* Laboratory for Cell Biology and Histology, Vrije Universiteit Brussel (VUB), Brussels, Belgium;
Evanston Northwestern Healthcare Research Institute, Illinois;
Department of Immunohematology and Bloodtransfusion, Leiden University Medical Center, The Netherlands; and
Institute for Biochemistry, BIL Biomedical Research Center, University of Lausanne, Epilanges, Switzerland
Correspondence: David Vermijlen, Laboratory for Cell Biology and Histology, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels, Belgium. E-mail: dvermijl{at}cyto.vub.ac.be
Hepatic natural killer (NK) cells are located in the liver sinusoids adherent to the endothelium. Human and rat hepatic NK cells induce cytolysis in tumor cells that are resistant to splenic or blood NK cells. To investigate the mechanism of cell death, we examined the capacity of isolated, pure (90%) rat hepatic NK cells to kill the splenic/blood NK-resistant mastocytoma cell line P815. Cell death was observed and quantified by fluorescence and transmission electron microscopy, DNA fragmentation, and 51Cr release. RNA and protein expression were determined by real time reverse transcription-polymerase chain reaction and Western blotting. Compared with splenic NK cells, hepatic NK cells expressed higher levels of perforin and granzyme B and readily induced apoptosis in P815 cells. Although P815 cells succumbed to recombinant Fas ligand (FasL) or isolated perforin/granzyme B, hepatic NK cells used only the granule pathway to kill this target. In addition, hepatic NK cells and sinusoidal endothelial cells strongly expressed the granzyme B inhibitor, protease inhibitor 9 (PI-9)/serine PI-6 (SPI-6), and P815 cells and hepatocytes were negative. Transfection of target cells with this inhibitor resulted in complete resistance to hepatic NK cell-induced apoptosis. In conclusion, hepatic NK cells kill splenic/blood NK-resistant/FasL-sensitive tumor cells exclusively by the perforin/granzyme pathway. Serine protease inhibitor PI-9/SPI-6 expression in liver sinusoidal endothelial cells may protect the liver microenvironment from this highly active perforin/granzyme pathway used to kill metastasizing cancer cells.
Key Words: Fas ligand • serine protease inhibitor PI-9/SPI-6 • liver sinusoidal endothelial cell • apoptosis • P815 • hepatocyte
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