Intracellular expression of toll-like receptor 4 in neuroblastoma cells and their unresponsiveness to lipopolysaccharide
© Hassan et al; licensee BioMed Central Ltd. 2006
Received: 30 June 2006
Accepted: 08 December 2006
Published: 08 December 2006
Recently it has been reported that, toll-like receptors (TLRs) are expressed on a series of tumor cells, such as colon cancer, breast cancer, prostate cancer, melanoma and lung cancer. Although some cancer cells like melanoma cells are known to respond to lipopolysaccharide (LPS) via TLR4, not all cancer cells are positive for TLR4. There is little information on the expression and function of TLR4 in neuroblastoma cells. In this study, we investigated the expression of TLR4 in human neuroblastoma NB-1 cell line.
Expression and localization of TLR4 were detected by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometric analysis, respectively. Activation of nuclear factor (NF)-κB by LPS was detected by degradation of IκB-α and NF-κB luciferase assay. Activation and expression of mitogen-activated protein (MAP) kinase and interferon regulatory factor (IRF)-3 was detected by immunoblot analysis.
Human NB-1 neuroblastoma cells expressed intracellular form of TLR4, but not the cell surface form. Further, NB-1 cells express CD14, MD2 and MyD88, which are required for LPS response. However, LPS did not significantly induce NF-κB activation in NB-1 cells although it slightly degraded IκB-α. NB-1 cells expressed no IRF-3, which plays a pivotal role on the MyD88-independent pathway of LPS signaling. Collectively, NB-1 cells are capable to avoid their response to LPS.
Although human NB-1 neuroblastoma cells possessed all the molecules required for LPS response, they did not respond to LPS. It might be responsible for intracellular expression of TLR4 or lack of IRF-3.
Recently it has been reported that, toll-like receptors (TLRs) are expressed on a series of tumor cells, such as colon cancer, breast cancer, prostate cancer, melanoma and lung cancer . In particular, melanoma cells are reported to express TLR-4, respond to lipopolysaccharide (LPS) and produce interleukin (IL)-8 . Thus, a direct link between tumor cell activation and LPS might be suggested because some tumor cells constitutively express TLR-4 . However, not all tumor cells are positive for TLR-4 and it seems to depend on the tissue from which tumor cells originated.
Among neural cells, microglia cells are resident immune cells of the brain and express almost all TLRs [3–6]. Apart from microglia, astrocytes also differentially express TLRs with a robust expression of TLR-2 and a low level of expression of TLR-1, 4, 5 and 9 . It is unclear whether neuroblasts express TLR-4 or not. Considering that neuroblastoma cells do not express TLR-4 , neuroblasts are unlikely to express TLR-4. In the present study, we examined two neuroblastoma cell lines to confirm the lack of TLR-4 expression in neuroblatoma cells. Surprisingly, we found that NB-1 neuroblastoma cells expressed intracellular TLR-4 but did not respond to LPS. Here, we discuss a possible mechanism of the unresponsiveness of intracellular TLR-4-expressing NB-1 neuroblastoma cells to LPS.
LPS from Esherichia coli O55:B5 was obtained from Sigma Chemicals, St. Louis, MO, USA. Phycoerythrin (PE)-conjugated anti-human TLR-4 antibody was obtained from eBioscience, San Diego, CA, USA. Antibody to human TLR-4 was purchased from Santa-Cruz biotechnology, Santa-Cruz, CA, USA. Antibody to phosphorylated interferon regulatory factor (IRF)-3 was from Upstate, Lake placid, New York, USA. Antibody to p38, c-Jun N-terminal kinase (JNK1/2), IκB, IRF-3, and phosphorylated form of p38 and JNK1/2 were purchased from Cell Signaling Technology, Beverly, MA, USA. Human interferon (IFN)-γ was purchased from Calbiochem, San Diego, CA, USA. Human IL-1β was purchased from R & D Inc, Concord, MA, USA.
