Respiratory epithelial cells require Toll-like receptor 4 for induction of Human β-defensin 2 by Lipopolysaccharide

Respiratory Research

Shane O'Neill¹  Noel McElvaney¹  Clifford C Taggart¹  Catherine Greene¹  Ruth MacRedmond¹ 

[1] Department of Respiratory Research, Royal College of Surgeons in Ireland, Beaumont Hospital, Dublin 9, Ireland

关键词: Human β-defensin 2.;    Lipopolysaccharide;    Toll-like Receptor 4;    Airway epithelium;   

Others  :  1222224
DOI  :  10.1186/1465-9921-6-116

 received in 2005-04-27, accepted in 2005-10-12,  发布年份 2005

【 摘 要 】

Background

The respiratory epithelium is a major portal of entry for pathogens and employs innate defense mechanisms to prevent colonization and infection. Induced expression of human β-defensin 2 (HBD2) represents a direct response by the epithelium to potential infection. Here we provide evidence for the critical role of Toll-like receptor 4 (TLR4) in lipopolysaccharide (LPS)-induced HBD2 expression by human A549 epithelial cells.

Methods

Using RTPCR, fluorescence microscopy, ELISA and luciferase reporter gene assays we quantified interleukin-8, TLR4 and HBD2 expression in unstimulated or agonist-treated A549 and/or HEK293 cells. We also assessed the effect of over expressing wild type and/or mutant TLR4, MyD88 and/or Mal transgenes on LPS-induced HBD2 expression in these cells.

Results

We demonstrate that A549 cells express TLR4 on their surface and respond directly to Pseudomonas LPS with increased HBD2 gene and protein expression. These effects are blocked by a TLR4 neutralizing antibody or functionally inactive TLR4, MyD88 and/or Mal transgenes. We further implicate TLR4 in LPS-induced HBD2 production by demonstrating HBD2 expression in LPS non-responsive HEK293 cells transfected with a TLR4 expression plasmid.

Conclusion

This data defines an additional role for TLR4 in the host defense in the lung.

