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Evaluation of toll-like receptor 4 gene expression of immortalized human liver cell lines
Evaluation of Toll-Like Receptor 4 Gene Expression of Immortalized Human Liver Cell Lines N. Kobayashi, M. Takesue, N. Kobayashi, T. Okitsu, T. Matsumura, T. Totsugawa, M. Maruyama, Y. Morimoto, T. Kunieda, N. Shibata, K. Ohmoto, S. Yamamoto, and N. Tanaka
C
URRENTLY a bioartificial liver (BAL) has been widely anticipated as a bridge to liver transplantation or liver regeneration.1 Cell– cell interactions between hepatocytes and liver nonparenchymal cells are important for the sophisticated functions of the organ. To develop a functional BAL, we have focused on the coculture system of liver parenchymal and nonparenchymal cell populations.2 The severe shortage of donor livers for cell isolation limits the use of human liver cells for BAL. Thus, we have established immortalized human liver cell lines as possible alternatives to normal cells.3–5 Patients with falling liver function often experience septic shock and multiple organ failure. Toll-like receptor (TLR)-4 has been identified as a receptor for enteric lipopolysaccharide (LPS).6 TLR-4 is involved in innate immune recognition and cellular activation in response to microbial antigens. Ligand engagement of TLRs results in activation of NF-B and induction of the cytokines and costimulatory molecules required for the activation of the adaptive immune response, as shown in Figure 1. Thus, it is of extreme importance to evaluate TLR-4 expression by the human liver cell lines that we have recently established to develop a BAL. MATERIALS AND METHODS Cells and Culture Conditions Human hepatocytes and human liver sinusoidal endothelial cells (HLSECs) were transduced with a retroviral vector SSR#69 encoding SV40T, as previously reported.3–5 One of the resulting hepatocyte clones, NKNT-3, and one of SV40T-transduced HLSEC clones, HNNT-2, were used in this work. TWNT-1, a human telomerase reverse transcriptase (hTERT)-immortalized clone derived from the hepatic fat-storing cell strain LI 90 was also used in the current TLR-4 study.7 Cells maintained chemically defined serum-free CS-C medium (Cell Systems. Co., Seattle, Wash) were examined by means of an inverted phase contrast microscope.
Reverse Transcriptase-Polymerase Chain Reaction Analysis for TLR-4 Total RNA was extracted from cells using a Qiagen kit (Valencia, Calif) following the manufacture’s instructions, and treated with RNase-free DNase. For the reverse transcriptase (RT) reaction, the MMLV preamplification system (Life Technologies, Inc. Rockville, Md) was applied, as previously reported.8 Polymerase chain reaction (PCR) amplification was done with Taq gold polymerase © 2003 by Elsevier Science Inc. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 35, 431– 432 (2003)
Fig 1. Diagrams representing the role of TLR-4. Recognition of LPS through TLR-4 receptor results in NF-B activation and subsequent production of various types of chomokines and cytokines. (Perkin Elmer, Foster City, Calif) for 32 cycles at 95 °C for 45 seconds 54 °C for 45 seconds, and 72 °C for 1 minute. The oligonucleotide primers for RT-PCR were: TLR-4, 5⬘-TGAGCAGTCGTGCTGGTATC and 5⬘-CAGGGCTTTTCTGAGTCGTC;9 for glyceraldehydes-3-phophate dehydrogenase (GAPDH), 5⬘-CGGAGTCAACGGATTTGGTCGTAT and 5⬘AGCCTTCTCCATGGTGGTGAAGAC. All PCR products were resolved on 1% agarose gel electrophoresis with DNA bands visualized by ethidium bromide staining.
RESULTS Expression Pattern of TLR-4 mRNA in Immortalized Liver Cells
RT-PCR analysis revealed that TLR-4 expression was detected in HNNT-2 cells (SV40T-transduced HLSECs) From the Division of Gastroenterology I, Department of Medicine, (N.K., M.T., T.K., N.S., K.O., S.Y.) Kawasaki Medical School, Kurashiki, Okayama; the Department of Surgery (N.K., T.O., T.M., T.T., M.M., Y.M., N.T.), Okayama University Graduate School of Medicine and Dentistry, Shikata-cho, Okayama, and The Japan Health Sciences Foundation Japan. Address reprint requests to Dr Naoya Kobayashi, Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. E-mail: [email protected] 0041-1345/03/$–see front matter doi:10.1016/S0041-1345(02)03800-9 431
432
Fig 2. Comparative expression of TIL-4 in human liver cell lines. The specific band for TLR-4 (167 bp) is detected in SV40-T transduced HLSEC line HNNT-2 and hTERT-immortalized hepatic fat-storing cell line TWNT-1. No corresponding band is detected in SV40-T immortalized human hepatocyte line NKNT-3. Detection of GAPDH mRNA showed equal loading of RNA (1, NKNT-3; 2, TWNT-1; 3, HNNT-2).
