Analysis of Cytokine Profiling in Hepatitis-Associated Aplastic Anemia.

In hepatitis-associated aplastic anemia (HAA), bone marrow failure follows an acute attack of seronegative hepatitis. Aplastic anemia (AA) is also associated with orthotopic liver transplantation for non-A, non-B, non-C hepatitis in young patients. Although the etiology of HAA is unknown, a clinical response to immunosuppressive therapy is observed in most clinical cases. The liver is the potential initial target organ of the immune response. We have previously determined the T-cell repertoire in the liver and peripheral blood lymphocytes in patients with HAA (Lu et al. Blood. 2004 Jun 15;103(12):4588–93). An antigen-driven T-cell expansion was implicated in the acute stage before bone marrow failure. In the present study, we measured serum cytokine levels in 22 HAA serum samples. Only interleukin (IL)-10 levels showed a significant increase compared to healthy controls (p<0.02); IFN-γ , TNF-α and IL-1, 2, 4, 5, 6, 8, 12 levels showed no significant difference. To further characterize the immune response in the liver, we performed RNAse protection assays and realtime PCR for RNA transcripts. In 4 HAA liver samples, 2 demonstrated increased IFN-γ by RNAse protection; TNF-α , IL-2, 3, 4, 5, 10, 15 transcripts were not different compared to transcript levels in 4 hepatitis B and/or C livers and 2 fulminant hepatitis (FH) samples. To confirm our preliminary data, we characterized the cytokine profile including CD4, CD8, CD69, TNF-α , IFN-γ and interleukins and compared them among the hepatitis B/C, FH and HAA liver samples by quantitative PCR for RNA transcripts. In 10 HAA liver samples, CD8 transcripts were increased and the CD4/CD8 ratio was decreased, but there was no significant difference between HAA and hepatitis B/C (p>0.05), confirming the important role of cellular immunity in HAA. IFN-γ transcripts were significantly overexpressed in both HAA and FH samples compared to hepatitis B/C samples (p<0.05), but without a difference between HAA and FH. CD69 transcripts were reduced in HAA but not significantly lower than in hepatitis B/C (p<0.05). There were no difference of TNF-α transcripts among HAA, hepatitis and FH samples (p>0.05). In summary, our data confirm that enhanced cellular immunity (increased IFN-γ level) is implicated in the pathogenesis of HAA, similar to that observed in the bone marrow of idiopathic AA, and in contrast to other liver diseases such as chronic hepatitis B/C. Quantitation of IFN-γ levels in acute hepatitis samples might be a predictive marker for the development of bone marrow failure after liver transplantation following seronegative hepatitis and FH.