- Open Access
Pit cells exclusively kill P815 tumor cells by the perforin/granzyme pathway
Comparative Hepatology volume 3, Article number: S58 (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
Hepatic NK cells induced nucleus condensation and fragmentation in P815 cells, as shown by fluorescent nuclear staining (data not shown) and TEM (Fig. 1). Chromatin was condensed into masses that abutted the inner surface of the nuclear envelope and was accompanied by nuclear fragmentation (Fig. 1). Using DNA fragmentation and 51Cr release we verified that hepatic and not splenic NK cells kill P815 cells .
We showed that P815 cells are sensitive to both the FasL and perforin/granzyme pathway and hepatic NK cells express FasL, perforin and granzyme B . Several approaches, distinguishing the FasL and perforin/granzyme pathway, were used to determine how hepatic NK cells induce apoptosis in P815 targets. Chelation of extracellular Ca2+ with EGTA (5 mmol/L), a treatment known to block granule exocytosis and the action of perforin , completely abolished DNA fragmentation and 51Cr release (Fig. 2). Preincubation of the effector cells with DCI (50 micromolar for 30 minutes), an inhibitor of granzymes in intact cells [11, 12], completely inhibited DNA fragmentation and substantially blocked 51Cr release (Fig. 2). Consistent with previous reports [13, 14], the general caspase inhibitor Z-VAD-FMK abrogated DNA fragmentation but 51Cr release was unaffected (Fig. 2). These results clearly demonstrate that P815 cells are exclusively killed by the granule pathway, whereas other cytotoxic lymphocytes can use both the FasL and perforin/granzyme pathway to kill this target [15–17].
We showed that hepatic NK cells and LSECs, which are in contact with the hepatic NK cells, are strongly positive for the granzyme B inhibitor, serine protease inhibitor PI-9/SPI-6, and that expression of this inhibitor in target cells results in complete resistance to hepatic NK cell-induced apoptosis . Based on these results, we put forward following model for hepatic NK cell-mediated killing (Figure 3): When a tumor cell enters a liver sinusoid, it is mechanically trapped and/or adheres to LSECs. Hepatic NK cells adhere to tumor cells by adhesion molecules like LFA-1. sFas produced by hepatocytes blocks FasL on the hepatic NK cells, preventing possible harmful effects on the FasL-sensitive LSECs and hepatocytes. On the other hand, highly expressed perforin and granzyme B, as a complex with serglycin as a scaffold , are released by granule exocytosis in the space formed between the NK-tumor conjugate. Damaging of other cells (e.g. hepatocytes) caused by leakage of granzyme B/perforin is prevented by the very efficient endocytic uptake of the granzyme B/serglycin/perforin complex by the HA-R expressed on LSECs . LSECs are protected from the action of granzyme B by strong expression of the granzyme B inhibitor PI-9/SPI-6 . Granzyme B, presumably taken up by the M6P-R , induces apoptosis in the tumor cell by activating the caspase cascade. On the other hand, cytolysis (51Cr release) is induced by a caspase-independent mechanism.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Berke G: The CTL's kiss of death. Cell. 1995, 81: 9-12. 10.1016/0092-8674(95)90365-8.
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.
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.
Sarin A, Haddad EK, Henkart PA: Caspase dependence of target cell damage induced by cytotoxic lymphocytes. J Immunol. 1998, 161: 2810-2816.
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.
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.
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.
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.
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.
–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.
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.
About this article
Cite this article
Vermijlen, D., Luo, D., Froelich, C.J. et al. Pit cells exclusively kill P815 tumor cells by the perforin/granzyme pathway. Comp Hepatol 3, S58 (2004). https://doi.org/10.1186/1476-5926-2-S1-S58
- Natural Killer Cell
- P815 Cell
- 51Cr Release
- Granule Exocytosis
- 51Cr Release Assay