Cd18/ICAM-1-dependent oxidafive NF-κB activation leading to nitric oxide production in rat Kupffer cells cocultured with syngeneic hepatoma cells

Iwao Kurose, Hidetsugu Saito, Soichiro Miura, Hirotoshi Ebinuma, Hajime Higuchi, Naoyuki Watanabe, Shigeyuki Zeki, Tetsuya Nakamura, Masaaki Takaishi, Hiromasa Ishii

研究成果: Article

42 引用 (Scopus)

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Previous studies have indicated that nitric oxide (NO) released from Kupffer cells modulates biological viability of cocultured hepatoma cells. This study was designed to evaluate the mechanisms by which Kupffer cells synthesize and release NO in response to cocultured hepatoma cells. Kupffer cells isolated from male Wistar rats were cocultured with rat hepatoma cell line, AH70 cells. The sum of nitrite and nitrate levels increased in the culture medium of Kupffer cells with AH70 cells as compared with those of Kupffer cells or AH70 cells alone. Increased expressions of iNOS and iNOS mRNA in Kupffer cells cocultured with AH70 cells were detected by an immunofluorescence staining and a fluorescence in situ hybridization study, respectively. A fluorescence in situ DNA-protein binding assay revealed that NF-κB activation occurs in Kupffer cells and activated NF-κB moved into the nuclei preceding to an increased production of NO. Oxidative stress indicated by dichlorofluorescein fluorescence was observed in Kupffer cells cocultured with AH70 cells. An increased calcium mobilization indicated as increased fluo-3-associated fluorescence was also induced in Kupffer cells after coculture with AH70 cells. Monoclonal antibodies directed against rat CD18 and ICAM-1, as well as TMB-8, a calcium inhibitor, prevented the calcium mobilization, active oxygen production, and NF-κB activation in addition to the increased production of NO. Pyrrolidine dithiocarbamate, an inhibitor of oxidative NF-κB activation, diphenylene iodonium, an NADPH oxidase inhibitor, and quinacrine, a phospholipase A2 inhibitor, significantly attenuated the increase in dichlorofluorescein fluorescence, NF-κB activation, and NO production. Therefore, this study suggests that CD18/ICAM- l-dependent cell-to-cell interaction with hepatoma cells causes calcium mobilization and oxidative activation of NF-κB, which may lead to the increased production of NO in Kupffer cells.

元の言語English
ページ(範囲)867-878
ページ数12
ジャーナルJournal of Clinical Investigation
99
発行部数5
出版物ステータスPublished - 1997 3 1

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Kupffer Cells
Intercellular Adhesion Molecule-1
Hepatocellular Carcinoma
Nitric Oxide
Fluorescence
Calcium
Quinacrine
NADPH Oxidase
DNA-Binding Proteins
Nitrites
Coculture Techniques
Fluorescence In Situ Hybridization
Cell Communication
Nitrates
Fluorescent Antibody Technique
Culture Media
Wistar Rats
Reactive Oxygen Species
Oxidative Stress
Monoclonal Antibodies

ASJC Scopus subject areas

  • Medicine(all)

これを引用

Cd18/ICAM-1-dependent oxidafive NF-κB activation leading to nitric oxide production in rat Kupffer cells cocultured with syngeneic hepatoma cells. / Kurose, Iwao; Saito, Hidetsugu; Miura, Soichiro; Ebinuma, Hirotoshi; Higuchi, Hajime; Watanabe, Naoyuki; Zeki, Shigeyuki; Nakamura, Tetsuya; Takaishi, Masaaki; Ishii, Hiromasa.

:: Journal of Clinical Investigation, 巻 99, 番号 5, 01.03.1997, p. 867-878.

研究成果: Article

Kurose, I, Saito, H, Miura, S, Ebinuma, H, Higuchi, H, Watanabe, N, Zeki, S, Nakamura, T, Takaishi, M & Ishii, H 1997, 'Cd18/ICAM-1-dependent oxidafive NF-κB activation leading to nitric oxide production in rat Kupffer cells cocultured with syngeneic hepatoma cells', Journal of Clinical Investigation, 巻. 99, 番号 5, pp. 867-878.
Kurose, Iwao ; Saito, Hidetsugu ; Miura, Soichiro ; Ebinuma, Hirotoshi ; Higuchi, Hajime ; Watanabe, Naoyuki ; Zeki, Shigeyuki ; Nakamura, Tetsuya ; Takaishi, Masaaki ; Ishii, Hiromasa. / Cd18/ICAM-1-dependent oxidafive NF-κB activation leading to nitric oxide production in rat Kupffer cells cocultured with syngeneic hepatoma cells. :: Journal of Clinical Investigation. 1997 ; 巻 99, 番号 5. pp. 867-878.
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abstract = "Previous studies have indicated that nitric oxide (NO) released from Kupffer cells modulates biological viability of cocultured hepatoma cells. This study was designed to evaluate the mechanisms by which Kupffer cells synthesize and release NO in response to cocultured hepatoma cells. Kupffer cells isolated from male Wistar rats were cocultured with rat hepatoma cell line, AH70 cells. The sum of nitrite and nitrate levels increased in the culture medium of Kupffer cells with AH70 cells as compared with those of Kupffer cells or AH70 cells alone. Increased expressions of iNOS and iNOS mRNA in Kupffer cells cocultured with AH70 cells were detected by an immunofluorescence staining and a fluorescence in situ hybridization study, respectively. A fluorescence in situ DNA-protein binding assay revealed that NF-κB activation occurs in Kupffer cells and activated NF-κB moved into the nuclei preceding to an increased production of NO. Oxidative stress indicated by dichlorofluorescein fluorescence was observed in Kupffer cells cocultured with AH70 cells. An increased calcium mobilization indicated as increased fluo-3-associated fluorescence was also induced in Kupffer cells after coculture with AH70 cells. Monoclonal antibodies directed against rat CD18 and ICAM-1, as well as TMB-8, a calcium inhibitor, prevented the calcium mobilization, active oxygen production, and NF-κB activation in addition to the increased production of NO. Pyrrolidine dithiocarbamate, an inhibitor of oxidative NF-κB activation, diphenylene iodonium, an NADPH oxidase inhibitor, and quinacrine, a phospholipase A2 inhibitor, significantly attenuated the increase in dichlorofluorescein fluorescence, NF-κB activation, and NO production. Therefore, this study suggests that CD18/ICAM- l-dependent cell-to-cell interaction with hepatoma cells causes calcium mobilization and oxidative activation of NF-κB, which may lead to the increased production of NO in Kupffer cells.",
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AU - Kurose, Iwao