Human neuroblastoma cell lines, NB-1 and SK-N-SH, and human monocytic cell line, U-937, were obtained from Riken Cell bank (Tsukuba, Japan). NB-1 and U-937 cells were maintained in RPMI containing 5% heat inactivated fetal calf serum (GIBCO-BRL, Gaithersburg, MD, USA) and SK-N-SH cells was maintained in MEM with 10% heat-inactivated fetal calf serum. Cells were cultured in the presence of antibiotics at 37°C under 5% CO2.
Laser flow cytometric analysis
NB-1 cells were seeded (1 × 106/ml) in 35 mm dish for overnight, trypsinized and washed with phosphate-buffed saline (PBS). To detect cell surface expression of TLR4, the cells were incubated with PE-conjugated anti-TLR4 antibody for 20 min in dark on ice, washed and resuspended in PBS. To detect intracellular expression of TLR4, the cells were fixed with a fixation buffer (eBioscience) in dark and treated with a permeabilization buffer (eBioscience) according to the instruction of the manufacturer. The cells were stained with PE-conjugated anti-TLR-4 antibody. Cell surface or intracellular TLR-4-positive cells were analyzed by a fluorescence activated cells sorter (BD FACS Calibur, San Jose, CA, USA). PE-conjugated anti-rat isotype IgG was used as a negative control for the experiments.
Reverse transcription-polymerase chain reaction (RT-PCR)
RT-PCR was performed as described previously . Briefly, NB-1 and SK-N-SH cells were seeded (1 × 106/ml) in 35 mm dish for overnight, trypsinized and washed with PBS. RNA was extracted from the cells with RNeasy mini kit (Qiagen, Valencia, CA, USA). Semi-quantative RT-PCR was carried out by using access quick RT-PCR system (Promega, Madison, WI, USA). Primer Sequence for TLR4, Forward 5 TGGATACGTTTCCTTATAAG 3 and Reverse 5 GAAATGGAGGCACCCCTTC 3 , CD14, forward 5 CTGCAACTTCTCCGAACCTC 3 and reverse 5 TAGGTCCTCGAGCGTCAGTT 5, MD2, forward 5 TTCCACCCTGTTTTCTTCCA 3 and reverse 5 AATCGTCATCAGATCCTCGC 3, MyD88, forward 5 GCACATGGGCACATACAGAC 3 and reverse 5 TGGGTCCTTTCCAGAGTTTG 3 . GAPDH for TLR4 and G3PDH for CD14, MD2, MyD88 was used as an equal loading control. Optimized reverse transcription and PCR conditions were as follows, TLR4 (48°C for 45 min followed by 95°C for 2 min and 30 cycles at 95°C 30 sec, 56°C for 30 sec, 72°C for 60 sec), CD14, MD2 and MyD88 (48°C for 45 min followed by 95°C for 2 min and 28 cycles at 95°C 30 sec, 60°C 30 sec, 72°C for 1 min). For each experiment RNA extracted from human monocyte U937 was used as a positive control for TLR4, CD14, MD2 and MyD88. PCR products were analyzed by electrophoresis on 2% agarose gel. The gels were stained with CYBR safe DNA gel stain (Molecular probe, Eugene, OR, USA) and visualized under an ultraviolet transilluminator. The 100 bp DNA size marker (Invitrogen, Carlsbad, CA, USA) was also run to determine approximate size of the product.
Immunoblotting was performed as described previously . To differentiate monocyte to machrophages, U937 cells were treated with PMA for 24 hours, washed and incubated for another 24 hours before treated with LPS as indicated below. NB-1 cells were directly seeded in 35 mm culture dish, treated with LPS (100 ng/ml) for various time. The cell lysates were extracted by lysis buffer containing 0.5 M Tris-HCL, 4% SDS and 2 mercaptoethanol, and boiled at 80°C for 5 min. The protein concentration of each sample was determined by BCA protein assay reagent (Pierce, Rockford, IL, USA). An equal amount of protein (40 μg) was analyzed by SDS-polyacrylamide gel electrophoresis under reducing conditions and transferred to a membrane filter. The membranes were treated with an appropriately diluted antibody for overnight. The immune complexes were detected with a 1:5000 dilution of horseradish peroxidase-conjugated protein G for 1 h and the bands were visualized with a chemiluminesent reagent (Pierce, Rockford, IL). The chemiluminescence was analyzed by a light capture system (AE6955, Atto Corp., Tokyo, Japan) with CS grab, version 1.0. Densimetric analysis of each band was analyzed with CS analyzer software, version 1.0 (Atto Corp., Tokyo, Japan).
