Pit cells exclusively kill P815 tumor cells by the perforin/granzyme pathway
© Vermijlen et al; licensee BioMed Central Ltd 2004
Published: 14 January 2004
Hepatic natural killer (NK) cells, also known as pit cells, are located in the liver sinusoids, adhering to the endothelial cells (LSECs), and are thus in a strategic position to kill arriving metastasizing tumor cells [1–3]. NK cells of different tissue origin (blood, spleen, liver) appear to have different levels of cytotoxicity. Lower levels can be enhanced by lymphokines such as interleukin-2 (IL-2) or IL-12, providing lymphokine-activated killer (LAK) cells . P815 mastocytoma cells were found to be resistant to the induction of cytolysis (quantified by 51Cr release) by NK cells from spleen or blood, but are sensitive to hepatic NK and LAK cells [[1, 3] and references therein]. Hepatic NK cells therefore might be considered as naturally activated LAK cells.
Cytotoxic lymphocytes (NK cells, LAK cells, cytotoxic T cells, NK-T cells) use the FasL and the perforin/granzyme pathway to kill target cells . FasL on effector cells binds Fas present on the target cell membrane, which results in oligomerization of Fas and activation of caspase 8. Perforin and granzymes, of which granzyme B is the most potent, reside in granules of the cytotoxic lymphocytes and are released by exocytosis. Intracellular delivery of granzyme B results in the initiation of the caspase cascade by proteolytic activation of caspase 3, either directly  or through a mitochondrium-dependent pathway . Caspases play a central role in the execution of apoptosis . In this study, we investigated the mechanism hepatic NK cells use to kill P815 cells.
P815, a mouse mastocytoma cell line, was maintained in culture medium consisting of DMEM (42430, GIBCO, Life Technologies, Belgium) supplemented with 10 % fetal bovine serum (Eurobiochem, Bierges, Belgium), sodium pyruvate (1 mmol/L), penicillin (100 U/ml), streptomycin (100 U/ml), and L-glutamine (0.2 mmol/L) (GIBCO, Life Technologies).
Transmission electron microscopy (TEM) was performed as described .
Quantitative DNA fragmentation assay was performed as described at an E/T ratio of 10/1 and 3 h co-incubation .
51Cr release assay
Cytolysis was measured in a 4 h 51Cr release assay as described previously . DCI (3,4-dichloroisocoumarin) and EGTA were purchased from ICN (Asse-Relegem, Belgium) and Z-VAD-FMK (Z-Val-Ala-Asp(OMe)-fluoromethylketone) from Bachem (Bubendorf, Switzerland).
Results and Discussion
- Wisse E, Luo D, Vermijlen D, Kanellopoulou C, De Zanger R, Braet F: On the function of pit cells, the liver-specific natural killer cells. Semin Liver Dis. 1997, 17: 265-86.View ArticlePubMedGoogle Scholar
- Wiltrout RH: Regulation and antimetastatic functions of liver-associated natural killer cells. Immunol Rev. 2000, 174: 63-76. 10.1034/j.1600-0528.2002.00014h.x.View ArticlePubMedGoogle Scholar
- Braet F, Luo D, Spector I, Vermijlen D, Wisse E: Endothelial and pit cells. In: The liver: biology and pathobiology. Edited by: Arias A, Boyer JL, Chisari FV, Fausto N, Schachter DA, Shafritz DA. 2001, Philadelphia, Lippincott Williams & Wilkins, 437-453.Google Scholar
- Yang X, Stennicke HR, Wang B, Green DR, Janicke RU, Srinivasan A, Seth P, Salvesen GS, Froelich CJ: Granzyme B mimics apical caspases. Description of a unified pathway for trans-activation of executioner caspase-3 and -7. J Biol Chem. 1998, 273: 34278-34283. 10.1074/jbc.273.51.34278.View ArticlePubMedGoogle Scholar
- MacDonald G, Shi L, Vande VC, Lieberman J, Greenberg AH: Mitochondria-dependent and -independent regulation of Granzyme B-induced apoptosis. J Exp Med. 1999, 189: 131-144. 10.1084/jem.189.1.131.PubMed CentralView ArticlePubMedGoogle Scholar
- Bouwens L, Remels L, Baekeland M, Van Bossuyt H, Wisse E: Large granular lymphocytes or "pit cells" from rat liver: isolation, ultrastructural characterization and natural killer activity. Eur J Immunol. 1987, 17: 37-42. 10.1002/eji.1830170107.View ArticlePubMedGoogle Scholar
