CL, Pier GB. Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect,2000.2(9):1051-1060.
[1]Janeway CA, Medzhitov R. Innate immune recognition. Annual Review of Immunology, 2002.20:197-216.
[2]Lu YC, Yeh WC, Ohashi PS. LP3/TLR4 signal transduction pathway. Cytokine,2008.42(2): 145-151.
[3]Gioannini T, Weiss JP. Regulation of interactions of Gram-negative bacterial endotoxins with mammalian cells. Immunol Res,2007.39(1-3):249-260.
[4]Miyake K. Innate immune sensing of pathogens and danger signals by cell surface Toll-like receptors. Semin Immunol,2007.19(1):3-10.
[5]钟文贤,孙士营,赵靖,等.1054例化脓性角膜炎的回顾性分析.中华眼科杂志,2007.43(3)：245-250.
[6]梁艳闯,王智群,李然,等.细菌性角膜炎病原学及耐药性分析.眼科研究,2007.25(3)：306-309.
[7]Houang E, Lam D, Fan D, et al.Microbial keratitis in Hong Kong:relationship to climate, environment and contact-lens disinfection. Transactions of the Royal Society of Tropical Medicine and Hygiene,2001.95(4):361-367.
[8]Fong CF, Hu FR, Tseng CH, et al.Antibiotic susceptibility of bacterial isolates from bacterial keratitis cases in a university hospital in Taiwan. American Journal of Ophthalmology, 2007.144(5):682-689.
[9]Tan DT, Lee CP, Lim AS.Corneal ulcers in two institutions in Singapore:analysis of causative factors, organisms and antibiotic resistance. Ann Acad Med Singapore,1995.24(6):823-829.
[10]Norina TJ, Raihan S, Bakiah S, et al.Microbial keratitis:aetiological diagnosis and clinical features in patients admitted to Hospital Universiti Sains Malaysia. Singapore Med J,2008.49(1): 67-71.
[11]Pachigolla G, Blomquist PH, Cavanagh HD. Microbial keratitis pathogens and antibiotic susceptibilities:a 5-year review of cases at an urban county hospital in north Texas. Eye Contact Lens,2007.33(1):45-49.
[12]Green M, Apel A, Stapleton F. Risk factors and causative organisms in microbial keratitis. Cornea,2008.27(1):22-27.
[13]Kipnis E, Sawa T, Wiener-Kronish J. Targeting mechanisms of Pseudomonas aeruginosa pathogenesis. Med Mal Infect,2006.36(2):78-91.
[14]Park BS, Song DH, Kim HM, et al. The structural basis of lipopolysaccharide recognition by the TLR4-MD-2 complex. Nature,2009.458(7242):1191-1195.
[15]Hyakushima N, Mitsuzawa H, Nishitani C, et al. Interaction of soluble form of recombinant extracellular TLR4 domain with MD-2 enables lipopolysaccharide binding and attenuates TLR4-mediated signaling. Journal of Immurology,2004.173(11):6940-6954.
[16]Hailman E, Lichenstein HS, Wurfel (?)M, et al. Lipopolysacc(?)ride (LPS)-binding protein accelerates the binding of LPS to CD 14. J Exp Med,1994.179(1):269-277.
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[18]Haziot A, Chen S, Ferrero E, et al. The monocyte differentiation antigen, CD 14, is anchored to the cell membrane by a phosphatidylinositol linkage. J Immunol,1988.141(2):547-552.
[19]Shimazu R, Akashi S, Ogata H, et al. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med,1999.189(11):1777-1782.
[20]Nagai Y, Shimazu R, Ogata H, et al. Requirement for MD-1 in cell surface expression of RP105/CD180 and B-cell responsiveness to lipopolysaccharide. Blood,2002.99(5):1699-1705.
[21]Inohara N, Nunez G ML-a conserved domain involved in innate immunity and lipid metabolism. Trends Biochem Sci,2002.27(5):219-221.
[22]Visintin A, Mazzoni A, Spitzer JA, et al. Secreted MD-2 is a large polymeric protein that efficiently confers lipopolysaccharide sensitivity to Toll-like receptor 4. Proc Natl Acad Sci U S A, 2001.98(21):12156-12161.