Luciferase reporter gene assay for NF-κB activation
The luciferase reporter gene assay was performed as described previously . Briefly, NB-1 cells (3 × 105/ml) were plated in a 35 mm plastic dish. On the following day, the cells were transfected with 0.5 mg/ml of pNF-κB-TA-luc luciferase reporter gene (Invitrogen, Carlsbad, CA, USA) and 0.05 mg of pRL-TK plasmid (Promega, Madison, WI, USA) by lipofectamine 2000 transfection reagent (Gibco-BRL). The transfected cells were incubated for 48 h, stimulated with LPS (100 ng/ml) and IL-1β (100 ng/ml) for 6 h, lysed with a lysis reagent from Promega, and the luciferase activity was determined by the dual luciferase assay kit (Promega). To confirm the activation of NF-κB, we used another plasmid pNiFty2-Luc from Invivogen, San-Diego, CA, USA.
Statistical significance was determined by Student's t test. Experimental result is expressed as the mean of triplicates ± SD in at least 3 independent experiments.
NB-1 cells express TLR4
Intracellular but not cell surface expression of TLR4 in NB-1 cells
NB-1 cells express CD14, MD2 and MyD88 which participate in TLR4 signaling pathway
LPS partially degrades IκB-α
LPS does not activate NF-κB in NB-1 cells
NB-1 cells do not express IRF-3
LPS does not activate MAP kinases in NB-1 cells
In the present study we have demonstrated that NB-1 and SK-N-SH neuroblastoma cells express intracellular TLR-4. On the other hand, SHSY-5Y neuroblastoma cells are reported to express no TLR4 at mRNA level . Therefore, there seemed to be a difference in TLR4 expression among various neuroblastoma cell lines. Although TLR4 is usually expressed on cell surface, there are a number of reports on intracellular expression of TLR4 in various cell types and cell lines, such as human placenta, coronary artery endothelial cells, dendritic cells, pulmonary epithelial cells, and intestinal epithelial cells m-ICc12 [11, 26–29]. Further, intracellular TLR4 is reported to responds to LPS [11, 26–29]. However, NB-1 cells expressing intracellular TLR4 did not respond to LPS.
NB-1 neuroblastoma cells express TLR4, MD2, CD14 and MyD88, which are required for LPS signaling. LPS slightly degraded IκB-α but did not significantly lead to NF-κB activation in NB-1 cells. On the other hand, NF-κB signaling acts normally because IL-1β induced the activation of NF-κB in those cells. Therefore, intracellular TLR4 expression might be involved in the unresponsiveness of NB-1 cells to LPS. LPS may not be internalized in NB-1 cells. Otherwise, internalized LPS may undergo deacylation by lysomal enzymes and become inactive before reaching Golgi apparatus where TLR4 reside. Further, we can not exclude a possibility that the LPS unresponsiveness may be due to deficiency of some molecule(s) required for LPS signaling. Interestingly, we found no expression of IRF-3 in NB-1 cells. Kawai et al. reported that IRF-3 plays a central role in MyD88-independent pathway . Therefore, the MyD88-independent pathway of LPS signaling may not occur in NB-1 cells. The impairment of MyD88-independent pathway with defected IFR-3 might be related to the unresponsiveness of NB-1 cells to LPS.
