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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 D, Vermijlen D, Kuppen PJ, Wisse E - Comp Hepatol (2002)

Bottom Line: RESULTS: 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(beta-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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Cell Biology and Histology, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels, Belgium. dvermijl@cyto.vub.ac.be

ABSTRACT
BACKGROUND: 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. RESULTS: 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(beta-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. CONCLUSIONS: MHC class I expression protects CC531s colon carcinoma cells from hepatic NK cell-mediated apoptosis and cytolysis, by blocking the perforin/granzyme pathway.

No MeSH data available.


Related in: MedlinePlus

Effect of anti-MHC I mAb OX18 on hepatic NK cell-mediated CC531s cell cytolysis (A, 51Cr release) and apoptosis (B, DNA fragmentation). (A), 51Cr-labeled CC531s cells were incubated at an E:T ratio of 10:1 with freshly isolated hepatic NK cells for 18 hours. Cytolysis was measured in a 51Cr-release assay. (B), [3H]-TdR labeled CC531s cells were incubated at an E:T ratio of 10:1 with hepatic NK cells for 3 hours. Apoptosis was measured in a quantitative DNA fragmentation assay. ConAb, control antibody. *p < 0.05, **p < 0.01 vs. the treatment with medium only (LSD test).
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Figure 1: Effect of anti-MHC I mAb OX18 on hepatic NK cell-mediated CC531s cell cytolysis (A, 51Cr release) and apoptosis (B, DNA fragmentation). (A), 51Cr-labeled CC531s cells were incubated at an E:T ratio of 10:1 with freshly isolated hepatic NK cells for 18 hours. Cytolysis was measured in a 51Cr-release assay. (B), [3H]-TdR labeled CC531s cells were incubated at an E:T ratio of 10:1 with hepatic NK cells for 3 hours. Apoptosis was measured in a quantitative DNA fragmentation assay. ConAb, control antibody. *p < 0.05, **p < 0.01 vs. the treatment with medium only (LSD test).

Mentions: The expression of MHC class I molecules on CC531s cells was examined by flow cytometry. In agreement with a previous study [22], CC531s cells expressed MHC class I molecules (data not shown). The mAb CC52, used as a negative control during functional assays, was shown to bind to CC531s cells, as has also been shown previously [28] (data not shown). When CC531s cells were preincubated with mAb OX18 against MHC class I molecules, the hepatic NK cell-mediated cytolysis against CC531s cells was increased in comparison with the lysis of untreated or control mAb treated tumour cells (Fig. 1A). Similarly, the preincubation of CC531s cells with mAb OX18 increased fragmented DNA (apoptosis) in CC531s cells when coincubated with hepatic NK cells (Fig. 1B). CC531s cells showed the typical morphological characteristics of apoptosis such as nuclear fragmentation, chromatin condensation (Fig. 2A), blebbing, and rounding up (Fig. 2B), when they were coincubated with hepatic NK cells. When CC531s cells were pretreated with mAb OX18, the number of apoptotic CC531s cells increased (Figs. 2C,2D,2G). It has been reported that anti-MHC class I antibody alone can induce apoptosis in cancer cells [29]. In order to address the question whether the enhanced apoptosis and cytolysis in CC531s cells was induced by the binding of mAb OX18, CC531s cells were incubated with mAb OX18 alone. After 3, 24 or 48 hours of incubation, no apoptosis or cytolysis in CC531s cells was observed (data not shown). This is the first time it is shown that, besides cytolysis, MHC class I molecules protect cancer cells from apoptosis induced by NK cells. This is relevant because it has been suggested that in vitro assays quantifying effector cell-mediated apoptosis, but not cytolysis, are in accordance with in vivo results [13].


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 D, Vermijlen D, Kuppen PJ, Wisse E - Comp Hepatol (2002)

Effect of anti-MHC I mAb OX18 on hepatic NK cell-mediated CC531s cell cytolysis (A, 51Cr release) and apoptosis (B, DNA fragmentation). (A), 51Cr-labeled CC531s cells were incubated at an E:T ratio of 10:1 with freshly isolated hepatic NK cells for 18 hours. Cytolysis was measured in a 51Cr-release assay. (B), [3H]-TdR labeled CC531s cells were incubated at an E:T ratio of 10:1 with hepatic NK cells for 3 hours. Apoptosis was measured in a quantitative DNA fragmentation assay. ConAb, control antibody. *p < 0.05, **p < 0.01 vs. the treatment with medium only (LSD test).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC149428&req=5

Figure 1: Effect of anti-MHC I mAb OX18 on hepatic NK cell-mediated CC531s cell cytolysis (A, 51Cr release) and apoptosis (B, DNA fragmentation). (A), 51Cr-labeled CC531s cells were incubated at an E:T ratio of 10:1 with freshly isolated hepatic NK cells for 18 hours. Cytolysis was measured in a 51Cr-release assay. (B), [3H]-TdR labeled CC531s cells were incubated at an E:T ratio of 10:1 with hepatic NK cells for 3 hours. Apoptosis was measured in a quantitative DNA fragmentation assay. ConAb, control antibody. *p < 0.05, **p < 0.01 vs. the treatment with medium only (LSD test).
Mentions: The expression of MHC class I molecules on CC531s cells was examined by flow cytometry. In agreement with a previous study [22], CC531s cells expressed MHC class I molecules (data not shown). The mAb CC52, used as a negative control during functional assays, was shown to bind to CC531s cells, as has also been shown previously [28] (data not shown). When CC531s cells were preincubated with mAb OX18 against MHC class I molecules, the hepatic NK cell-mediated cytolysis against CC531s cells was increased in comparison with the lysis of untreated or control mAb treated tumour cells (Fig. 1A). Similarly, the preincubation of CC531s cells with mAb OX18 increased fragmented DNA (apoptosis) in CC531s cells when coincubated with hepatic NK cells (Fig. 1B). CC531s cells showed the typical morphological characteristics of apoptosis such as nuclear fragmentation, chromatin condensation (Fig. 2A), blebbing, and rounding up (Fig. 2B), when they were coincubated with hepatic NK cells. When CC531s cells were pretreated with mAb OX18, the number of apoptotic CC531s cells increased (Figs. 2C,2D,2G). It has been reported that anti-MHC class I antibody alone can induce apoptosis in cancer cells [29]. In order to address the question whether the enhanced apoptosis and cytolysis in CC531s cells was induced by the binding of mAb OX18, CC531s cells were incubated with mAb OX18 alone. After 3, 24 or 48 hours of incubation, no apoptosis or cytolysis in CC531s cells was observed (data not shown). This is the first time it is shown that, besides cytolysis, MHC class I molecules protect cancer cells from apoptosis induced by NK cells. This is relevant because it has been suggested that in vitro assays quantifying effector cell-mediated apoptosis, but not cytolysis, are in accordance with in vivo results [13].

Bottom Line: RESULTS: 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(beta-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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Cell Biology and Histology, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090 Brussels, Belgium. dvermijl@cyto.vub.ac.be

ABSTRACT
BACKGROUND: 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. RESULTS: 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(beta-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. CONCLUSIONS: MHC class I expression protects CC531s colon carcinoma cells from hepatic NK cell-mediated apoptosis and cytolysis, by blocking the perforin/granzyme pathway.

No MeSH data available.


Related in: MedlinePlus