MHC class I expression protects rat colon carcinoma cells from hepatic natural killer cell-mediated apoptosis and cytolysis, by blocking the perforin/granzyme pathway
© Luo et al; licensee BioMed Central Ltd. 2002
Received: 28 August 2002
Accepted: 20 November 2002
Published: 20 November 2002
Hepatic natural killer (NK) cells, the most cytotoxic cells of the natural occurring NK cells, are located in the liver sinusoids and are thus in a strategic position to kill arriving metastasising tumour cells, like colon carcinoma cells. It is known that major histocompatibility complex (MHC) class I on tumour cells negatively regulates NK cell-mediated cytolysis, but this is found using blood- or spleen-derived NK cells. Therefore, using isolated rat hepatic NK cells and the syngeneic colon carcinoma cell line CC531s, we investigated whether this protective role of MHC class I is also operative in hepatic NK cells, and addressed the mechanism of MHC class I protection.
When MHC class I on CC531s cells was masked by preincubation with monoclonal antibody OX18, hepatic NK cell-mediated cytolysis (51Cr release) as well as apoptosis (DNA fragmentation, nucleus condensation and fragmentation) increased. When hepatic NK cells were preincubated with the granzyme inhibitor 3,4-dichloroisocoumarin, or when extracellular Ca2+ was chelated by ethylene glycol-bis(β-aminoethyl ether)-N, N-tetraacetic acid, the enhanced cytolysis and apoptosis were completely inhibited. The involvement of the perforin/granzyme pathway was confirmed by showing that the enhanced cytolysis was caspase-independent.
MHC class I expression protects CC531s colon carcinoma cells from hepatic NK cell-mediated apoptosis and cytolysis, by blocking the perforin/granzyme pathway.
Natural killer (NK) cells are large granular lymphocytes that have the ability to kill cells without prior sensitisation and therefore play an important role in host defence . NK cell-mediated target cell killing is mainly implemented by two pathways, namely the perforin/granzyme pathway and the Fas ligand (FasL) pathway [2–5]. In the latter pathway, FasL on effector cells binds Fas present on the target cells which results in oligomerization of Fas and activation of caspase 8. Perforin and granzymes, of which granzyme B is the most potent one, reside in the granules of NK cells and are released by exocytosis after conjugation between the effector and target cell [4, 5]. Inside the cytoplasm of the target cell, granzyme B activates caspase 3 directly  or indirectly, via a mitochondrion-dependent pathway . Caspases play an essential role in the execution of apoptosis .
NK cells display two types of surface receptors: (i) activation receptors, such as the CD161 molecule that recognises structures on target cells and triggers NK cells to kill; (ii) inhibitory receptors, such as Ly-49 molecules, that recognise target cell MHC class I molecules and inhibit killing by NK cells [8, 9]. When MHC class I molecules are absent or expressed in reduced amounts, the NK cells proceed with their attack . The mechanism of MHC class I protection is not fully understood. MHC class I molecules do not block target cell recognition by NK cells . A recent study shows that H-2Dd MHC class I molecules on target cells partially inhibit granzyme A release from mouse Ly-49A+ NK cells . However, it is unclear whether such partial inhibition of granzyme A release is sufficient to protect target cells. Moreover, the assay used in the past to detect cytotoxicity by cytolysis is the release of 51Cr from loaded target cells. A recent study questioned the relevance of the 51Cr release assay compared to what occurs in vivo, whereas the DNA fragmentation assay to measure apoptosis coincided with in vivo results . Therefore, it is needed to explore whether the protective role of MHC class I is also operative in apoptosis induced by NK cells.
Compared with NK cells from spleen and peripheral blood, hepatic NK cells, also called pit cells , are much more cytotoxic [15, 16]. Strategically located in the liver sinusoids, they constitute a first line of cellular defence against invading cancer cells, like colon carcinoma cells [15, 17–20]. In this study, using freshly isolated hepatic NK cells and CC531s, a syngeneic Fas ligand-resistant colon carcinoma cell line , we (i) demonstrated that MHC class I protects colon carcinoma cells from hepatic NK cell-mediated killing; and (ii) showed the involvement of the perforin/granzyme pathway in the mechanism of MHC class I protection.