AU - Saito, Hidetsugu

AU - Miura, Soichiro

AU - Ebinuma, Hirotoshi

AU - Higuchi, Hajime

AU - Watanabe, Naoyuki

AU - Zeki, Shigeyuki

AU - Nakamura, Tetsuya

AU - Takaishi, Masaaki

AU - Ishii, Hiromasa

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N2 - Previous studies have indicated that nitric oxide (NO) released from Kupffer cells modulates biological viability of cocultured hepatoma cells. This study was designed to evaluate the mechanisms by which Kupffer cells synthesize and release NO in response to cocultured hepatoma cells. Kupffer cells isolated from male Wistar rats were cocultured with rat hepatoma cell line, AH70 cells. The sum of nitrite and nitrate levels increased in the culture medium of Kupffer cells with AH70 cells as compared with those of Kupffer cells or AH70 cells alone. Increased expressions of iNOS and iNOS mRNA in Kupffer cells cocultured with AH70 cells were detected by an immunofluorescence staining and a fluorescence in situ hybridization study, respectively. A fluorescence in situ DNA-protein binding assay revealed that NF-κB activation occurs in Kupffer cells and activated NF-κB moved into the nuclei preceding to an increased production of NO. Oxidative stress indicated by dichlorofluorescein fluorescence was observed in Kupffer cells cocultured with AH70 cells. An increased calcium mobilization indicated as increased fluo-3-associated fluorescence was also induced in Kupffer cells after coculture with AH70 cells. Monoclonal antibodies directed against rat CD18 and ICAM-1, as well as TMB-8, a calcium inhibitor, prevented the calcium mobilization, active oxygen production, and NF-κB activation in addition to the increased production of NO. Pyrrolidine dithiocarbamate, an inhibitor of oxidative NF-κB activation, diphenylene iodonium, an NADPH oxidase inhibitor, and quinacrine, a phospholipase A2 inhibitor, significantly attenuated the increase in dichlorofluorescein fluorescence, NF-κB activation, and NO production. Therefore, this study suggests that CD18/ICAM- l-dependent cell-to-cell interaction with hepatoma cells causes calcium mobilization and oxidative activation of NF-κB, which may lead to the increased production of NO in Kupffer cells.

AB - Previous studies have indicated that nitric oxide (NO) released from Kupffer cells modulates biological viability of cocultured hepatoma cells. This study was designed to evaluate the mechanisms by which Kupffer cells synthesize and release NO in response to cocultured hepatoma cells. Kupffer cells isolated from male Wistar rats were cocultured with rat hepatoma cell line, AH70 cells. The sum of nitrite and nitrate levels increased in the culture medium of Kupffer cells with AH70 cells as compared with those of Kupffer cells or AH70 cells alone. Increased expressions of iNOS and iNOS mRNA in Kupffer cells cocultured with AH70 cells were detected by an immunofluorescence staining and a fluorescence in situ hybridization study, respectively. A fluorescence in situ DNA-protein binding assay revealed that NF-κB activation occurs in Kupffer cells and activated NF-κB moved into the nuclei preceding to an increased production of NO. Oxidative stress indicated by dichlorofluorescein fluorescence was observed in Kupffer cells cocultured with AH70 cells. An increased calcium mobilization indicated as increased fluo-3-associated fluorescence was also induced in Kupffer cells after coculture with AH70 cells. Monoclonal antibodies directed against rat CD18 and ICAM-1, as well as TMB-8, a calcium inhibitor, prevented the calcium mobilization, active oxygen production, and NF-κB activation in addition to the increased production of NO. Pyrrolidine dithiocarbamate, an inhibitor of oxidative NF-κB activation, diphenylene iodonium, an NADPH oxidase inhibitor, and quinacrine, a phospholipase A2 inhibitor, significantly attenuated the increase in dichlorofluorescein fluorescence, NF-κB activation, and NO production. Therefore, this study suggests that CD18/ICAM- l-dependent cell-to-cell interaction with hepatoma cells causes calcium mobilization and oxidative activation of NF-κB, which may lead to the increased production of NO in Kupffer cells.

KW - dichlorofluorescein

KW - fluo-3

KW - fluorescence in situ DNA-protein binding assay

KW - laser scanning confocal microscopy

KW - mRNA fluorescence in situ hybridization

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