【 授权许可】

   
2005 MacRedmond et al; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

【 参考文献 】

• [1]Bowie A, O'Neill LA: The interleukin-1 receptor/Toll-like receptor superfamily: signal generators for pro-inflammatory interleukins and microbial products. J Leukoc Biol 2000, 67:508-514.
• [2]May MJ, Ghosh S: Signal transduction through NF-kappa B. Immunol Today 1998, 19:80-88.
• [3]Yamamoto M, Takeda K, Akira S: TIR domain-containing adaptors define the specificity of TLR signaling. Mol Immunol 2004, 40:861-868.
• [4]Yang D, Chertov O, Bykovskaia SN, Chen Q, Buffo MJ, Shogan J, Anderson M, Schroder JM, Wang JM, Howard OM, Oppenheim JJ: Beta-defensins: linking innate and adaptive immunity through dendritic and T cell CCR6. Science 1999, 286:525-528.
• [5]Ganz T: Antimicrobial polypeptides in host defense of the respiratory tract. J Clin Invest 2002, 109:693-697.
• [6]Bals R, Hiemstra PS: Innate immunity in the lung: how epithelial cells fight against respiratory pathogens. Eur Respir J 2004, 23:327-333.
• [7]Ganz T: Defensins: antimicrobial peptides of innate immunity. Nat Rev Immunol 2003, 3:710-720.
• [8]Raj PA, Dentino AR: Current status of defensins and their role in innate and adaptive immunity. FEMS Microbiol Lett 2002, 206:9-18.
• [9]Schutte BC, Mitros JP, Bartlett JA, Walters JD, Jia HP, Welsh MJ, Casavant TL, McCray PBJ: Discovery of five conserved beta -defensin gene clusters using a computational search strategy. Proc Natl Acad Sci U S A 2002, 99:2129-2133.
• [10]Harder J, Meyer-Hoffert U, Teran LM, Schwichtenberg L, Bartels J, Maune S, Schroder JM: Mucoid Pseudomonas aeruginosa, TNF-alpha, and IL-1beta, but not IL-6, induce human beta-defensin-2 in respiratory epithelia. Am J Respir Cell Mol Biol 2000, 22:714-721.
• [11]Singh PK, Jia HP, Wiles K, Hesselberth J, Liu L, Conway BA, Greenberg EP, Valore EV, Welsh MJ, Ganz T, Tack BF, McCray PBJ: Production of beta-defensins by human airway epithelia. Proc Natl Acad Sci U S A 1998, 95:14961-14966.
• [12]Hertz CJ, Wu Q, Porter EM, Zhang YJ, Weismuller KH, Godowski PJ, Ganz T, Randell SH, Modlin RL: Activation of Toll-like receptor 2 on human tracheobronchial epithelial cells induces the antimicrobial peptide human beta defensin-2. J Immunol 2003, 171:6820-6826.
• [13]Sethi S, Murphy TF: Bacterial infection in chronic obstructive pulmonary disease in 2000: a state-of-the-art review. Clin Microbiol Rev 2001, 14:336-363.
• [14]Parad RB, Gerard CJ, Zurakowski D, Nichols DP, Pier GB: Pulmonary outcome in cystic fibrosis is influenced primarily by mucoid Pseudomonas aeruginosa infection and immune status and only modestly by genotype. Infect Immun 1999, 67:4744-4750.
• [15]Vora P, Youdim A, Thomas LS, Fukata M, Tesfay SY, Lukasek K, Michelsen KS, Wada A, Hirayama T, Arditi M, Abreu MT: Beta-defensin-2 expression is regulated by TLR signaling in intestinal epithelial cells. J Immunol 2004, 173:5398-5405.
• [16]Becker MN, Diamond G, Verghese MW, Randell SH: CD14-dependent lipopolysaccharide-induced beta-defensin-2 expression in human tracheobronchial epithelium. J Biol Chem 2000, 275:29731-29736.
• [17]Monick MM, Yarovinsky TO, Powers LS, Butler NS, Carter AB, Gudmundsson G, Hunninghake GW: Respiratory syncytial virus up-regulates TLR4 and sensitizes airway epithelial cells to endotoxin. J Biol Chem 2003, 278:53035-53044.
• [18]Jia HP, Kline JN, Penisten A, Apicella MA, Gioannini TL, Weiss J, McCray PBJ: Endotoxin responsiveness of human airway epithelia is limited by low expression of MD-2. Am J Physiol Lung Cell Mol Physiol 2004, 287:L428-37.
• [19]Greene C, Lowe G, Taggart C, Gallagher P, McElvaney N, O'Neill S: Tumor necrosis factor-alpha-converting enzyme: its role in community-acquired pneumonia. J Infect Dis 2002, 186:1790-1796.
• [20]Griffin S, Taggart CC, Greene CM, O'Neill S, McElvaney NG: Neutrophil elastase up-regulates human beta-defensin-2 expression in human bronchial epithelial cells. FEBS Lett 2003, 546:233-236.
• [21]Frantz S, Kobzik L, Kim YD, Fukazawa R, Medzhitov R, Lee RT, Kelly RA: Toll4 (TLR4) expression in cardiac myocytes in normal and failing myocardium. J Clin Invest 1999, 104:271-280.
• [22]Re F, Strominger JL: Separate functional domains of human MD-2 mediate toll-like receptor 4-binding and lipopolysaccharide responsiveness. J Immunol 2003, 171:5272-5276.
• [23]Guillot L, Medjane S, Le-Barillec K, Balloy V, Danel C, Chignard M, Si-Tahar M: Response of human pulmonary epithelial cells to lipopolysaccharide involves Toll-like receptor 4 (TLR4)-dependent signaling pathways: evidence for an intracellular compartmentalization of TLR4. J Biol Chem 2004, 279:2712-2718.
