Dataset Information

Regulation of indoleamine 2,3-dioxygenase in primary human saphenous vein endothelial cells.

ABSTRACT: BACKGROUND: Indoleamine 2,3-dioxygenase (IDO) is an enzyme associated with the regulation of immune responses. Cytokines such as IFN? induce its expression in endothelial cells originating from immune-privileged sites. In this study, we investigate regulators of IDO in primary endothelial cells from a non-immune-privileged site and determine whether IDO expression affects immune cell behavior. METHODS: IDO expression was determined using real-time quantitative polymerase chain reaction and immunoblotting. IDO activity was estimated using an IDO enzyme assay. Primary cells were transfected using microporation, and T-cell migration was determined using a cell transmigration assay. RESULTS: IDO is expressed in human saphenous vein endothelial cells after stimulation with IFN? but not after treatment with TNF?, IL-1?, IL-2, IL-4, IL-6, or IL-10. VEGF? and heparin negatively regulate IFN?-driven increases in IDO. Overexpression of IDO in endothelial cells does not affect transmigration of T-cells. CONCLUSION: IDO is expressed in human saphenous vein endothelial cells after stimulation with IFN?. Heparin and angiogenesis stimulators such as VEGF? negatively regulate its expression.

SUBMITTER: Mouratidis PX

PROVIDER: S-EPMC4446016 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Regulation of indoleamine 2,3-dioxygenase in primary human saphenous vein endothelial cells.

Mouratidis Petros Xe PX George Andrew Jt AJ

Journal of inflammation research 20150521

<h4>Background</h4>Indoleamine 2,3-dioxygenase (IDO) is an enzyme associated with the regulation of immune responses. Cytokines such as IFNγ induce its expression in endothelial cells originating from immune-privileged sites. In this study, we investigate regulators of IDO in primary endothelial cells from a non-immune-privileged site and determine whether IDO expression affects immune cell behavior.<h4>Methods</h4>IDO expression was determined using real-time quantitative polymerase chain react ...[more]

PMID: 26056484

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Project description:Indoleamine 2,3-dioxygenase catalyzes the O(2)-dependent oxidation of L-tryptophan (L-Trp) to N-formylkynurenine (NFK) as part of the kynurenine pathway. Inhibition of enzyme activity at high L-Trp concentrations was first noted more than 30 years ago, but the mechanism of inhibition has not been established. Using a combination of kinetic and reduction potential measurements, we present evidence showing that inhibition of enzyme activity in human indoleamine 2,3-dioxygenase (hIDO) and a number of site-directed variants during turnover with L-tryptophan (L-Trp) can be accounted for by the sequential, ordered binding of O(2) and L-Trp. Analysis of the data shows that at low concentrations of L-Trp, O(2) binds first followed by the binding of L-Trp; at higher concentrations of L-Trp, the order of binding is reversed. In addition, we show that the heme reduction potential (E(m)(0)) has a regulatory role in controlling the overall rate of catalysis (and hence the extent of inhibition) because there is a quantifiable correlation between E(m)(0) (that increases in the presence of L-Trp) and the rate constant for O(2) binding. This means that the initial formation of ferric superoxide (Fe(3+)-O(2)(•-)) from Fe(2+)-O(2) becomes thermodynamically less favorable as substrate binds, and we propose that it is the slowing down of this oxidation step at higher concentrations of substrate that is the origin of the inhibition. In contrast, we show that regeneration of the ferrous enzyme (and formation of NFK) in the final step of the mechanism, which formally requires reduction of the heme, is facilitated by the higher reduction potential in the substrate-bound enzyme and the two constants (k(cat) and E(m)(0)) are shown also to be correlated. Thus, the overall catalytic activity is balanced between the equal and opposite dependencies of the initial and final steps of the mechanism on the heme reduction potential. This tuning of the reduction potential provides a simple mechanism for regulation of the reactivity, which may be used more widely across this family of enzymes.

Dataset Information

Regulation of indoleamine 2,3-dioxygenase in primary human saphenous vein endothelial cells.

Publications

Regulation of indoleamine 2,3-dioxygenase in primary human saphenous vein endothelial cells.

Similar Datasets

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