Cytotoxic effect of an anti-liver monoclonal autoantibody obtained after neonatal thymectomy in mice.

A monoclonal autoantibody, LSA-1, against murine liver antigen was obtained by fusing spleen cells from a neonatally thymectomized BALB/c mouse with SP2/0 murine myeloma cells. The LSA-1 isotype was IgG2b and kappa. LSA-1 was specific to the liver, especially, to a liver-specific membrane lipoprotein (LSP) fraction. By Western blotting analysis, LSA-1 mainly detected a 100 kDa protein of LSP fraction. LSA-1 stained cytoplasm of the cryostat sections of liver in immunohistochemical analysis. Furthermore, the antigen recognized with LSA-1 was highly expressed on the surface of a murine hepatoma cell line, MH134, slightly on a murine normal liver cell line, C1469, and on freshly prepared hepatocytes, but not on spleen cells. LSA-1 had a cytotoxic activity on liver cell lines in the presence of a complement in vitro. Furthermore, injection of LSA-1 into mice-induced liver injury. These results suggest that anti-liver autoantibody plays an important role in the induction of autoimmune hepatitis. Accordingly, this antibody will be a useful tool for the analysis of the pathogenesis of autoimmune hepatitis.

Dysfunction of immune system and induction of autoantibodies to liver antigens by neonatal thymectomy in mice.

We examined the development of autoantibodies to liver proteins and hepatitis in BALB/c mice thymectomized 2 days after birth and attempted to characterized the immune function of these mice. Autoantibodies to crude liver proteins detected by ELISA were found in 21 (84%) of 25 mice thymectomized 2 days after birth. In these mice, sera of 11 animals showed reactivity with both liver specific proteins (LSP) and the second fraction of crude liver proteins; sera of 3 mice showed reactivity with only the second fraction but sera showed reactivity with only LSP. By Western-immunoblotting, sera of BALB/c mice which showed high autoantibody level to liver proteins detected a strong band around 150kD in the second fraction of crude liver proteins. Still more, hepatic inflammation; mononuclear cell infiltration in the portal area, was induced in mice with apparently high autoantibody level to crude liver proteins. These results in BALB/c mice corresponded with our previous reports which employed C3H/HeN mice. Next, we examined immune functions of mice thymectomized 2 days after birth. In thymectomized mice, the proportion of Thy-1, L3T4 and Lyt-2 positive cells (T cells) decreased and the proportion of B220 positive cells (B cells) increased. The proliferative response of lymph nodes lymphocytes cultured with mitomycin C-treated syngeneic spleen cells was lower, and the total IgG level in the sera was higher when compared with control normal mice. Anti-nuclear antibody (ANA) also appeared in the sera of thymectomized mice 2 days after birth. All these results suggest that the dysfunction of T cell and polyclonal activation of B cell were induced in neonatally thymectomized mice and resulted in the production of ANA and autoantibodies to liver proteins.

Induction of autoimmune hepatitis and autoantibodies to liver antigens by neonatal thymectomy in mice.

We examined development of autoimmune hepatitis in neonatally thymectomized C3H/HeN mice and tried to characterize the nature of liver antigens recognized by the autoantibodies at the molecular level. Autoantibodies to crude liver proteins detected by ELISA were found in 12(67%) of 18 mice thymectomized 2 days after birth. However, autoantibodies were not detected in mice thymectomized 7 days after birth. The autoantibodies mainly consisted of IgG and reached the maximum level 8 weeks after birth. Hepatic inflammation, mononuclear cell infiltration in the portal area, was seen in 5 (28%) of 18 mice thymectomized 2 days after birth, but not in mice thymectomized 7 days after birth. Most infiltrating cells were Thy-1+ lymphocytes. The serum autoantibody level to crude liver proteins in mice with hepatitis was much higher than that in mice without hepatitis. We fractionated crude liver proteins by a Sepharose 6B column and examined the reactivity against the autoantibodies. The autoantibodies of three of five mice with hepatitis reacted with th approximately 150kD liver proteins other than liver-specific protein (LSP). By Western immunoblotting of SCS-PAGE using LSP and fractionated liver proteins, we found that the molecular weights of the target antigens were 52kD in LSP and 150kD (strong band), 138, 128, 120 and 110kD (weak band) in fractionated liver proteins other than LSP. This 150-kD target molecule in crude liver proteins was found only in liver. These results indicate that hepatitis and autoantibodies to liver proteins are induced spontaneously by neonatal thymectomy in mice, and the candidates of autoantigen in this hepatitis model are 52-kD protein in LSP and 150-kD liver proteins different from LSP. Still more, we regard the 150-kD molecule as a new autoantigen related to hepatitis.

B cell mitogenic activity of Toxocara canis adult worm antigen.

The effect of Toxocara (T.) canis antigen (TcAg) on lymphocytes was studied in vitro using normal murine spleen cells and human peripheral blood lymphocytes. TcAg prepared from adult worms stimulated murine spleen cells to proliferate at concentrations of 1-125 micrograms/ml. The responder cells TcAg are B cells, because the response was depleted by the treatment of spleen cells with anti-immunoglobulin (Ig) antibody and complement and after separation on a nylon wool column. This response was not due to the contamination of lipopolysaccharide (LPS), because TcAg could stimulate C3H/HeJ spleen cells which are low responders to LPS. Not only the proliferative response but also polyclonal IgG and IgE production were stimulated with TcAg. TcAg also stimulated macrophages to produce interleukin-1 and could stimulate human B cells. These results suggest that TcAg is a potent B cell mitogen and this activity may be relevant to the alteration of immunological functions in hosts infected with T. canis.