title: 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 alpha (IFN alpha) 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 IFN gamma. 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 IFN gamma-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 IFN alpha. In contrast to IFN gamma-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 IFN alpha does not preferentially modulate the Fas-L-mediated killing by NK cells.
authors.affiliation: Department of Microbiology and Immunobiology, UCLA School of Medicine 900951747, USA.
authors.forename: S
authors.initials: S
authors.lastname: Mori
authors.forename: A
authors.initials: A
authors.lastname: Jewett
authors.forename: K
authors.initials: K
authors.lastname: Murakami-Mori
authors.forename: M
authors.initials: M
authors.lastname: Cavalcanti
authors.forename: B
authors.initials: B
authors.lastname: Bonavida
mesh_terms: D003602:Cytotoxicity, Immunologic; D053222:Fas Ligand Protein; D005786:Gene Expression Regulation; D053804:Granzymes; D019073:HT29 Cells / Q000276:immunology; D015395:Histocompatibility Antigens Class I / Q000032:analysis; D006801:Humans; D016898:Interferon-alpha / Q000494:pharmacology; D018664:Interleukin-12 / Q000494:pharmacology; D019169:Jurkat Cells / Q000276:immunology; D007694:Killer Cells, Natural / Q000187:drug effects / Q000276:immunology*; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D012333:RNA, Messenger / Q000096:biosynthesis; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000235:genetics; D015398:Signal Transduction; D014407:Tumor Cells, Cultured / Q000276:immunology*
chemical_list: C505661:FASLG protein, human; D053222:Fas Ligand Protein; D015395:Histocompatibility Antigens Class I; D016898:Interferon-alpha; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D012333:RNA, Messenger; D054353:Perforin; D018664:Interleukin-12; C509213:GZMB protein, human; D053804:Granzymes; D012697:Serine Endopeptidases; C509212:GZMA protein, human