Thieblemont et al. demonstrated that LPS is transported to Golgi apparatus . Further, Hornef et al. reported that TLR4 resides in the Golgi apparatus and cellular trafficking of LPS to Golgi apparatus is important for innate immune response . On the other hand, there are several contradictory reports on it. Latz et al. reported that LPS traffics to the Golgi apparatus with TLR4-CD14-MD2 complex but does not initiate the signal transduction pathway and that TLR4 must reside on cell surface to trigger the signaling pathway, . Even when LPS was artificially introduced into the cytoplasm, human coronary epithelial cells were failed to activate due to its intracellular localization of TLR4 . Our present study supports a possibility that LPS might fail in triggering NF-κB activation in NB-1 cells through intracellular TLR4 expression or impaired MyD88-independent signaling.
In this report we for the first time demonstrate that neuroblastoma cells express TLR4 at the cytoplasm but not at the cell surface. We further showed that, neuroblastoma cell line NB-1 does not respond to LPS. We conclude that unresponsiveness to LPS might be due to intracellular localization of TLR4 or lack of IRF-3 expression. Further studies must be necessary to confirm the expression and role of TLR4 in neuroblastoma cells and neuroblasts.
This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. We are grateful to K. Takahashi and A. Morikawa for the technical assistance.
- Huang B, Zhao J, Li X, He KL, Chen Y, Mayer L, Unkeless JC, Xiong H: Toll like receptors on tumor cells facilitate evasion of immune surveillance. Can Res. 2005, 65: 5009-5014. 10.1158/0008-5472.CAN-05-0784.View ArticleGoogle Scholar
- Molteni M, Marabella D, Orlandi C, Rossetti C: Melanoma cell lines are responsive in vitro to lipopolysaccharide and express TLR-4. Can letter. 2006, 235: 75-83. 10.1016/j.canlet.2005.04.006.View ArticleGoogle Scholar
- Lehnardt S, Massilon L, Follett P, Jensen FE, Ratan R, Rosenberg PA, Volpe JJ, Vartanian T: Activation of innate immunity in the CNS triggers neurodegeneration through a Toll-like receptor 4 dependent pathway. Proc Natl Acad Sci USA. 2003, 111: 8514-8519. 10.1073/pnas.1432609100.View ArticleGoogle Scholar
- Iliev AI, Stringaris AK, Nau R, Neumann H: Neuronal injury mediated via stimulation of microglial toll-like receptor-9 (TLR9). FASEB J. 2004, 18: 412-424.PubMedGoogle Scholar
- Jack CS, Arbour N, Manusow J, Montgrain , Blain VM, McCrea E, Shapiro A, Antel JP: TLR signaling tailors innate immune responses in human microglia and astrocytes. J Immunol. 2005, 175: 4320-43330.View ArticlePubMedGoogle Scholar
- Zhang Z, Trautmann K, Schluesener HJ: Microglia activation in rat spinal cord by systemic injection of TLR3 and TLR7/8 agonists. J Neuroimmunol. 2005, 164: 154-160. 10.1016/j.jneuroim.2005.03.014.View ArticlePubMedGoogle Scholar
- Hassan F, Islam S, Mu MM, Ito H, Koide N, Mori I, Yoshida T, Yokochi T: Lipopolysaccharide Prevents Doxorubicin-InducedApoptosis in RAW 264.7 Macrophage Cells by Inhibiting p53 Activation. Mol Cancer Res. 2005, 3: 373-379. 10.1158/1541-7786.MCR-05-0046.View ArticlePubMedGoogle Scholar
- Smith PD, Smythies LE, Mosteller-Barnum M, Sibley DA, Russell MW, Merger M, Sellers MT, Orenstein JM, Shimada T, Graham MF, Kubagawa H: Intestinal macrophages lack CD14 and CD89 and consequently are down-regulated for LPS- and IgA-mediated activities. J Immunol. 2001, 167: 2651-2656.View ArticlePubMedGoogle Scholar