Results and Discussion
Protection of target cells from NK cell lysis by expression of MHC class I molecules has been demonstrated in different experimental systems in human , mouse  and rat [10, 22]. In rat, several MHC class I genes have been identified, i.e., RT1.A, RT1.C and RT1.E . It has been shown that transfection of RT1.A and RT1.C protects target cells from lysis by NK cells . However, other data indicate that RT1.A molecules inhibit NK cells, whereas RT1.C region molecules activate natural killing [24, 25]. Masking of RT1.A, RT1.C, or both alleles on target cells with allele-specific mAbs, has no effect on lysis by NK cells . In view of these facts, mAb OX18 was chosen to investigate the mechanism of MHC class I protection of CC531s target cells from hepatic NK cell-mediated killing. It has been found that (i) mAb OX18 binds total rat MHC class I , (ii) masking of MHC class I molecules on target cells by mAb OX18 or F(ab')2 fragments of OX18 enhances the syngeneic target cell cytolysis by rat NK cells , and (iii) the enhanced NK cell-mediated target lysis by mAb OX18 is not caused by antibody dependent cellular cytotoxicity (ADCC) .
To address the mechanism of protection of MHC class I on CC531s cells, we used several approaches to assess the granule exocytosis pathway: granzyme inhibition by DCI, Ca2+ chelation by EGTA and caspase inhibition by Z-VAD-FMK.
The perforin/granzyme pathway is Ca2+-dependent, involving extracellular Ca2+ at three distinct steps: (i) granule exocytosis; (ii) binding of secreted perforin to the membrane of target cells; and (iii) perforin polymerisation . When extracellular Ca2+ was chelated by EGTA during the co-incubation, the anti-MHC class I mAb-enhanced cytolysis and apoptosis of CC531s cells by hepatic NK cells was completely inhibited (Figs. 2, 3).
MHC class I expression protects colon carcinoma cells from apoptosis and cytolysis induced by hepatic NK cells, by blocking the perforin/granzyme pathway. This mechanism of immune escape could possibly contribute to the incomplete killing by hepatic NK cells of arriving colon carcinoma cells in the liver sinusoids, resulting in the formation of liver metastases.
Materials and Methods
Isolation and purification of hepatic NK cells
Hepatic NK cells were isolated and purified from 3- to 5-month old male Wag/Rij rats (RT1u, a Wistar-derived inbred strain, Harlan, The Netherlands) according to the method described before . The purity of the isolated hepatic NK cells was at least 90%, as evaluated by light microscopy using May-Giemsa staining cytospins and by flow cytometric analysis using mAb 3.2.3, which recognises CD161A molecules on the surface of rat NK cells . The viability of the recovered cells was more than 95%, as determined by trypan blue exclusion. The procedures used in this study were approved by the local ethical committee (license no. LA1230212).
Tumour cell line
CC531s, a dimethylhydrazine-induced colon carcinoma of Wag/Rij rats , was maintained in culture medium RPMI-1640 (Gibco, Life Technologies, Gent, Belgium), supplemented with 10% fetal calf serum (Eurobiochem, Bierges, Belgium), penicillin (100 U/ml), streptomycin (100 μg/ml), and glutamin (0.2 mM) (Gibco, Life Technologies, Gent, Belgium).
Reagents and antibodies
3,4-dichloroisocoumarin (DCI) and ethylene glycol-bis(β-aminoethyl ether)-N, N-tetraacetic acid (EGTA), were purchased from ICN (Asse-Relegem, Belgium). The monoclonal antibody (mAb) OX18 (anti-rat MHC class I, IgG1)  was purchased from ECACC (Porton Down, Salisbury, UK). MAb CC52 (IgG1)  was developed in the Department of Surgery and Pathology, Leiden University Medical Center, The Netherlands . Z-Val-Ala-Asp(OMe)-fluoromethylketone (Z-VAD-FMK) was obtained from Bachem (Bubendorf, Switzerland).
The expression of MHC class I molecules on the CC531s cells was determined by one-colour flow cytometric analysis, as described previously . Briefly, 0.5 × 106 cells were incubated (30 minutes, 4°C) with the primary antibody OX18. Cells were then washed three times with cold phosphate-buffered saline (PBS), containing 1% bovine serum albumin and 0.02% sodium azide. Subsequently, cells were incubated with FITC-conjugated antimouse IgG1 (Gilbertsville, PA). After incubation and washing, cells were fixed with 2% paraformaldehyde in PBS and analysed (FACSort; Becton Dickinson, Mountain View, CA, USA). Isotype-matched irrelevant antibody was used as a negative control.
