A Stanford1, Y Chen, X R Zhang, R Hoffman, R Zamora, H R Ford. 1. Department of Surgery, Children's Hospital of Pittsburgh and the University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
Abstract
BACKGROUND: Dendritic cells (DCs) play a crucial role in the amplification of the immune response by promoting antigen presentation, T-lymphocyte proliferation, and proinflammatory cytokine and nitric oxide (NO) production. We have previously shown that the exogenous NO donor, s-nitroso-N-acetyl-penicillamine, promotes DC apoptosis by disrupting the mitochondrial membrane potential, which induces cytochrome-C release and activates caspase 3. To further elucidate the signaling pathway, we examined the expression of cellular inhibitors of apoptosis proteins (cIAPs) and poly (ADP-ribose) polymerase cleavage (PARP), a terminal event in the apoptotic cascade. METHODS: DC2.4 were exposed to 250 micromol/L s-nitroso-N-acetyl-penicillamine for various intervals. Apoptosis and necrosis were measured by terminal deoxynucleotidyl transferase nick-end labeling assay or flow cytometry with Annexin V and propidium iodide. DC2.4 were cultured with the pan-caspase inhibitor, ZVAD (100 micromol/L). cIAP, pro-caspases, and PARP expression or activation was measured by Western blot. Caspase enzyme activity was confirmed with the use of specific substrates. RESULTS: NO-induced DC apoptosis correlated with the downregulation of cIAP expression. Caspase 3 and 6 were upregulated by SNAP and significantly inhibited by ZVAD. Maximal PARP cleavage occurred at 8 hours and coincided with the downregulation of cIAP and peak caspase 3 and near maximal caspase 6 activity. CONCLUSIONS: NO-induced DC apoptosis is associated with the downregulation of cIAP expression, which facilitates caspase cascade activation and subsequent PARP cleavage.
BACKGROUND: Dendritic cells (DCs) play a crucial role in the amplification of the immune response by promoting antigen presentation, T-lymphocyte proliferation, and proinflammatory cytokine and nitric oxide (NO) production. We have previously shown that the exogenous NO donor, s-nitroso-N-acetyl-penicillamine, promotes DC apoptosis by disrupting the mitochondrial membrane potential, which induces cytochrome-C release and activates caspase 3. To further elucidate the signaling pathway, we examined the expression of cellular inhibitors of apoptosis proteins (cIAPs) and poly (ADP-ribose) polymerase cleavage (PARP), a terminal event in the apoptotic cascade. METHODS: DC2.4 were exposed to 250 micromol/L s-nitroso-N-acetyl-penicillamine for various intervals. Apoptosis and necrosis were measured by terminal deoxynucleotidyl transferase nick-end labeling assay or flow cytometry with Annexin V and propidium iodide. DC2.4 were cultured with the pan-caspase inhibitor, ZVAD (100 micromol/L). cIAP, pro-caspases, and PARP expression or activation was measured by Western blot. Caspase enzyme activity was confirmed with the use of specific substrates. RESULTS: NO-induced DC apoptosis correlated with the downregulation of cIAP expression. Caspase 3 and 6 were upregulated by SNAP and significantly inhibited by ZVAD. Maximal PARP cleavage occurred at 8 hours and coincided with the downregulation of cIAP and peak caspase 3 and near maximal caspase 6 activity. CONCLUSIONS: NO-induced DC apoptosis is associated with the downregulation of cIAP expression, which facilitates caspase cascade activation and subsequent PARP cleavage.
Authors: Daniela Giordano; Chang Li; Mehul S Suthar; Kevin E Draves; Daphne Y Ma; Michael Gale; Edward A Clark Journal: J Leukoc Biol Date: 2010-12-22 Impact factor: 4.962
Authors: Karina R B Bastos; Luciana de Deus Vieira de Moraes; Cláudia A Zago; Cláudio R F Marinho; Momtchilo Russo; José M M Alvarez; Maria R D'Império Lima Journal: Immunology Date: 2005-04 Impact factor: 7.397