and TWNT-1 cells (hTERT-immortalized hepatic stallete cells). A more intense band for TLR-4 was observed in HNNT-2 cells compared to TWNT-1 cells (Fig 2). In contrast, SV40T-immortalized human hepatocyte NKNT-3 showed no TLR-4 expression. Equal loading of RNA was verified by detecting GAPDH mRNA.
KOBAYASHI, TAKESUE, KOBAYASHI ET AL
BAL to treat such patients. Our current finding that the transduced HLSEC line HNNT-2 showed more intense TLR-4 expression compared to immortalized hepatocytes and fat-storing cells is consistent with the fact that vascular endothelial cells, like monocytes and macrophages, are a critical target for LPS and many cytokines. Regulation of TLR-4 expression and production of soluble cytokines and chemokines in immortalized liver cells exposed to various concentrations of LPS must be analyzed before their use in BAL. In conclusion, the present study demonstrates that innate TLR-4 gene expression is well preserved in transduced HLSECs and fat-storing cells, suggesting that such cells are capable of directly sensing the presence of LPS thus performing an important role in hepatic sinusoidal linings as an integral component of a functional BAL. ACKNOWLEDGMENT We thank Dr T. Matsuura (Jikei University School of Medicine, Tokyo, Japan) and Dr K. Murakami (Tohoku Nenkin Hospital, Sendai, Japan) for providing LI90 cells. This work was supported in part by grants from the Ministry of Education, Science, and Culture, Japan; by grants from the Ministry of Economy and Industry, Japan.
DISCUSSION
The liver sinusoid is an important interface between the host and the external environment. Endothelial cells that line the hepatic sinusoid have a primary role in the immune and inflammatory systems. Vertebrates initiate a series of defense mechanisms, including a network of host-derived inflammatory mediators, by sensing the presence of pathogen-associated molecular patterns of microbial antigens, such as Gram-negative bacterial LPS and Gram-positive bacterial staphylococcal enterotoxin B.10,11 LPS is a potent activator of cells in the immune and inflammatory systems as well as endothelial cells, contributing to the systemic changes observed in septic shock. Host organisms have developed receptors that recognize specific pathogen-related molecular patterns.12 The human TLRs are a recently discovered family of receptors involved in innate immune recognition and cellular activation in response to bacterial antigens.13–15 To date, eight mammalian TLR family members have been documented, but only the ligands for TLR-2 and TLR-4 have been identified.6 TLR superfamily members are found in many species; those with known functions are involved in host responses to injury and infection. Patients with disturbed liver function often demonstrate serious infections, such as septic shock that eventually result in multi-organ failure.16 Thus, expression of TLRs must be documented on human liver cell lines that will be used in
REFERENCES 1. Watanabe FD, Mullon CJ, Hewitt WR, et al: Ann Surg 225:484, 1997 2. Begue JM, Guguen-Guillouzo C, Pasdeloup N, et al: Hepatology 4:839, 1984 3. Kobayashi N, Fujiwara T, Westerman KA, et al: Science 287:1258, 2000 4. Noguchi H, Kobayashi N, Westerman KA, et al: Hum Gene Ther 13:321, 2002 5. Watanabe T, Kobayashi N, Hoguchi H, et al: ASAIO J 47:166, 2001 6. Faure E, Equlis O, Sieling PA, et al: J Biol Chem 275:11058, 2000 7. Murakami K, Abe T, Miyazazwa M, et al: Lab Invest 72:731, 1995 8. Yoshioka T, Morimoto Y, Iwagaki H, et al: J Int Med Res 29:409, 2001 9. Tapping RI, Akashi S, Miyake K, et al: J Immunol 165:5780, 2000 10. Huemann D, Glauser MP: Calandra: Curr Opin Microbiol 1:49, 1998 11. Roberts AI, Blumberg RS, Christ AD, et al: Immunology 101:185, 2000 12. Medzhitov R, Janeway CA Jr: Curr Opin Immunol 9:4, 1997 13. Medzhitov R, Preston-Hurlburt P, Janeway CA: Nature 388:394, 1997 14. Wright SD: Exp Med 189:605, 1999 15. Chow JC, Young DW, Golenbock DT, et al: J Biol Chem 274:10689, 1999 16. Williams R: Semin Liver Dis 16:343, 1996
C