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[31]Mitsuzawa H, Nishitani C, Hyakushima N, et al. Recombinant soluble forms of extracellular TLR4 domain and MD-2 inhibit lipopolysaccharide binding on cell surface and dampen lipopolysaccharide-induced pulmonary inflammation in mice. J Immunol,2006.177(11): 8133-8139.
[32]Lyczak JB, Cannon CL, Pier GB. Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect,2000.2(9):1051-1060.
[33]Kurpakus-Wheater M, Kernacki KA, Hazlett LD. Maintaining corneal integrity how the "window" stays clear-Introduction. Progress in Histochemistry and Cytochemistry,2001.36(3): 185-259.
[34]Raoust E, Balloy V, Garcia-Verdugo I, et al. Pseudomonas aeruginosa LPS or Flagellin Are Sufficient to Activate TLR-Dependent Signaling in Murine Alveolar Macrophages and Airway Epithelial Cells. Plos One,2009.4(10):.e7259.
[35]Miyake K. Innate recognition of lipopolysaccharide by CD14 and toll-like receptor 4-MD-2: unique roles for MD-2. International Immunopharmacology,2003.3(1):119-128.
[36]Song PI, Abraham TA, Park Y, et al. The expression of functional LPS receptor proteins CD14 and toll-like receptor 4 in human corneal cells. Investigative Ophthalmology & Visual Science,2001.42(12):2867-2877.
[37]Ueta M, Nochi T, et al. Intracellularly expressed TLR2s and TLR4s contribution to an immunosilent environment at the ocular mucosal epithelium. Journal of Immunology,2004.173(5): 3337-3347.
[38]Zhang J, Kumar A, Wheater M, et al. Lack of MD-2 expression in human corneal epithelial cells is an underlying mechanism of lipopolysaccharide (LPS) unresponsiveness. Immunol Cell Biol,2009.87(2):141-148.
[2]梁艳闯,王智群,李然,等.细菌性角膜炎病原学及耐药性分析.眼科研究,2007.25(3)：306-309.
[3]Houang E, Lam D, Fan D, et al. Microbial keratitis in Hong Kong:relationship to climate, environment and contact-lens disinfection. Transactions of the Royal Society of Tropical Medicine and Hygiene,2001.95(4):361-367.
[4]Fong CF, Hu FR, Tseng CH, et al. Antibiotic susceptibility of bacterial isolates from bacterial keratitis cases in a university hospital in Taiwan. American Journal of Ophthalmology, 2007.144(5):682-689.
[5]Tan DT, Lee CP, Lim AS.Corneal ulcers in two institutions in Singapore:analysis of causative factors, organisms and antibiotic resistance. Ann Acad Med Singapore,1995.24(6):823-829.
[6]Norina TJ, Raihan S, Bakiah S, et al. Microbial keratitis:aetiological diagnosis and clinical features in patients admitted to Hospital Universiti Sains Malaysia. Singapore Med J,2008.49(1): 67-71.
[7]Pachigolla G, Blomquist PH, Cavanagh HD. Microbial keratitis pathogens and antibiotic susceptibilities:a 5-year review of cases at an urban county hospital in north Texas. Eye Contact Lens,2007.33(1):45-49.
[8]Green M, Apel A, Stapleton F. Risk factors and causative organisms in microbial keratitis. Cornea,2008.27(1):22-27.
[9]王丹,孙旭光.铜绿假单胞菌性角膜炎.国际眼科纵览,2006.30(6)：398-401.
[10]Hazlett LD. Corneal response to Pseudomonas aeruginosa infection. Prog Retin Eye Res, 2004.23(1):1-30.
[11]Ueta M, Nochi T, Jang MH, et al. Intracellularly expressed TLR2s and TLR4s contribution to an immunosilent environment at the ocular mucosal epithelium. Journal of Immunology, 2004.173(5):3337-3347.
[12]Song PI, Abraham TA, Park Y, et al. The expression of functional LPS receptor proteins CD14 and toll-like receptor 4 in human corneal cells. Investigative Ophthalmology & Visual Science,2001.42(12):2867-2877.
