ABSTRACT
Respiratory infections, including influenza in humans, are often accompanied by a hepatitis that is usually mild and self-limiting. The mechanism of this kind of liver damage is not well understood. In the present study, we show that influenza-associated hepatitis occurs due to the formation of inflammatory foci that include apoptotic hepatocytes, antigen-specific CD8(+) T cells, and Kupffer cells. Serum aminotransaminase levels were elevated, and both the histological and serum enzyme markers of hepatitis were increased in secondary influenza infection, consistent with a primary role for antigen-specific T cells in the pathogenesis. No virus could be detected in the liver, making this a pure example of "collateral damage" of the liver. Notably, removal of the Kupffer cells prevented the hepatitis. Such hepatic collateral damage may be a general consequence of expanding CD8(+) T-cell populations during many extrahepatic viral infections, yielding important implications for liver pathobiology.
Subject(s)
Hepatitis/etiology , Influenza, Human/complications , Kupffer Cells/immunology , Adolescent , Adult , Alanine Transaminase/blood , Animals , CD8-Positive T-Lymphocytes/immunology , Hepatitis/pathology , Hepatitis/virology , Hepatocytes/immunology , Hepatocytes/pathology , Hepatocytes/virology , Humans , Influenza A Virus, H1N1 Subtype , Influenza, Human/immunology , Killer Cells, Natural/immunology , Killer Cells, Natural/pathology , Kupffer Cells/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Middle AgedABSTRACT
BACKGROUND: Transgenic TCR mice are often used experimentally as a source of T cells of a defined specificity. One of the most widely used transgenic TCR models is the OT-1 transgenic mouse in which the CD8+ T cells express a TCR specific for the SIINFEKL peptide of ovalbumin presented on kb. Although OT-1 CD8+ can be used in a variety of different experimental settings, we principally employ adoptive transfer and peptide-driven expansion of OT-1 cells in order to explore the distribution and fate of these antigen-specific OT-1 T cells. We set out to develop a quantitative PCR assay for OT-1 cells in order to assess the distribution of OT-1 CD8+ T cells in tissues that are either intrinsically difficult to dissociate for flow cytometric analysis or rendered incompatible with flow cytometric analysis through freezing or fixation. RESULTS: We show excellent correlation between flow cytometric assessment of OT-1 cells and OT-1 signal by qPCR assays in cell dilutions as well as in in vivo adoptive transfer experiments. We also demonstrate that qPCR can be performed from archival formalin-fixed paraffin-embedded tissue sections. In addition, the non-quantitative PCR using the OT-1-specific primers without the real-time probe is a valuable tool for OT-1 genotyping, obviating the need for peripheral blood collection and subsequent flow cytometric analysis. CONCLUSION: An OT-1 specific qPCR assay has been developed to quantify adoptively transferred OT-1 cells. OT-1 qPCR to determine cell signal is a valuable adjunct to the standard flow cytometric analysis of OT-1 cell number, particularly in experimental settings where tissue disaggregation is not desirable or in tissues which are not readily disassociated.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Egg Proteins/immunology , H-2 Antigens/immunology , Ovalbumin/immunology , Polymerase Chain Reaction/methods , Receptors, Antigen, T-Cell, alpha-beta/genetics , Transgenes , Adoptive Transfer , Animals , Base Sequence , CD8-Positive T-Lymphocytes/transplantation , Flow Cytometry , Mice , Mice, Inbred C57BL , Mice, Transgenic , Molecular Sequence Data , Peptide Fragments , Receptors, Antigen, T-Cell, alpha-beta/immunology , Sensitivity and Specificity , T-Cell Antigen Receptor Specificity , VDJ ExonsABSTRACT
At the end of an immune response, activated lymphocyte populations contract, leaving only a small memory population. The deletion of CD8(+) T cells from the periphery is associated with an accumulation of CD8(+) T cells in the liver, resulting in both CD8(+) T cell apoptosis and liver damage. After adoptive transfer and in vivo activation of TCR transgenic CD8(+) T cells, an increased number of activated CD8(+) T cells was observed in the lymph nodes, spleen, and liver of mice treated with anti-TNF-alpha. However, caspase activity was decreased only in CD8(+) T cells in the liver, not in those in the lymphoid organs. These results indicate that TNF-alpha is responsible for inducing apoptosis in the liver and suggest that CD8(+) T cells escaping this mechanism of deletion can recirculate into the periphery.