• [24]Devaney JM, Greene CM, Taggart CC, Carroll TP, O'Neill SJ, McElvaney NG: Neutrophil elastase up-regulates interleukin-8 via toll-like receptor 4. FEBS Lett 2003, 544:129-132.
• [25]Lee JH, Del Sorbo L, Uhlig S, Porro GA, Whitehead T, Voglis S, Liu M, Slutsky AS, Zhang H: Intercellular adhesion molecule-1 mediates cellular cross-talk between parenchymal and immune cells after lipopolysaccharide neutralization. J Immunol 2004, 172:608-616.
• [26]Tsutsumi-Ishii Y, Nagaoka I: Modulation of human beta-defensin-2 transcription in pulmonary epithelial cells by lipopolysaccharide-stimulated mononuclear phagocytes via proinflammatory cytokine production. J Immunol 2003, 170:4226-4236.
• [27]Keicho N, Elliott WM, Hogg JC, Hayashi S: Adenovirus E1A upregulates interleukin-8 expression induced by endotoxin in pulmonary epithelial cells. Am J Physiol 1997, 272:L1046-52.
• [28]Krakauer T: Stimulant-dependent modulation of cytokines and chemokines by airway epithelial cells: cross talk between pulmonary epithelial and peripheral blood mononuclear cells. Clin Diagn Lab Immunol 2002, 9:126-131.
• [29]Kaper JB, Nataro JP, Mobley HL: Pathogenic Escherichia coli. Nat Rev Microbiol 2004, 2:123-140.
• [30]Schulz C, Farkas L, Wolf K, Kratzel K, Eissner G, Pfeifer M: Differences in LPS-induced activation of bronchial epithelial cells (BEAS-2B) and type II-like pneumocytes (A-549). Scand J Immunol 2002, 56:294-302.
• [31]Erridge C, Bennett-Guerrero E, Poxton IR: Structure and function of lipopolysaccharides. Microbes Infect 2002, 4:837-851.
• [32]Kulshin VA, Zahringer U, Lindner B, Jager KE, Dmitriev BA, Rietschel ET: Structural characterization of the lipid A component of Pseudomonas aeruginosa wild-type and rough mutant lipopolysaccharides. Eur J Biochem 1991, 198:697-704.
• [33]Lyczak JB, Cannon CL, Pier GB: Establishment of Pseudomonas aeruginosa infection: lessons from a versatile opportunist. Microbes Infect 2000, 2:1051-1060.
• [34]Koyama S, Sato E, Nomura H, Kubo K, Miura M, Yamashita T, Nagai S, Izumi T: The potential of various lipopolysaccharides to release IL-8 and G-CSF. Am J Physiol Lung Cell Mol Physiol 2000, 278:L658-66.
• [35]Wurfel MM, Wright SD: Lipopolysaccharide-binding protein and soluble CD14 transfer lipopolysaccharide to phospholipid bilayers: preferential interaction with particular classes of lipid. J Immunol 1997, 158:3925-3934.
• [36]Viriyakosol S, Tobias PS, Kitchens RL, Kirkland TN: MD-2 binds to bacterial lipopolysaccharide. J Biol Chem 2001, 276:38044-38051.
• [37]Muir A, Soong G, Sokol S, Reddy B, Gomez M, Van Heeckeren A, Prince A: Toll like receptors in normal and cystic fibrosis airway epithelial cells. Am J Respir Cell Mol Biol 2003.
• [38]Azghani AO, Miller EJ, Peterson BT: Virulence factors from Pseudomonas aeruginosa increase lung epithelial permeability. Lung 2000, 178:261-269.
• [39]Diamond G, Russell JP, Bevins CL: Inducible expression of an antibiotic peptide gene in lipopolysaccharide-challenged tracheal epithelial cells. Proc Natl Acad Sci U S A 1996, 93:5156-5160.
• [40]Haziot A, Chen S, Ferrero E, Low MG, Silber R, Goyert SM: The monocyte differentiation antigen, CD14, is anchored to the cell membrane by a phosphatidylinositol linkage. J Immunol 1988, 141:547-552.
• [41]Tsutsumi-Ishii Y, Nagaoka I: NF-kappa B-mediated transcriptional regulation of human beta-defensin-2 gene following lipopolysaccharide stimulation. J Leukoc Biol 2002, 71:154-162.
• [42]O'Neill LA, Dunne A, Edjeback M, Gray P, Jefferies C, Wietek C: Mal and MyD88: adapter proteins involved in signal transduction by Toll-like receptors. J Endotoxin Res 2003, 9:55-59.
• [43]Takeda K, Akira S: TLR signaling pathways. Semin Immunol 2004, 16:3-9.
• [44]Graham NM: The epidemiology of acute respiratory infections in children and adults: a global perspective. Epidemiol Rev 1990, 12:149-178.
• [45]Renshaw M, Rockwell J, Engleman C, Gewirtz A, Katz J, Sambhara S: Cutting edge: impaired Toll-like receptor expression and function in aging. J Immunol 2002, 169:4697-4701.
• [46]Ohta T, Yamashita N, Maruyama M, Sugiyama E, Kobayashi M: Cigarette smoking decreases interleukin-8 secretion by human alveolar macrophages. Respir Med 1998, 92:922-927.
• [47]Hasday JD, Bascom R, Costa JJ, Fitzgerald T, Dubin W: Bacterial endotoxin is an active component of cigarette smoke. Chest 1999, 115:829-835.
• [48]Iwami KI, Matsuguchi T, Masuda A, Kikuchi T, Musikacharoen T, Yoshikai Y: Cutting edge: naturally occurring soluble form of mouse Toll-like receptor 4 inhibits lipopolysaccharide signaling. J Immunol 2000, 165:6682-6686.