[13]Zhang J, Xu K, Ambati B, et al. Toll-like receptor 5-mediated corneal epithelial inflammatory responses to Pseudomonas aeruginosa flagellin. Invest Ophthalmol Vis Sci,2003.44(10): 4247-4254.
[14]Lyczak JB, Cannon CL, Pier GB. Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect,2000.2(9):1051-1060.
[15]Kurpakus-Wheater M, Kernacki KA, Hazlett LD. Maintaining corneal integrity how the "window" stays clear-Introduction. Progress in Histochemistry and Cytochemistry,2001.36(3): 185-259.
[16]Kipnis E, Sawa T, Wiener-Kronish J. Targeting mechanisms of Pseudomonas aeruginosa pathogenesis. Med Mal Infect,2006.36(2):78-91.
[17]Pier GB. Pseudomonas aeruginosa lipopolysaccharide:a major virulence factor, initiator of inflammation and target for effective immunity. Int J Med Microbiol,2007.297(5):277-295.
[18]Lu YC, Yeh WC, Ohashi PS. LPS/TLR4 signal transduction pathway. Cytokine,2008.42(2): 145-151.
[19]Raoust E, Balloy V, Garcia-Verdugo I, et al. Pseudomonas aeruginosa LPS or Flagellin Are Sufficient to Activate TLR-Dependent Signaling in Murine Alveolar Macrophages and Airway Epithelial Cells. Plos One,2009.4(10):e7259.
[20]Miyake K. Innate recognition of lipopolysaccharide by CD14 and toll-like receptor 4-MD-2: unique roles for MD-2. International Immunopharmacology,2003.3(1):119-128.
[21]Visintin A, Mazzoni A, Spitzer JH, et al. Secreted MD-2 is a large polymeric protein that efficiently confers lipopolysaccharide sensitivity to Toll-like receptor 4. Proc Natl Acad Sci U S A, 2001.98(21):12156-12161.
[22]Shimazu R, Akashi S, Ogata H, et al. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med,1999.189(11):1777-1782.
[23]Hailman E, Lichenstein HS, Wurfel MM, et al. Lipopolysaccharide (LPS)-binding protein accelerates the binding of LPS to CD14. J Exp Med,1994.179(1):269-277.
[24]Wurfel MM, Hailman E, Wright SD, Soluble Cd14 Acts as a Shuttle in the Neutralization of Lipopolysaccharide (Lps) by Lps-Binding Protein and Reconstituted High-Density-Lipoprotein. Journal of Experimental Medicine,1995.181 (5):1743-1754.
[25]Haziot A, Chen S, Ferrero E, et al. The monocyte differentiation antigen, CD 14, is anchored to the cell membrane by a phosphatidylinositol linkage. J Immunol,1988.141(2):547-552.
[26]Takeda K, Akira S. Microbial recognition by Toll-like receptors. J Dermatol Sci,2004.34(2): 73-82.
[27]Takeda K, Akira S. TLR signaling pathways. Semin Immunol,2004.16(1):3-9.
[28]Nagai Y, Shimazu R, Ogata H, et al. Requirement for MD-1 in cell surface expression of RP105/CD180 and B-cell responsiveness to lipopolysaccharide. Blood,2002.99(5):1699-1705.
[29]Inohara N, Nunez G. ML--a conserved domain involved in innate immunity and lipid metabolism. Trends Biochem Sci,2002.27(5):219-221.
[30]Ohnishi T, Muroi M, Tanamoto K. MD-2 is necessary for the toll-like receptor 4 protein to undergo glycosylation essential for its translocation to the cell surface. Clin Diagn Lab Immunol,2003.10(3):405-410.
[31]Zhang J, Kumar A, Wheatcr M, et al. Lack of MD-2 expression in human corneal epithelial cells is an underlying mechanicm of lipopolysaccharide (LPS) unresponsiveness. Immunol Cell Biol,2009.87(2):141-148.
[1]钟文贤,孙士营,赵靖,等.1054例化脓性角膜炎的回顾性分析.中华眼科杂志,2007.43(3)：245-250.
[2]梁艳闯,王智群,李然,等,细菌性角膜炎病原学及耐药性分析.眼科研究,2007.25(3)：306-309.
