- Open Access
Pit cells exclusively kill P815 tumor cells by the perforin/granzyme pathway
© Vermijlen et al; licensee BioMed Central Ltd 2004
- Published: 14 January 2004
- Natural Killer Cell
- P815 Cell
- 51Cr Release
- Granule Exocytosis
- 51Cr Release Assay
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).
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