Increased bcl-2 expression in lymphocytes and its association with hepatocellular damage in patients with autoimmune hepatitis

Abstract

The proto-oncogene product bcl-2 is known to inhibit apoptotic cell death, and its dysregulation might play a critical role in the development of autoimmune disease. To elucidate the role of bcl-2 in autoimmune hepatitis (AIH), its expression in peripheral blood mononuclear cells (PBMC) and in liver-infiltrating lymphocytes (LIL) was investigated. Increased bcl-2 expression in PBMC was found in AIH patients compared with that in chronic hepatitis C (CHC) patients and in healthy controls. The level of bcl-2 expression significantly correlated with serum ALT level. Further analysis showed that CD4+ T cells are enriched in bcl-2-expressing PBMC. To characterize the Th1/Th2 profile of bcl-2-expressing CD4+ T cells, intracellular interferon-gamma (IFN-gamma) and IL-4 were analysed. The results revealed that most of the bcl-2-expressing cells were found to be IFN-gamma-secreting Th1 cells. In three patients for whom their clinical courses could be followed, bcl-2 expression was decreased after the initiation of immunosuppressive therapy with corticosteroids. However, the level of IFN-gamma + cells was not altered. Immunohistochemical analysis also showed that large amounts of bcl-2+ cells were observed in periportal area in the liver. In conclusion, bcl-2-expressing cells were shown to be increased in peripheral blood and liver in AIH and the bcl-2 product was expressed mainly in CD4+ Th1-type cells, suggesting that these cells might promote the cellular immune response and contribute to the development of hepatitis and hepatocellular damage in AIH.

Fig 1

Mean fluorescence intensity (MFI) of bcl-2 expression in peripheral blood mononuclear cells (PBMC) from autoimmune hepatitis (AIH) patients, chronic hepatitis C (CHC) patients and healthy controls. After isolated PBMC were permeabilized to make anti-bcl-2 antibody access to nuclei, cells were reacted with anti-bcl-2 antibody and analysed by flow cytometry. Each error bar shows the mean MFI ± s.d. *Significant difference from control group (P < 0.001) and CHC group (P < 0.005) calculated by one-way anova.

Fig 2

Immunohistochemical analysis of bcl-2⁺ cells in the liver. Liver biopsy specimen taken from two autoimmune hepatitis (AIH) patients before steroid therapy were subjected to immunohistochemical staining with bcl-2 antigen as described in Patients and Methods. Bcl-2⁺ cells were widely observed in infiltrating lymphocytes located in the central part of the portal area of both patients.

Fig 3

Correlation between mean fluorescence intensity (MFI) of bcl-2 expression and serum ALT level. MFI and ALT level of each datum are plotted. Four autoimmune hepatitis (AIH) patients with high bcl-2 expression and high transaminase levels had not been given any treatment (•). The correlation coefficient is shown in the Figure.

Fig 4

Relation between bcl-2 expression and inflammatory activity in liver specimen in autoimmune hepatitis (AIH) and chronic hepatitis C (CHC) patients. The inflammatory activity was expressed using the grade of necroinflammatory activity in European Classification (A0∼A3). The group with severe inflammatory activity showed significantly higher bcl-2 expression than that with none or mild inflammation in AIH patients (○) (P < 0.01, (*)). All patients before immunosuppressive therapy (•) were grade A3 and showed high bcl-2 expression. There was no correlation in CHC patients (□).

Fig 5

The change of the level of bcl-2⁺ peripheral blood mononuclear cells (PBMC) before and after corticosteroid administration. Each bar shows the percentage of bcl-2⁺ cells. ALT levels at the time of measurement are shown besides each bar. Bcl-2 expression in case 1 was analysed before and 20 days after administration of prednisone (30 mg/day). Case 2 received steroid pulse therapy (1 g/day) for 3 days and prednisone (40 mg/day) was administrated thereafter. Bcl-2 expression was evaluated 14 days after steroid therapy had started. In case 3, bcl-2 expression was evaluated at 14 days after the increase of prednisone dose from 5 to 30 mg/day. Percentage of bcl-2⁺ cells (abscissa) remarkably decreased as the ALT level decreased after the administration or increase of corticosteroid in all patients.