Induction of nitric oxide synthase is involved in the mechanism of Fas- mediated apoptosis in haemopoietic cells

Abstract

Induction of nitric oxide synthase (iNOS) and production of the toxic metabolite nitric oxide (NO) is one of the interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α) regulated effector mechanisms that can lead to apoptosis of haemopoietic progenitor cells. Fas-receptor (Fas-R) expression can be stimulated by IFN-γ and TNF-α. Transactivation of iNOS, and possibly Fas-R promoters, by interferon regulatory factor-1 expressed in response to IFN-γ may be a part of the INOS transduction pathway. We investigated whether the effects of Fas-R triggering in haemopoietic cells were mediated by NO. On Western blotting, we observed that Fas-receptor agonist, monoclonal antibody CH11, enhanced expression of iNOS. As shown by the reverse transcription polymerase chain reaction, CH11 also induced INOS mRNA expression in purified CD34+ cells. To determine whether NO was involved in Fas-mediated apoptosis we inhibited INOS-catalysed production of NO using anti-sense (AS) oligodeoxynucleotides (ODN) directed against iNOS mRNA. After culture of haemopoietic cells in the presence of AS-ODN, INOS expression decreased and was no longer enhanced by Fas. This effect was associated with the prevention of Fas-mediated apoptosis, as determined by a DNA fragmentation and terminal deoxynucleotidyl transferase staining. In colony assays, specific AS-oligonucleotides prevented FAS-mediated inhibition of colony formation by total bone marrow and CD34+ progenitor cells. Our data suggest that the inhibitory effects of Fas, including induction of apoptosis, are mediated by effector mechanisms that may be similar to those described for IFN-γ and TNF-α.