The participation of the Fas-mediated cytotoxic pathway by natural killer cells is tumor-cell-dependent

Abstract

 Cytotoxic T lymphocytes and natural killer (NK) cells kill target cells by two main mechanisms, namely, the perforin/granzymes and the Fas ligand (Fas-L) pathways. The preferential activation of either of these two mechanisms by target cells is not known. This study examined whether various NK stimuli regulate preferentially the perforin/granzyme or the Fas pathways during the NK-cell-mediated cytotoxic reaction (NK-CMC). Purified peripheral-blood-derived NK cells were stimulated with interleukin-2 (IL-2), IL-12, or interferon α (IFNα) and their response was analyzed by the reverse transcriptase/polymerase chain reaction (RT-PCR) for NK-associated gene expression and by the ⁵¹Cr-release assay for cytotoxic function. RT-PCR data revealed that the perforin, granzyme A and granzyme B mRNAs were constitutively expressed in unstimulated NK cells and the level of perforin mRNA was augmented following activation. IL-2 enhanced the level of Fas-L mRNA in NK cells; however, the Fas-L level was much lower than that obtained in activated T cells. NK-CMC against Fas-sensitive cells was examined in the presence of neutralizing anti-(Fas antigen receptor) (Fas-R) antibody (ZB-4) or EGTA/Mg²⁺, which inhibits the perforin/granzyme pathway but not the Fas Fas-L interaction. The human colon adenocarcinoma HT-29 cells were sensitized to anti-Fas-R antibody (CH-11) cytotoxicity following treatment with IFNγ. NK-CMC against untreated HT-29 cells was completely inhibited by EGTA/Mg²⁺ and was unaffected by ZB-4, while both EGTA/Mg²⁺ and ZB-4 partially inhibited NK-CMC against IFNγ-treated HT-29 cells. Similar findings to those obtained with untreated NK cells were observed with NK cells stimulated with IL-2, IL-2 plus IL-12 or IFNα. In contrast to IFNγ-treated HT-29 cells, the neutralizing anti-Fas antibody ZB-4 did not inhibit NK-CMC against Fas-sensitive U937, CEM or Jurkat tumor cells. These findings demonstrate that the Fas pathway is involved in NK-CMC against certain target cells but not all. Further, the data demonstrate that activation of NK cells by IL-2, IL-2 plus IL-12 or IFNα does not preferentially modulate the Fas-L-mediated killing by NK cells.