High-affinity uptake of kynurenine and nitric oxide-mediated inhibition of indoleamine 2,3-dioxygenase in bone marrow-derived myeloid dendritic cells.

Indoleamine 2,3-dioxygenase (IDO)-initiated tryptophan metabolism along the kynurenine (Kyn) pathway in some dendritic cells (DC) such as plasmacytoid DC (pDC) regulates T-cell responses. It is unclear whether bone marrow-derived myeloid DC (BMDC) express functional IDO. The IDO expression was examined in CD11c(+)CD11b(+) BMDC differentiated from mouse bone marrow cells using GM-CSF. CpG oligodeoxynucleotides (CpG) induced the expression of IDO protein with the production of nitric oxide (NO) in BMDC in cultures for 24h. In the enzyme assay using cellular extracts of BMDC, the IDO activity of BMDC stimulated with CpG was enhanced by the addition of a NO synthase (NOS) inhibitor, suggesting that IDO activity was suppressed by NO production. On the other hand, the concentration of Kyn in the culture supernatant of BMDC was not increased by stimulation with CpG. Exogenously added Kyn was taken up by BMDC independently of CpG stimulation and NO production, and the uptake of Kyn was inhibited by a transport system L-specific inhibitor or high concentrations of tryptophan. The uptake of tryptophan by BMDC was markedly lower than that of Kyn. In conclusion, IDO activity in BMDC is down-regulated by NO production, whereas BMDC strongly take up exogenous Kyn.

Diazotization of kynurenine by acidified nitrite secreted from indoleamine 2,3-dioxygenase-expressing myeloid dendritic cells.

Indoleamine 2,3-dioxygenase (IDO)-initiated tryptophan metabolism along the kynurenine (Kyn) pathway regulates T-cell responses in some dendritic cells (DC) such as plasmacytoid DC. A Kyn assay using HPLC showed that samples were frequently deproteinized with trichloroacetic acid (TCA). In the present study, bone marrow-derived myeloid DC (BMDC) were differentiated from mouse bone marrow cells with GM-CSF. CpG oligodeoxynucleotides (CpG) induced the expression of IDO protein with NO production in BMDC cultured for 24 h. The concentrations of Kyn in the culture supernatants were not increased by stimulation with CpG but rather decreased by based on the Kyn assay after deproteinization with TCA. The level of Kyn exogenously added into the cell-free culture supernatant of BMDC stimulated with CpG was severely decreased by deproteinization with TCA but not methanol, and the decrease was prevented when BMDC was stimulated with CpG in the presence of a NOS inhibitor. Under acidic conditions, Kyn reacted with nitrite produced by BMDC, and generated a new compound that was not detected by Ehrlich reagent reacting with the aromatic amino residue of Kyn. An analysis by mass spectrometry showed the new compound to be a diazotization form of Kyn. In conclusion, the deproteinization of samples by acidic treatment should be avoided for the Kyn assay when NO is produced.

Cinnabarinic acid generated from 3-hydroxyanthranilic acid strongly induces apoptosis in thymocytes through the generation of reactive oxygen species and the induction of caspase.

3-Hydroxyanthranilic acid (3HAA) is one of the tryptophan metabolites along the kynurenine pathway and induces apoptosis in T cells. We investigated the mechanism of 3HAA-induced apoptosis in mouse thymocytes. The optimal concentration of 3HAA for apoptosis induction was 300-500 microM. The induction of apoptosis by a suboptimal concentration (100 microM) of 3HAA was enhanced by superoxide dismutase (SOD) as well as MnCl2 and further promoted in the presence of catalase. The 3HAA-mediated generation of intracellular reactive oxygen species (ROS) was enhanced by SOD or MnCl2 and inhibited by catalase. Corresponding to apoptosis induction, the generation of cinnabarinic acid (CA) through the oxidation of 3HAA was enhanced by SOD or MnCl2 in the presence of catalase. The synthesized CA possessed more than 10 times higher apoptosis-inducing activity than 3HAA. The intracellular ROS generation was induced by CA within 15 min and decreased to the control levels within 4 h, whereas the 3HAA-induced ROS generation increased gradually up to 4 h. Corresponding to ROS generation, the mitochondrial membrane potential was downregulated within 15 min and retained by the CA treatment. Apoptosis induction by 3HAA or CA was dependent on caspases, and caspase-3 was much more strongly activated by CA than 3HAA. In conclusion, the CA generated from 3HAA possesses a strong apoptosis-inducing activity in thymocytes through ROS generation, the loss of mitochondrial membrane potential, and caspase activation.