[3]Houang E, Lam D, Fan D, et al. Microbial keratitis in Hong Kong:relationship to climate, environment and contact-lens disinfection. Transactions of the Royal Society of Tropical Medicine and Hygiene,2001.95(4):361-367.
[4]Fong CF, Hu FR, Tseng CH, et al. Antibiotic susceptibility of bacterial isolates from bacterial keratitis cases in a university hospital in Taiwan. American Journal of Ophthalmology,2007.144(5):682-689.
[5]Tan DT, Lee CP, Lim AS.Corneal ulcers in two institutions in Singapore:analysis of causative factors, organisms and antibiotic resistance. Ann Acad Med Singapore,1995.24(6):823-829.
[6]Norina TJ, Raihan S, Bakiah S, et al. Microbial keratitis:aetiological diagnosis and clinical features in patients admitted to Hospital Universiti Sains Malaysia. Singapore Med J,2008.49(l): 67-71.
[7]Pachigolla G, Blomquist PH, Cavanagh HD. Microbial keratitis pathogens and antibiotic susceptibilities:a 5-year review of cases at an urban county hospital in north Texas. Eye Contact Lens,2007.33(1):45-49.
[8]Green M, Apel A, Stapleton F. Risk factors and causative organisms in microbial keratitis. Cornea,2008.27(1):22-27.
[9]Kipnis E, Sawa T, Wiener-Kronish J. Targeting mechanisms of Pseudomonas aeruginosa pathogenesis. Med Mal Infect,2006.36(2):78-91.
[10]Pier GB. Pseudomonas aeruginosa lipopolysaccharide:a major virulence factor, initiator of inflammation and target for effective immunity. Int J Med Microbiol,2007.297(5):277-295.
[11]Lu YC, Yeh WC, Ohashi PS. LPS/TLR4 signal transduction pathway. Cytokine,2008.42(2): 145-151.
[12]Raoust E, Balloy V, Garcia-Verdugo I, et al. Pseudomonas aeruginosa LPS or Flagellin Are Sufficient to Activate TLR-Dependent Signaling in Murine Alveolar Macrophages and Airway Epithelial Cells. Plos One,2009.4(10):e7259.
[13]Miyake K. Innate recognition of lipopolysaccharide by CD14 and toll-like receptor 4-MD-2: unique roles for MD-2. International Immunopharmacology,2003.3(1):119-128.
[14]Visintin A, Mazzoni A, Spitzer JH, et al. Secreted MD-2 is a large polymeric protein that efficiently confers lipopolysaccharide sensitivity to Toll-like receptor 4. Proc Natl Acad Sci U S A, 2001.98(21):12156-12161.
[15]Shimazu R, Akashi S, Ogata H, et al. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med,1999.189(11):1777-1782.
[16]Ueta M, Nochi T, Jang MH, et al. Intracellularly expressed TLR2s and TLR4s contribution to an immunosilent environment at the ocular mucosal epithelium. Journal of Immunology,2004.173(5):3337-3347.
[17]Song PI, Abraham TA, Park Y, et al. The expression of functional LPS receptor proteins CD14 and toll-like receptor 4 in human corneal cells. Investigative Ophthalmology & Visual Science,2001.42(12):2867-2877.
[18.]Zhang J, Kumar A, Wheater M, et al. Lack of MD-2 expression in human corneal epithelial cells is an underlying mechanism of lipopolysaccharide (LPS) unresponsiveness. Immunol Cell Biol,2009.87(2):141-148.
[19]Lyczak JB, Cannon CL, Pier GB. Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect,2000.2(9):1051-1060.
[20]Kurpakus-Wheater M, Kernacki KA, Hazlett LD. Maintaining corneal integrity how the "window" stays clear-Introduction. Progress in Histochemistry and Cytochemistry,2001.6(3): 185-259.
[21]Janeway CA, Medzhitov R. Innate immune recognition. Annual Review of Immunology, 2002.20:197-216.
[22]Singh H, Sen R, Baltimore D, et al. A nuclear factor that binds to a conserved sequence motif in transcriptional control elements of immunoglobulin genes. Nature,1986.319(6049):154-158.