- Hirata T, Osuga Y, Hirota Y, Koga K, Yoshino O, Harada M, Morimoto C, Yano T, Nishii O, Tsutsumi O, Taketani Y: Evidence for the Presence of Toll-Like Receptor 4 System in the Human Endometrium. J Clin Endocrin & Metab. 2005, 90: 548-556. 10.1210/jc.2004-0241.View ArticleGoogle Scholar
- Islam S, Hassan F, Mu MM, Ito H, Koide N, Mori I, Yoshida T, Yokochi T: Piceatannol Prevents Lipopolysacharide (LPS) induced nitric oxide (NO) production and nuclear factor (NF)-κB activation through inhibiting Iκ-β kinasa (IKK). Micro Immunol. 2004, 48: 729-736.View ArticleGoogle Scholar
- Hornef MW, Henriques N, Vandewalle A, Normark S: Intracellular recognition of lipopolysaccahirde by toll like receptor 4 in intestinal epithelial cells. J Exp Med. 2003, 198: 1225-1235. 10.1084/jem.20022194.View ArticlePubMedPubMed CentralGoogle Scholar
- Akira S, Takeda K: Toll like receptor signaling. Nat Rev Immunol. 2004, 4: 499-511. 10.1038/nri1391.View ArticlePubMedGoogle Scholar
- Ahmad-Nejad P, Hacker H, Rutz M, Bauer S, Vabulas RM, Wagner H: Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments. Eur J Immunol. 2002, 32: 1958-1968. 10.1002/1521-4141(200207)32:7<1958::AID-IMMU1958>3.0.CO;2-U.View ArticlePubMedGoogle Scholar
- Heil F, Ahmad-Naiad P, Hemmi H, Hochrein H, Ampenberger E, Gellet T, Dietrich H, Lipford G, Takeda K, Akira S, Wagner H, Bauer S: The Toll-like receptor 7 (TLR7)-specific stimulus loxoribine uncovers a strong relationship within the TLR7, 8 and 9 subfamily. Eur J Immunol. 2003, 33: 2987-2997. 10.1002/eji.200324238.View ArticlePubMedGoogle Scholar
- Matsumoto M, Funami K, Tanabe M, Oshiumi H, Shingai M, Seto Y, Yamamoto A, Seya T: Subcellular localization of Toll-like receptor 3 in human dendritic cells. J Immunol. 2003, 171: 3154-3162.View ArticlePubMedGoogle Scholar
- Latz E, Schoenemeyer A, Visintin A, Fitzgerald KA, Monks BG, Knetter CF, Lien E, Nilsen NJ, Espevik T, Golenbock DT: TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat Immunol. 2004, 5: 190-198. 10.1038/ni1028.View ArticlePubMedGoogle Scholar
- Wright SD, Ramos RA, Tobias PS, Ulvitch RJ, Mathison JC: CD14, a receptor for complexes of LPS and LBP binding protein. Science. 1990, 249: 1431-1433. 10.1126/science.1698311.View ArticlePubMedGoogle Scholar
- Shimazu R, Akashi S, Ogata H, Nagai Y, Fukudome K, Miyake K, Kimoto M: MD-2, a molecule that confers LPS responsiveness on toll like receptor 4. J Exp Med. 1999, 189: 1777-1782. 10.1084/jem.189.11.1777.View ArticlePubMedPubMed CentralGoogle Scholar
- Li Q, Verma IM: NF-kB regulation in the immune system. Nat Rev Immunol. 2002, 2: 725-734. 10.1038/nri910.View ArticlePubMedGoogle Scholar
- Fiebich BL, Mueksch B, Boehringer M, Hull M: Interleukin-1 β induces cyclooxygenase-2 and prostaglandin E(2) synthesis in human neuroblastoma cells: involvement of p38 mitogen-activated protein kinase and nuclear factor-kappa B. J Neurochem. 2000, 75: 2020-2028. 10.1046/j.1471-4159.2000.0752020.x.View ArticlePubMedGoogle Scholar
- Kawai T, Adachi O, Ogawa T, Takeda K, Akira S: Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity. 2000, 11: 115-122. 10.1016/S1074-7613(00)80086-2.View ArticleGoogle Scholar
- Doyle SE, Vaidya SA, O'Connell R, Dadgostar H, Dempsey PW, Ting-Ting Wu, Rao G, Sun R, Haberland ME, Modlin RL, Cheng G: IRF3 Mediates a TLR3/TLR4-Specific Antiviral Gene Program. Immunity. 2002, 17: 251-263. 10.1016/S1074-7613(02)00390-4.View ArticlePubMedGoogle Scholar
