Establishment of murine model for auto immune hepatitis (AIH)

• Project/Area Number: 14570523
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Gastroenterology
• Research Institution: Aichi Medical University
• Principal Investigator: OKUMURA Akihiko Aichi Medical University, School of Medicine, Assistant Professor, 医学部, 講師 (70288512)
• Co-Investigator(Kenkyū-buntansha): ISHIKAWA Tetsuya Aichi Medical University, School of Medicine, Assistant Professor, 医学部, 講師 (10288508) ; KAKUMU Shinichi Aichi Medical University, School of Medicine, Professor, 医学部, 教授 (10115545)
• Project Period (FY): 2002 – 2003
• Project Status: Completed(Fiscal Year 2003)
• Budget Amount: ¥3,500,000 (Direct Cost : ¥3,500,000); Fiscal Year 2003 : ¥1,000,000 (Direct Cost : ¥1,000,000); Fiscal Year 2002 : ¥2,500,000 (Direct Cost : ¥2,500,000)
• Keywords: murine hepatitis model / CYP2D6 / CD4^+-T cell / CD8^+-T cell / CD4^+ CD25^+-T cell (T-reg) / liver infiltrating lymphocytes / 肝内浸潤リンパ球(LIL) / CYP2D6 / ベクター / 融合蛋白 / CD4+T細胞 / CD8+T細胞 / 特異的免疫応答 / 肝障害 / 抗体産生

Research Abstract

Background : We established murine hepatitis model by immunizing mice with human P450IID6 (CYP2D6), the target molecule of LKM-1 antibody. In the present study, we investigated the mechanism of liver cell injury in AIH, including the dynamics of T-reg, using our murine hepatitis model. Materials and methods : Eight week old C57BL/6 male mice were immunized with β-gal-CYP2D6 (0.1μmol), the fusion protein of β-gal and CYP2D6, intraperitoneally. Serum levels of ALT and anti-CYP2D6, and histologic changes in the liver were tested weekly until 4 week after immunization. To determine the proportion of CD4^+-T cell, CD8^+-T cell, and T-reg, liver infiltrating lymphocytes (LIL) and splenocytes (SP) were collected, stained with anti-CD4-PE in combination with anti-CD8-FITC or anti-CD25-FITC, and run on FACS flow cytometer, collecting data on 1x10^4 cells, analyzed using Cell Quest software. Results : Serum ALT level reached the peak at 2 week after immunization and returned to normal level at 4 week in the mice immunized with β-gal-CYP2D6. Histologically, periportal infiltration of the inflammatory cells and focal necrosis in the lobule was observed in the livers of the β-gal-CYP2D6-immunized mice. When LIL were used for the FACS analysis, the ratio of CD4/CD8 decreased at 1 week after immunization, synchronized with the elevation of ALT levels, then recovered gradually in parallel with the resolution of ALT flare. When SP were used, however, the change of CD4/CD8 was marginal, indicating the different dynamics of CD4^+- and CD8^+-T cells in SP from that in LIL during the course of hepatitis. The frequency of CD4^+ CD25^+-T cell (T-reg) in SP were similar to LIL. The frequency of T-reg in LIL decreased immediately at 1 week after immunization, and gradually increased to the same level as pre-immunization. T-reg in SP decreased more slowly than that in LIL, reached minimum at 2 week after immunization, then increased similarly to T-reg in LIL. Conclusions : Immunization of C57BL/6 mice with β-gal-CYP2D6 resulted in inflammation in the liver. Our results using FACS analysis indicated the predominance of CD8^+T cells in the livers with hepatitis. Further, the frequency of T-reg changed drastically during the flare of ALT levels, implies the possibility that T-reg play some roles on the course of hepatitis.