[23]Baldwin AS Jr. The NF-kappa B and I kappa B proteins:new discoveries and insights. Annu Rev Immunol,1996.14:649-683.
[24]Driscoll KE, Hassenbein DQ Carter JM,et al. Cloning, expression, and functional characterization of rat MIP-2:a neutrophil chemoattractant and epithelial cell mitogen. J Leukoc Biol,1995.58(3):359-364.
[25]Roebuck KA, Rahman A, Lakshminarayanan V, et al. H2O2 and tumor necrosis factor-alpha activate intercellular adhesion molecule 1 (ICAM-1) gene transcription through distinct cis-regulatory elements within the ICAM-1 promoter. J Biol Chem,1995.270(32):18966-18974.
[26]Liang Y, Zhou Y, Shen P. NF-kappaB and its regulation on the immune system. Cell Mol Immunol,2004.1(5):343-350.
[27]Hazlett LD. Corneal response to Pseudomonas aeruginosa infection. Prog Retin Eye Res, 2004.23(1):1-30.
[28]Carlson EC, Drazba J, Yang X, et al. Visualization and characterization of inflammatory cell recruitment and migration through the corneal stroma in endotoxin-induced keratitis. Invest Ophthalmol Vis Sci,2006.47(l):241-248.
[29]Yoshimura T, Matsushima K, Oppenheim JJ, et al. Neutrophil chemotactic factor produced by lipopolysaccharide (LPS)-stimulated human blood mononuclear leukocytes:partial characterization and separation from interleukin 1 (IL 1). J Immunol,1987.139(3):788-93.
[30]Baggiolini M. Chemokines in pathology and medicine. J Intern Med,2001.250(2):91-104.
[31]Cario E, Rosenberg IM, Brandwein SL, et al. Lipopolysaccharide activates distinct signaling pathways in intestinal epithelial cell lines expressing Toll-like receptors. J Immunol,2000.164(2): 966-972.
[32]Becker MN, Diamond G, Verghese MW, et al. CD14-dependent lipopolysaccharide-induced beta-defensin-2 expression in human tracheobronchial epithelium. J Biol Chem,2000.275(38): 29731-29736.
[33]Wolfs TG, Buurman WA, van Schadewijk A, et al. In vivo expression of Toll-like receptor 2 and 4 by renal epithelial cells:IFN-gamma and TNF-alpha mediated up-regulation during inflammation. J Immunol,2002.168(3):1286-1293.
[34]Schilling JD, Mulvey MA, Vincent CD, et al. Bacterial invasion augments epithelial cytokine responses to Escherichia coli through a lipopolysaccharide-dependent mechanism. J Immunol,2001.166(2):1148-1155.
[35]Uehara A, Sugawara S, Takada H. Priming of human oral epithelial cells by interferon-gamma to secrete cytokines in response to lipopolysaccharides, lipoteichoic acids and peptidoglycans. J Med Microbiol,2002.51(8):626-634.
[36]Talreja J, Dileepan K, Puri S, et al. Human conjunctival epithelial cells lack lipopolysaccharide responsiveness due to deficient expression of MD2 but respond after interferon-gamma priming or soluble MD2 supplementation. Inflammation,2005.29(4-6): 170-181.
[37]Ohnishi T, Muroi M, Tanamoto K. MD-2 is necessary for the toll-like receptor 4 protein to undergo glycosylation essential for its translocation to the cell surface. Clin Diagn Lab Immunol,2003.10(3):405-410.
[1]Talreja J, Dileepan K, Puri S, et al. Human conjunctival epithelial cells lack lipopolysaccharide responsiveness due to deficient expression of MD2 but respond after interferon-gamma priming or soluble MD2 supplementation. Inflammation,2005.29(4-6): 170-181.
[2]Shimazu R, Akashi S, Ogata H, et al. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med,1999.189(11):1777-1782.
[3]Nagai Y, Shimazu R, Ogata H, et al. Requirement for MD-1 in cell surface expression of RP105/CD180 and B-cell responsiveness to lipopolysaccharide.Blood,2002.99(5):1699-1705.
[4]Inohara N, Nunez G. ML — a conserved domain involved in innate immunity and lipid metabolism. Trends Biochem Sci,2002.27(5):219-221.
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