URRENTLY a bioartificial liver (BAL) has been widely anticipated as a bridge to liver transplantation or liver regeneration.1 Cell– cell interactions between hepatocytes and liver nonparenchymal cells are important for the sophisticated functions of the organ. To develop a functional BAL, we have focused on the coculture system of liver parenchymal and nonparenchymal cell populations.2 The severe shortage of donor livers for cell isolation limits the use of human liver cells for BAL. Thus, we have established immortalized human liver cell lines as possible alternatives to normal cells.3–5 Patients with falling liver function often experience septic shock and multiple organ failure. Toll-like receptor (TLR)-4 has been identified as a receptor for enteric lipopolysaccharide (LPS).6 TLR-4 is involved in innate immune recognition and cellular activation in response to microbial antigens. Ligand engagement of TLRs results in activation of NF-B and induction of the cytokines and costimulatory molecules required for the activation of the adaptive immune response, as shown in Figure 1. Thus, it is of extreme importance to evaluate TLR-4 expression by the human liver cell lines that we have recently established to develop a BAL. MATERIALS AND METHODS Cells and Culture Conditions Human hepatocytes and human liver sinusoidal endothelial cells (HLSECs) were transduced with a retroviral vector SSR#69 encoding SV40T, as previously reported.3–5 One of the resulting hepatocyte clones, NKNT-3, and one of SV40T-transduced HLSEC clones, HNNT-2, were used in this work. TWNT-1, a human telomerase reverse transcriptase (hTERT)-immortalized clone derived from the hepatic fat-storing cell strain LI 90 was also used in the current TLR-4 study.7 Cells maintained chemically defined serum-free CS-C medium (Cell Systems. Co., Seattle, Wash) were examined by means of an inverted phase contrast microscope.
Reverse Transcriptase-Polymerase Chain Reaction Analysis for TLR-4 Total RNA was extracted from cells using a Qiagen kit (Valencia, Calif) following the manufacture’s instructions, and treated with RNase-free DNase. For the reverse transcriptase (RT) reaction, the MMLV preamplification system (Life Technologies, Inc. Rockville, Md) was applied, as previously reported.8 Polymerase chain reaction (PCR) amplification was done with Taq gold polymerase © 2003 by Elsevier Science Inc. 360 Park Avenue South, New York, NY 10010-1710 Transplantation Proceedings, 35, 431– 432 (2003)
Fig 1. Diagrams representing the role of TLR-4. Recognition of LPS through TLR-4 receptor results in NF-B activation and subsequent production of various types of chomokines and cytokines. (Perkin Elmer, Foster City, Calif) for 32 cycles at 95 °C for 45 seconds 54 °C for 45 seconds, and 72 °C for 1 minute. The oligonucleotide primers for RT-PCR were: TLR-4, 5⬘-TGAGCAGTCGTGCTGGTATC and 5⬘-CAGGGCTTTTCTGAGTCGTC;9 for glyceraldehydes-3-phophate dehydrogenase (GAPDH), 5⬘-CGGAGTCAACGGATTTGGTCGTAT and 5⬘AGCCTTCTCCATGGTGGTGAAGAC. All PCR products were resolved on 1% agarose gel electrophoresis with DNA bands visualized by ethidium bromide staining.
RESULTS Expression Pattern of TLR-4 mRNA in Immortalized Liver Cells
RT-PCR analysis revealed that TLR-4 expression was detected in HNNT-2 cells (SV40T-transduced HLSECs) From the Division of Gastroenterology I, Department of Medicine, (N.K., M.T., T.K., N.S., K.O., S.Y.) Kawasaki Medical School, Kurashiki, Okayama; the Department of Surgery (N.K., T.O., T.M., T.T., M.M., Y.M., N.T.), Okayama University Graduate School of Medicine and Dentistry, Shikata-cho, Okayama, and The Japan Health Sciences Foundation Japan. Address reprint requests to Dr Naoya Kobayashi, Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. E-mail: [email protected] 0041-1345/03/$–see front matter doi:10.1016/S0041-1345(02)03800-9 431
432
Fig 2. Comparative expression of TIL-4 in human liver cell lines. The specific band for TLR-4 (167 bp) is detected in SV40-T transduced HLSEC line HNNT-2 and hTERT-immortalized hepatic fat-storing cell line TWNT-1. No corresponding band is detected in SV40-T immortalized human hepatocyte line NKNT-3. Detection of GAPDH mRNA showed equal loading of RNA (1, NKNT-3; 2, TWNT-1; 3, HNNT-2).
and TWNT-1 cells (hTERT-immortalized hepatic stallete cells). A more intense band for TLR-4 was observed in HNNT-2 cells compared to TWNT-1 cells (Fig 2). In contrast, SV40T-immortalized human hepatocyte NKNT-3 showed no TLR-4 expression. Equal loading of RNA was verified by detecting GAPDH mRNA.
