The role of serpinb9/serine protease inhibitor 6 in preventing granzyme B-dependent hepatotoxicity.

Natural killer (NK) and natural killer T (NKT) cells are prominent components of the resident liver lymphocyte population.1 In addition, increased numbers of activated NK cells and cytotoxic T lymphocytes (CTLs) are present in the liver during systemic2 and localized immune responses.3 These cytotoxic effector cells play an essential role in the clearance of noncytopathic viruses from infected tissues.4,5 In addition, NK and NKT cells amplify hepatocellular injury initiated by noninfectious causes of liver injury.6 Mice with defects in perforin/granzyme effector mechanisms have been found to have delayed clearance of a number of viral infections from extrahepatic sites but exhibit no delay in the clearance of recombinant adenoviral vectors from the liver.7 In contrast, mice with defects in the Fas ligand (FasL)/Fas or other death receptor pathways exhibit delayed clearance of adenoviral vectors from the liver but not from the lungs.8 This appears to be related to the resistance of virally infected hepatocytes to killing by perforin-dependent and granzyme-dependent cytotoxic effector pathways, which results in a more prominent role for FasL and tumor necrosis factor (TNF) receptor—mediated killing mechanisms in the clearance of viral infection in the liver.7,9,10 Human proteinase inhibitor 9 (PI-9) and the murine orthologue, serine protease inhibitor 6 (SPI-6), are members of a family of ovalbumin serpin inhibitors11-13 present in immune-privileged cells, antigen-presenting cells, and cytotoxic T cells that afford protection against the actions of granzyme B.14-20 The absence of SPI-6 during viral infection results in a breakdown in the cytotoxic granule integrity and increased levels of cytoplasmic granzyme B, which result in higher levels of T cell apoptosis.21 When expressed by tumor cell lines, PI-912,17,22,23 and SPI-612,22 confer resistance to perforin/granzyme B—mediated killing by CTL and NK cells. PI-9 and SPI-6 selectively inhibit granzyme B but do not affect Fas-mediated and TNF-mediated apoptosis.24 Thus, one potential explanation for hepatocyte resistance to the perforin and granzyme cytotoxicity pathway is the expression of PI-9/SPI-6 in the liver. Initial evaluations of human or mouse tissues for PI-9 or SPI-6 expression revealed high levels of expression in the spleen, the lungs, and a variety of immune-privileged sites, but they did not reveal high levels of PI-9 or SPI-6 expression in normal liver.11,12 Subsequent studies conducted in our laboratory, however, noted the up-regulation of PI-9 and SPI-6 expression in human and mouse hepatocytes during viral infection and in mouse liver after an in vitro treatment with interferon alpha (IFN-α).25 Therefore, the induction of PI-9/SPI-6 during viral hepatitis may serve as a mechanism to protect infected hepatocytes from excessively vigorous perforin/granzyme B—mediated killing by NK cells and CTLs.25,26 To evaluate this hypothesis, the current studies were performed to assess the effects of inhibiting SPI-6 expression in hepatocytes in the presence or absence of viral infection of the liver.