Apoptosis in NK Cells. Natural killer (NK) cells exhibited ligand- induced death similar to activated T cells, when primed by interleukin 2 (IL-2), IL-12 and IL-15 and triggered by occupancy of the CD16 receptor. We have examined the mechanisms involved in NK apoptosis and compared these cells to the reported studies performed in T cells. Selected genes have been reported as critical for T-cell apoptosis, such as Nur77, c-myc, and BCL2; however, these appeared not to be regulated and required during NK cell apoptosis. Metabolic [Act. D, Wortmannin] and transport [Nac, CsA, FK506, MgEGTA] inhibitors were examined for interference with the CD16-induced apoptosis, with only Nac inhibiting cell death. ICE proteases, shown important for Fas-induced T-cell apoptosis, were examined for their effect on NK lysis and apoptosis. CD16-induced apoptosis was not blocked by ZVAD, whereas Fas and Staursporin induced death was blocked by this ICE inhibitory peptide. By contrast, CD16-induced death was potently blocked by a cathepsin B inhibitor, ZFA. Therefore, NK cells undergo rapid apoptotic episode when CD16 is cross-linked. However, the Fas receptor pathway in NK and T cells has similar requirements. NK target cellular lysis, mediated by the exocytosis of perforin and granzymes, was not blocked by either ZVAD or ZVA peptides. These results suggest a complex balance of pathways that can be used by NK cells to kill targets and to be regulated via apoptosis by the immune system. Monocyte Regulation. The antineoplastic agent bryostatin-1 (bryo-1) can potently induce the production of pro- inflammatory cytokines from human peripheral blood monocytes. Stimulation of monocytes increased levels of IL-8 mRNA and induced the expression of IL-1beta, tumor necrosis factor-alpha (TNF-alpha), and IL- 6 mRNA and the secretion of all four proinflammatory cytokines. Bryo-1 synergized with IL-2 in part, based on the ability of bryo-1 to upregulate IL-2Rgamma chain expression. The effects of bryo-1 could be blocked by the protein kinase C (PKC) inhibitors. These results show for the first time that bryo-1 is a powerful activator of human monocytes and suggest that stimulation of monokine secretion by bryo-1 may represent at least one of the mechanisms responsible for the in vivo antitumor activity of this drug. We have previously reported that a 19- base pair (bp) element of the 5'-flanking region of the iNOS gene containing a sequence homology to a hypoxia-responsive enhancer (iNOS- HRE), mediates hypoxia-induced activation of the iNOS promoter in IFN- gamma-treated murine macrophages. The iron chelator desferrioxamine (DFX) was examined for regulation of the iNOS promoter and iNOS gene expression. DFX induced DNA-binding activity to the hypoxia-inducible factor 1 (HIF-1) consensus sequence of the iNOS promoter and activated the iNOS-HRE. Functional analysis of the 5'-flanking region of the iNOS gene demonstrated that IFN-gamma plus DFX activated the full-length iNOS promoter and that the iNOS-HRE was required for DFX-induced iNOS transcriptional activity. These data establish that DFX is a costimulus for the transcriptional activation of the iNOS gene in IFN-gamma-treated macrophages and they provide evidence that the iNOS-HRE is required for the DFX-dependent activation of the iNOS promoter. Furthermore, our results indicate that the iNOS-HRE is a regulatory element of the iNOS promoter responsive to iron chelation.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Intramural Research (Z01)
Project #
1Z01BC009256-15
Application #
6161051
Study Section
Special Emphasis Panel (LEI)
Project Start
Project End
Budget Start
Budget End
Support Year
15
Fiscal Year
1997
Total Cost
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
United States
Zip Code