KOBAYASHI, TAKESUE, KOBAYASHI ET AL
BAL to treat such patients. Our current finding that the transduced HLSEC line HNNT-2 showed more intense TLR-4 expression compared to immortalized hepatocytes and fat-storing cells is consistent with the fact that vascular endothelial cells, like monocytes and macrophages, are a critical target for LPS and many cytokines. Regulation of TLR-4 expression and production of soluble cytokines and chemokines in immortalized liver cells exposed to various concentrations of LPS must be analyzed before their use in BAL. In conclusion, the present study demonstrates that innate TLR-4 gene expression is well preserved in transduced HLSECs and fat-storing cells, suggesting that such cells are capable of directly sensing the presence of LPS thus performing an important role in hepatic sinusoidal linings as an integral component of a functional BAL. ACKNOWLEDGMENT We thank Dr T. Matsuura (Jikei University School of Medicine, Tokyo, Japan) and Dr K. Murakami (Tohoku Nenkin Hospital, Sendai, Japan) for providing LI90 cells. This work was supported in part by grants from the Ministry of Education, Science, and Culture, Japan; by grants from the Ministry of Economy and Industry, Japan.
DISCUSSION
The liver sinusoid is an important interface between the host and the external environment. Endothelial cells that line the hepatic sinusoid have a primary role in the immune and inflammatory systems. Vertebrates initiate a series of defense mechanisms, including a network of host-derived inflammatory mediators, by sensing the presence of pathogen-associated molecular patterns of microbial antigens, such as Gram-negative bacterial LPS and Gram-positive bacterial staphylococcal enterotoxin B.10,11 LPS is a potent activator of cells in the immune and inflammatory systems as well as endothelial cells, contributing to the systemic changes observed in septic shock. Host organisms have developed receptors that recognize specific pathogen-related molecular patterns.12 The human TLRs are a recently discovered family of receptors involved in innate immune recognition and cellular activation in response to bacterial antigens.13–15 To date, eight mammalian TLR family members have been documented, but only the ligands for TLR-2 and TLR-4 have been identified.6 TLR superfamily members are found in many species; those with known functions are involved in host responses to injury and infection. Patients with disturbed liver function often demonstrate serious infections, such as septic shock that eventually result in multi-organ failure.16 Thus, expression of TLRs must be documented on human liver cell lines that will be used in
REFERENCES 1. Watanabe FD, Mullon CJ, Hewitt WR, et al: Ann Surg 225:484, 1997 2. Begue JM, Guguen-Guillouzo C, Pasdeloup N, et al: Hepatology 4:839, 1984 3. Kobayashi N, Fujiwara T, Westerman KA, et al: Science 287:1258, 2000 4. Noguchi H, Kobayashi N, Westerman KA, et al: Hum Gene Ther 13:321, 2002 5. Watanabe T, Kobayashi N, Hoguchi H, et al: ASAIO J 47:166, 2001 6. Faure E, Equlis O, Sieling PA, et al: J Biol Chem 275:11058, 2000 7. Murakami K, Abe T, Miyazazwa M, et al: Lab Invest 72:731, 1995 8. Yoshioka T, Morimoto Y, Iwagaki H, et al: J Int Med Res 29:409, 2001 9. Tapping RI, Akashi S, Miyake K, et al: J Immunol 165:5780, 2000 10. Huemann D, Glauser MP: Calandra: Curr Opin Microbiol 1:49, 1998 11. Roberts AI, Blumberg RS, Christ AD, et al: Immunology 101:185, 2000 12. Medzhitov R, Janeway CA Jr: Curr Opin Immunol 9:4, 1997 13. Medzhitov R, Preston-Hurlburt P, Janeway CA: Nature 388:394, 1997 14. Wright SD: Exp Med 189:605, 1999 15. Chow JC, Young DW, Golenbock DT, et al: J Biol Chem 274:10689, 1999 16. Williams R: Semin Liver Dis 16:343, 1996