- Kanellopoulou C, Seynaeve C, Crabb– E, Baekeland M, Vermijlen D, Vermoesen A, Braet F, De Zanger R, Wisse E: Isolation of pure pit cells with a magnetic cell sorter and effect of contaminating T cells on their cytolytic capability against CC531. In: Cells of the Hepatic Sinusoid 6. Edited by: Wisse E, Knook DL, Balabaud C. 1997, Leiden, The Kupffer Cell Foundation, 471-473.Google Scholar
- Vermijlen D, Luo D, Froelich CJ, Medema JP, Kummer AJ, Willems E, Braet F, Wisse E: Hepatic natural killer cells exclusively kill splenic/blood natural killer-resistant tumor cells by the perforin/granzyme pathway. J Leukoc Biol. 2002, 72: 668-676.PubMedGoogle Scholar
- Luo D, Vermijlen D, Vanderkerken K, Kuppen PJ, Seynaeve C, Eddouks M, Baekeland M, Wisse E: Involvement of LFA-1 in hepatic NK cell (pit cell)-mediated cytolysis and apoptosis of colon carcinoma cells. J Hepatol. 1999, 31: 110-116. 10.1016/S0168-8278(99)80170-6.View ArticlePubMedGoogle Scholar
- Berke G: The CTL's kiss of death. Cell. 1995, 81: 9-12. 10.1016/0092-8674(95)90365-8.View ArticlePubMedGoogle Scholar
- Hudig D, Allison NJ, Pickett TM, Winkler U, Kam CM, Powers JC: The function of lymphocyte proteases. Inhibition and restoration of granule-mediated lysis with isocoumarin serine protease inhibitors. J Immunol. 1991, 147: 1360-1368.PubMedGoogle Scholar
- Odake S, Kam CM, Narasimhan L, Poe M, Blake JT, Krahenbuhl O, Tschopp J, Powers JC.: Human and murine cytotoxic T lymphocyte serine proteases : subsite mapping with peptide thioester substrates and inhibition of enzyme activity and cytolysis by isocoumarins. Biochemistry. 1991, 30: 2217-2227. 10.1021/bi00222a027.View ArticlePubMedGoogle Scholar
- Sarin A, Haddad EK, Henkart PA: Caspase dependence of target cell damage induced by cytotoxic lymphocytes. J Immunol. 1998, 161: 2810-2816.PubMedGoogle Scholar
- Trapani JA, Davis J, Sutton VR, Smyth MJ: Proapoptotic functions of cytotoxic lymphocyte granule constituents in vitro and in vivo. Curr Opin Immunol. 2000, 12: 323-329. 10.1016/S0952-7915(00)00094-7.View ArticlePubMedGoogle Scholar
- Bradley M, Zeytun A, Rafi-Janajreh A, Nagarkatti PS, Nagarkatti M: Role of spontaneous and interleukin-2-induced natural killer cell activity in the cytotoxicity and rejection of Fas+ and Fas-tumor cells. Blood. 1998, 92: 4248-4255.PubMedGoogle Scholar
- Hodgson PD, Grant MD, Michalak TI: Perforin and Fas/Fas ligand-mediated cytotoxicity in acute and chronic woodchuck viral hepatitis. Clin Exp Immunol. 1999, 118: 63-70. 10.1046/j.1365-2249.1999.01010.x.PubMed CentralView ArticlePubMedGoogle Scholar
- Schr–ter M, Lowin B, Borner C, Tschopp J: Regulation of Fas(Apo-1/CD95)- and perforin-mediated lytic pathways of primary cytotoxic T lymphocytes by the protooncogene bcl-2. Eur J Immunol. 1995, 25: 3509-3513. 10.1002/eji.1830251245.View ArticleGoogle Scholar
- Metkar SS, Wang B, Aguilar-Santelises M, Raja SM, Uhlin-Hansen L, Podack E, Trapani JA, Froelich CJ: Cytotoxic cell granule-mediated apoptosis: perforin delivers granzyme B-serglycin complexes into target cells without plasma membrane pore formation. Immunity. 2002, 16: 417-428. 10.1016/S1074-7613(02)00286-8.View ArticlePubMedGoogle Scholar
- –ynebr–ten I, Hansen B, Smedsr–d B, Uhlin-Hansen L: Serglycin secreted by leukocytes is efficiently eliminated from the circulation by sinusoidal scavenger endothelial cells in the liver. J Leukoc Biol. 2000, 67: 183-188.Google Scholar
- Motyka B, Korbutt G, Pinkoski MJ, Heibein JA, Caputo A, Hobman M, Barry M, Shostak I, Sawchuk , Holmes CF, Gauldie J, Bleackley RC: Mannose 6-phosphate/insulin-like growth factor II receptor is a death receptor for granzyme B during cytotoxic T cell-induced apoptosis. Cell. 2000, 103: 491-500. 10.1016/S0092-8674(00)00140-9.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.