Nitric oxide mediates dendritic cell apoptosis by downregulating inhibitors of apoptosis proteins and upregulating effector caspase activity.
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.
Stanford
A
A
Department of Surgery, Children's Hospital of Pittsburgh and the University of Pittsburgh School of Medicine, Pittsburgh, PA 15213, USA.
Chen
Y
Y
Zhang
X R
XR
Hoffman
R
R
Zamora
R
R
Ford
H R
HR
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis / Q000276:immunology*; D001854:Bone Marrow Cells / Q000166:cytology; D053148:Caspase 3; D053178:Caspase 6; D020169:Caspases / Q000378:metabolism*; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000276:immunology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology / Q000378:metabolism; D015536:Down-Regulation / Q000276:immunology; D004789:Enzyme Activation / Q000187:drug effects; D020287:In Situ Nick-End Labeling; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000494:pharmacology; D010396:Penicillamine / Q000031:analogs & derivatives / Q000494:pharmacology; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism; D015854:Up-Regulation / Q000276:immunology
D000590:Amino Acid Chloromethyl Ketones; D015853:Cysteine Proteinase Inhibitors; D020030:Nitric Oxide Donors; C110881:S-nitro-N-acetylpenicillamine; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D009569:Nitric Oxide; D011065:Poly(ADP-ribose) Polymerases; C505409:Casp3 protein, mouse; C505516:Casp6 protein, mouse; D053148:Caspase 3; D053178:Caspase 6; D020169:Caspases; D010396:Penicillamine