Transgenic mice expressing osteopontin in hepatocytes as a model of autoimmune hepatitis

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Abstract

Osteopontin, a crucial factor for Th1 immune response, is expressed in stellate cells and macrophages activated in injured liver. To clarify the role of osteopontin in inflammatory changes in the liver, we attempted to establish transgenic mice expressing osteopontin in hepatocytes. Mouse osteopontin cDNA, cloned from concanavalin-A-stimulated spleen cells in C57BL/6 mice, was constructed into the vector containing serum amyloid-P component promoter. This construction was microinjected into fertilized eggs of C57BL/6 mice, and 4 lines of the transgenic mice were obtained. Western blotting and immunohistochemistry revealed that osteopontin was expressed in hepatocytes, but not in non-parenchymal cells, in the transgenic mice. The mean osteopontin concentrations in the liver and plasma in the mice were 13 and 2.6 times higher than those in negative littermates. Antinuclear antibody was positive in the plasma in 50% of the transgenic mice. In the transgenic mice later than 12 weeks of age, mononuclear cell infiltration in the liver developed, and these cells were positive for CD8 and HLA-DR. Plasma ALT activity was increased with focal necrosis in hepatic lobules in the transgenic mice later than 24 weeks of age. The transgenic mice expressing osteopontin in hepatocytes may be useful as a model of autoimmune hepatitis.

Section snippets

Materials and methods

Construction of osteopontin transgene. Mouse osteopontin cDNA (0.9 kb) was isolated by RT-polymerase chain reaction (PCR) using total RNA prepared from concanavalin-A-activated spleen cells of C57BL/6 mouse (Japan SLC, Hamamatsu, Japan). HindIII site in the open reading frame of mouse osteopontin cDNA was abolished by silent mutation using a standard PCR method. The mutated osteopontin cDNA was then cloned into the EcoRI site of pLG1-SAP vector [11] that contains human serum amyloid P component

Transgenic mouse lines expressing osteopontin

Among 52 mice brought up after the transplantation, 6 mice (3 males and 3 females) carried the transgene. These 6 mice were mated with C57BL/6 mice at 8 weeks of age, and the transgene was transmitted to the offspring of 4 lines; the SAP-osteopontin 17, 35, 43, and 73 lines. The transgenic mice were mated each other in the same lines, and the offspring carrying the transgene derived from the SAP-osteopontin 17, 43, and 73 lines were used in the experiments. The offspring from the

Discussion

In the present study, four lines of osteopontin transgenic mice were obtained. We used a pLG1-SAP vector [11] that contains the human SAP promoter consisting of a motif putatively recognized by hepatocyte nuclear factor (HNF)-1 [14] and the exon and intron of rabbit β-globin gene, because osteopontin may be expressed exclusively in hepatocytes in such transgenic mice. In fact, Western blot analysis using isolated liver cells and immunohistochemical examination revealed that osteopontin protein

Acknowledgements

We thank Ms. Kayoko Naiki (Department of Internal Medicine, Saitama Medical School) for her technical help.

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