Quantitative DNA fragmentation assay
DNA fragmentation in the CC531s cells was determined as described previously . In short, [methyl-3H]thymidine ([3H]-TdR) labeled CC531s cells were preincubated with the mAb OX18 (final concentration was 10 μg/ml) for 15 minutes, at room temperature, and freshly isolated hepatic NK cells were preincubated with 50 μM DCI for 30 minutes. It was shown that mAb CC52 binds to CC531s cells  and is used in this study as a negative control in the functional assays. The cells were then washed twice. EGTA (final concentration was 5 mM) and Z-VAD-FMK (final concentration 80 μM) were present during coincubation. The hepatic NK cells (105 cells in 100 μl) and CC531s cells (104 cells in 100 μl) were placed in triplicate in 1.5 ml microcentrifuge tubes. After 3 hours coincubation and centrifugation, the incubation medium was removed from the tubes. Subsequently, the pelleted cells were lysed. The lysates were ultracentrifuged (10,000 g for 15 minutes at 4°C) to separate fragmented DNA from intact DNA. Radioactivity (cpm) in the incubation medium, in the 10,000 g supernatant and in the 10,000 pellet was determined in a β counter (Beckman, Fullerton, CA, USA). The percentage of fragmented DNA was calculated using the following formula:
in which: cpmfr = the radioactivity in the incubation medium plus the cpm in the 10,000 g supernatant; cpmtotal = cpmfr + radioactivity in the 10,000 g pellet; exp = experimental (target cells with effector cells); spont = spontaneous (target cells and medium only).
Hoechst 33342/propidium iodide staining
CC531s cells and freshly isolated hepatic NK cells were treated as mentioned above. CC531s cells (104 cells in 100 μl), were coincubated with hepatic NK cells (105 cells in 100 μl) in a flat-bottom 96-multiwell plate, for 3 hours at 37°C. After coincubation, nuclei of the cells were stained with Hoechst 33342 and propidium iodide, as described previously . Preparations were studied with a Leica DM IRB/E inverted fluorescence microscope (Leica, Heidelberg, Germany) with ultraviolet excitation, at 340 to 380 nm. The images were recorded and the number of apoptotic CC531s cells was determined by the characteristic morphological changes of the apoptotic nucleus, i.e., condensation of chromatin and nuclear fragmentation. CC531s cells, not coincubated with hepatic NK cells, were used as a control. The fragmented nuclei were counted in at least 300 cells in each preparation and the percentage of apoptotic CC531s cells was calculated using the following formula:
Cytolysis was measured in a 18 hour 51Cr-release assay using 96-multiwell plates, as described previously . Briefly, 51Cr-labeled CC531s cells and hepatic NK cells were treated with the mAb or DCI or EGTA or Z-VAD-FMK, as mentioned above. CC531s cells at a concentration of 104 cells/well were coincubated with hepatic NK cells at an effector-to-target (E:T) ratio of 10:1 in a final volume of 200 μl. After 18 hours coincubation, the supernatant of each well was aspirated and radioactivity was determined in a γ counter. The cpm usually ranged from 1200 cpm (spontaneous release) to 5000 cpm (maximal release). Results were expressed as percentage of specific lysis according to the formula:
in which: exp = experimental (target cells with effector cells); spont = spontaneous (target cells and medium only); max = maximal, obtained after the addition of SDS (1% final concentration). All data are triplicate determinations.
Statistical analysis was performed by one-way ANOVA (n = 3 in each group, unless otherwise indicated) with post-hoc multiple comparison analysis made by LSD (the least-significant difference) test, using SPSS statistical package (SPSS Inc., Chicago, IL, USA). Variances were assumed to be homogeneous. Statistically significant difference between two groups was considered at the level of p < 0.05.
List of abbreviations used
antibody dependent cellular cytotoxicity
counts per minute
ethylene glycol-bis(β-aminoethyl ether)-N, N-tetraacetic acid
major histocompatibility complex
Supported by grants G038000 and G000599 from the Fund for Scientific Research – Flanders and grants OZR230 and OZR492 from the Research Council of the Free University of Brussels.
We thank Carine Seynaeve and Marijke Baekeland for their technical help, Karen Crits for her technical assistance in flow cytometry, Chris Derom for her photographic support and Ronald de Zanger for his assistance in statistics.