- Toshchakov V, Jones BW, Perera PY, Thomas K, Cody MJ, Zhang S, Major J, Hamilton TA, Fenton MJ, Vogel SN: TLR4, but not TLR2, mediates IFN-beta-induced STAT1alpha/beta-dependent gene expression in macrophages. Nat Immunol. 2002, 3: 392-398. 10.1038/ni774.View ArticlePubMedGoogle Scholar
- Hoshino K, Kaisho T, Iwabe T, Takeuchi O, Akira S: Differential involvement of IFN-β in Toll like receptor-stimulated dendritic cell activation. Int Imunol. 2002, 14: 1225-1231. 10.1093/intimm/dxf089.View ArticleGoogle Scholar
- Wang C, Deng L, Hong M, Akkaraju GR, Inoue J, Chen ZJ: TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature. 2001, 412: 346-351. 10.1038/35085597.View ArticlePubMedGoogle Scholar
- Beijar EC, Mallard C, Powell TL: Expression and subcellular localization of TLR-4 in term and first triester human placentra. Placentra. 2006, 27: 322-326. 10.1016/j.placenta.2004.12.012.View ArticleGoogle Scholar
- Dunzendorfer S, Lee HK, Soldau K, Tobias PS: Toll like receptor 4 functions intracellularly in human coronary artery endothelial cells; roles of LBP and sCD14 in mediating LPS responses. FASEB J. 2004, 18: 1117-1129.PubMedGoogle Scholar
- Uronen-Hansson H, Allen J, Osman J, Squires G, Klein N, Callard RE: Toll like receptor 2 (TLR2) and TLR4 are present inside human dendritic cells, associated with microtubules and the golgi apparatus but are not detectable on the cell surface: integrity of microtubule is required for interleukin-12 production in response to internalized bacteria. Immunology. 2004, 111: 173-178. 10.1111/j.0019-2805.2003.01803.x.View ArticlePubMedPubMed CentralGoogle Scholar
- Guillot L, Medjane S, Le-Barillec K, Balloy V, Danel C, Chingard M, Si-Tahar M: Response of human pulmonary epithelial cells to lipopolysaccharide involves toll like receptpr 4 (TLR4)-dependent signaling pathway; evidence for an intracellular compartmentalizatioin of TLR4. J Biol Chem. 2004, 279: 2712-2718. 10.1074/jbc.M305790200.View ArticlePubMedGoogle Scholar
- Kawai T, Takeuchi O, Fujita T, Jun-ichiro I, Muhldadt PE, Sato S, Hoshino K, Akira S: Lipopolysaccharide stimulates the MyD88-independent pathway and results in the activation of IFN-regulatory factor 3 and the expression of a subset of Lipopolysaccharide-inducibel genes. J Immunol. 2001, 167: 5887-5894.View ArticlePubMedGoogle Scholar
- Thieblemont N, Wright SD: Transport of bacterial lypopolysaccharide to the golgi apparatus. J Exp Med. 1999, 4: 523-534. 10.1084/jem.190.4.523.View ArticleGoogle Scholar
- Latz E, Visintin A, Lien E, Fitzgeralk KA, Monks BG, Kurt-Jones EA, Golenbock DT, Espevik T: lypopolysaccharide rapidly traffics to and from the golgi apparatus with the toll like receptor4-MD-2-CD14 complex in a process that is distinct from the initiation of signal transduction. J Biol Chem. 2002, 277: 47834-47843. 10.1074/jbc.M207873200.View ArticlePubMedGoogle Scholar
- Ueta M, Nochi T, Jang MO, Park EJ, Igarashi O, Hino A, Kawasaki S, Shikina T, Hiroi T, Kinoshita S, Kiyuono H: Intracellularly expressed TLR3s and TLR4s contribution to an immunosilent environment at the ocular mucosal epithelium. J Immunol. 2004, 173: 3337-3347.View ArticlePubMedGoogle Scholar
- The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/6/281/prepub
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.