- Trinchieri G: Biology of natural killer cells. Adv Immunol. 1989, 47: 187-376.View ArticlePubMedGoogle Scholar
- Montel AH, Bochan MR, Hobbs JA, Lynch DH, Brahmi Z: Fas involvement in cytotoxicity mediated by human NK cells. Cell Immunol. 1995, 166: 236-246. 10.1006/cimm.1995.9974.View ArticlePubMedGoogle Scholar
- Oshimi Y, Oda S, Honda Y, Nagata S, Miyazaki S: Involvement of Fas ligand and Fas-mediated pathway in the cytotoxicity of human natural killer cells. J Immunol. 1996, 157: 2909-2915.PubMedGoogle Scholar
- Shresta S, Macivor DM, Heusel JW, Russell JH, Ley TJ: Natural killer and lymphokine-activated killer cells require granzyme B for the rapid induction of apoptosis in susceptible target cells. Proc Natl Acad Sci U S A. 1995, 92: 5679-5683.PubMed CentralView ArticlePubMedGoogle Scholar
- Berke G: The CTL's kiss of death. Cell. 1995, 81: 9-12.View ArticlePubMedGoogle 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 Velde C, 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
- Yokoyama WM, Seaman WE: The Ly-49 and NKR-P1 gene families encoding lectin-like receptors on natural killer cells: the NK gene complex. Annu Rev Immunol. 1993, 11: 613-635. 10.1146/annurev.immunol.11.1.613.View ArticlePubMedGoogle Scholar
- Imboden J: Turning off natural killers. Innate immunity. Curr Biol. 1996, 6: 1070-1072.View ArticlePubMedGoogle Scholar
- Kraus E, Lambracht D, Wonigeit K, Hunig T: Negative regulation of rat natural killer cell activity by major histocompatibility complex class I recognition. Eur J Immunol. 1996, 26: 2582-2586.View ArticlePubMedGoogle Scholar
- Kaufman DS, Schoon RA, Leibson PJ: MHC class I expression on tumour targets inhibits natural killer cell-mediated cytotoxicity without interfering with target recognition. J Immunol. 1993, 150: 1429-1436.PubMedGoogle Scholar
- Kim S, Yokoyama WM: NK cell granule exocytosis and cytokine production inhibited by Ly-49A engagement. Cell Immunol. 1998, 183: 106-112. 10.1006/cimm.1998.1252.View ArticlePubMedGoogle Scholar
- Motyka B, Korbutt G, Pinkoski MJ, Heibein JA, Caputo A, Hobman M, Barry M, Shostak I, Sawchuk T, Holmes CF, et al: 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.View ArticlePubMedGoogle Scholar
- Wisse E, van't Noordende JM, van der Meulen J, Daems WT: The pit cell: description of a new type of cell occurring in rat liver sinusoids and peripheral blood. Cell Tissue Res. 1976, 173: 423-435.View ArticlePubMedGoogle Scholar
- Vanderkerken K, Bouwens L, Wisse E: Characterization of a phenotypically and functionally distinct subset of large granular lymphocytes (pit cells) in rat liver sinusoids. Hepatology. 1990, 12: 70-75.View ArticlePubMedGoogle Scholar
- Luo D, Vanderkerken K, Chen MC, Vermijlen D, Asosingh K, Willems E, Triantis V, Eizirik DL, Kuppen PJ, Wisse E: Rat hepatic natural killer cells (pit cells) express mRNA and protein similar to in vitro interleukin-2 activated spleen natural killer cells. Cell Immunol. 2001, 210: 41-48. 10.1006/cimm.2001.1803.View ArticlePubMedGoogle Scholar
- 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: 265-286.View ArticlePubMedGoogle Scholar
- Winnock M, Garcia-Barcina M, Huet S, Bernard P, Saric J, Bioulac-Sage P, Gualde N, Balabaud C: Functional characterization of liver-associated lymphocytes in patients with liver metastasis. Gastroenterology. 1993, 105: 1152-1158.PubMedGoogle 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
- Vermijlen D, Luo D, Robaye B, Seynaeve C, Baekeland M, Wisse E: Pit cells (Hepatic natural killer cells) of the rat induce apoptosis in colon carcinoma cells by the perforin/granzyme pathway. Hepatology. 1999, 29: 51-56.View ArticlePubMedGoogle Scholar
- Vermijlen D, Froelich CJ, Luo D, Suarez-Huerta N, Robaye B, Wisse E: Perforin and granzyme B induce apoptosis in FasL-resistant colon carcinoma cells. Cancer Immunol Immunother. 2001, 50: 212-217. 10.1007/s002620100191.View ArticlePubMedGoogle Scholar
- Smits KM, Kuppen PJ, Eggermont AM, Tamatani T, Miyasaka M, Fleuren GJ: Rat interleukin-2-activated natural killer (A-NK) cell-mediated lysis is determined by the presence of CD18 on A-NK cells and the absence of major histocompatibility complex class I on target cells. Eur J Immunol. 1994, 24: 171-175.View ArticlePubMedGoogle Scholar
- Rolstad B, Vaage JT, Naper C, Lambracht D, Wonigeit K, Joly E, Butcher GW: Positive and negative MHC class I recognition by rat NK cells. Immunol Rev. 1997, 155: 91-104.View ArticlePubMedGoogle Scholar
- Naper C, Rolstad B, Wonigeit K, Butcher GW, Vaage JT: Genes in two MHC class I regions control recognition of a single rat NK cell allodeterminant. Int Immunol. 1996, 8: 1779-1785.View ArticlePubMedGoogle Scholar
- Vaage JT, Naper C, Lovik G, Lambracht D, Rehm A, Hedrich HJ, Wonigeit K, Rolstad B: Control of rat natural killer cell-mediated allorecognition by a major histocompatibility complex region encoding nonclassical class I antigens. J Exp Med. 1994, 180: 641-651.View ArticlePubMedGoogle Scholar
- Giezeman-Smits KM, Kuppen PJ, Ensink NG, Eggermont AM, Stals F, Wonigeit K, Fleuren GJ: The role of MHC class I expression in rat NK cell-mediated lysis of syngeneic tumour cells and virus-infected cells. Immunobiology. 1996, 195: 286-299.View ArticlePubMedGoogle Scholar
- Fukumoto T, McMaster WR, Williams AF: Mouse monoclonal antibodies against rat major histocompatibility antigens. Two Ia antigens and expression of Ia and class I antigens in rat thymus. Eur J Immunol. 1982, 12: 237-243.View ArticlePubMedGoogle Scholar
- Kuppen PJ, Eggermont AM, Smits KM, Van Eendenburg JD, Lazeroms SP, van de Velde CJ, Fleuren GJ: The development and purification of a bispecific antibody for lymphokine-activated killer cell targeting against the rat colon carcinoma CC531. Cancer Immunol Immunother. 1993, 36: 403-408.View ArticlePubMedGoogle Scholar
- Woodle ES, Smith DM, Bluestone JA, Kirkman WM, Green DR, Skowronski EW: Anti-human class I MHC antibodies induce apoptosis by a pathway that is distinct from the Fas antigen-mediated pathway. J Immunol. 1997, 158: 2156-2164.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.View ArticlePubMedGoogle Scholar
- Sarin A, Williams MS, Alexander-Miller MA, Berzofsky JA, Zacharchuk CM, Henkart PA: Target cell lysis by CTL granule exocytosis is independent of ICE/Ced-3 family proteases. Immunity. 1997, 6: 209-215.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
- 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
- Chambers WH, Vujanovic NL, DeLeo AB, Olszowy MW, Herberman RB, Hiserodt JC: Monoclonal antibody to a triggering structure expressed on rat natural killer cells and adherent lymphokine-activated killer cells. J Exp Med. 1989, 169: 1373-1389.View ArticlePubMedGoogle Scholar
- Marquet RL, Westbroek DL, Jeekel J: Interferon treatment of a transplantable rat colon adenocarcinoma: importance of tumour site. Int J Cancer. 1984, 33: 689-692.View ArticlePubMedGoogle Scholar
- Hagenaars M, Koelemij R, Ensink NG, Van Eendenburg JD, Van Vlierberghe RL, Eggermont AM, van de Velde CJ, Fleuren GJ, Kuppen PJ: The development of novel mouse monoclonal antibodies against the CC531 rat colon adenocarcinoma. Clin Exp Metastasis. 2000, 18: 281-289. 10.1023/A:1011062002851.View ArticlePubMedGoogle Scholar
- Luo D, Vanderkerken K, Bouwens L, Kuppen PJ, Baekeland M, Seynaeve C, Wisse E: The role of adhesion molecules in the recruitment of hepatic natural killer cells (pit cells) in rat liver. Hepatology. 1996, 24: 1475-1480.View ArticlePubMedGoogle Scholar
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