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Tryptophan-derived catabolites are responsible for inhibition of T and natural killer cell proliferation induced by indoleamine 2,3-dioxygenase.

Frumento G, Rotondo R, Tonetti M, Damonte G, Benatti U, Ferrara GB - J. Exp. Med. (2002)

Bottom Line: Macrophages exposed to macrophage colony-stimulating factor acquire the capacity to suppress T cell proliferation; this effect is associated with de novo expression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO).Inhibition of cell proliferation induced by the tryptophan catabolites resulting from IDO activity was selective, applying only to cells undergoing activation.We suggest that IDO exerts its effect on cell proliferation by (i) starting the cascade of biochemical reactions that produce the three catabolites and by (ii) enhancing their inhibitory potential by depriving the extracellular microenvironment of tryptophan.

View Article: PubMed Central - PubMed

Affiliation: Immunogenetics Laboratory, National Cancer Research Institute, University of Genoa, Largo Rosanna Benzi 10, 16132 Genoa, Italy. guido.frumento@istge.it

ABSTRACT
Macrophages exposed to macrophage colony-stimulating factor acquire the capacity to suppress T cell proliferation; this effect is associated with de novo expression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). We have purified IDO and tested its activity in in vitro models of T cell activation. IDO was able to inhibit proliferation of CD4(+) T lymphocytes, CD8(+) T lymphocytes, and natural killer (NK) cells; proliferation of B lymphocytes was not affected. The inhibitory role of tryptophan and of its catabolites was then tested. In the presence of tryptophan, only L-kynurenine and picolinic acid inhibit cell proliferation. In a tryptophan-free medium cell proliferation was not affected. In the absence of tryptophan inhibition induced by L-kynurenine and picolinic acid was observed at concentrations below the lowest concentration that was effective in the presence of tryptophan, and quinolinic acid acquired some inhibitory capacity. Inhibition of cell proliferation induced by the tryptophan catabolites resulting from IDO activity was selective, applying only to cells undergoing activation. Resting cells were not affected and could subsequently activate normally. We suggest that IDO exerts its effect on cell proliferation by (i) starting the cascade of biochemical reactions that produce the three catabolites and by (ii) enhancing their inhibitory potential by depriving the extracellular microenvironment of tryptophan.

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Effect of tryptophan catabolites on T cell proliferation. (A) Increasing concentrations of l-kynurenine (▪––▪), picolinic acid (•- - -•), or quinolinic acid (▴…▴) were added to PHA-activated PBLs. Cell culture was stopped after 96 h and proliferation was evaluated by measuring 3[H]thymidine incorporation. (B) PHA-activated PBLs were incubated with the indicated combinations of 250 μM l-kynurenine, picolinic acid, or quinolinic acid. Control is represented by cells incubated with medium alone. Cell culture was stopped after 96 h and proliferation was evaluated by measuring 3[H]thymidine incorporation. (C) PHA-activated PBLs were incubated with 250 μM of l-kynurenine, picolinic acid, or quinolinic acid, respectively. Control is represented by untreated PHA-blasts. Cell culture was stopped after 96 h and the cells in the cell cycle were counted. m ± 1 SD, n = 3.
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fig4: Effect of tryptophan catabolites on T cell proliferation. (A) Increasing concentrations of l-kynurenine (▪––▪), picolinic acid (•- - -•), or quinolinic acid (▴…▴) were added to PHA-activated PBLs. Cell culture was stopped after 96 h and proliferation was evaluated by measuring 3[H]thymidine incorporation. (B) PHA-activated PBLs were incubated with the indicated combinations of 250 μM l-kynurenine, picolinic acid, or quinolinic acid. Control is represented by cells incubated with medium alone. Cell culture was stopped after 96 h and proliferation was evaluated by measuring 3[H]thymidine incorporation. (C) PHA-activated PBLs were incubated with 250 μM of l-kynurenine, picolinic acid, or quinolinic acid, respectively. Control is represented by untreated PHA-blasts. Cell culture was stopped after 96 h and the cells in the cell cycle were counted. m ± 1 SD, n = 3.

Mentions: Tryptophan is converted by IDO to N-formylkynurenine; this substance is further catabolized to kynurenine and then to the terminal metabolites picolinic acid or quinolinic acid. To understand the mechanisms of the inhibitory effect of the enzyme on PBL proliferation, it is mandatory to elucidate the effects that reduction of tryptophan and/or increase of downstream catabolites in the medium have on cell proliferation. Therefore, we started to investigate the effects exerted on PBL proliferation by three tryptophan-derived catabolites: l-kynurenine, picolinic acid, and quinolinic acid. The test was performed in complete culture medium, that contains 26 μM tryptophan, and the substances were added at the beginning of the test (Fig. 4 A). At concentrations above 1 mM a toxic effect was detected for all the three substances. Below this threshold, a dose-dependent inhibition of proliferation was observed with l-kynurenine and, to a lesser extent, with picolinic acid; instead, quinolinic acid was ineffective. Upon coincubation, l-kynurenine and picolinic acid showed a clear synergistic effect (Fig. 4 B). The two substances exerted their effect by blocking cell cycle in the mid-G1 phase (Fig. 4 C).


Tryptophan-derived catabolites are responsible for inhibition of T and natural killer cell proliferation induced by indoleamine 2,3-dioxygenase.

Frumento G, Rotondo R, Tonetti M, Damonte G, Benatti U, Ferrara GB - J. Exp. Med. (2002)

Effect of tryptophan catabolites on T cell proliferation. (A) Increasing concentrations of l-kynurenine (▪––▪), picolinic acid (•- - -•), or quinolinic acid (▴…▴) were added to PHA-activated PBLs. Cell culture was stopped after 96 h and proliferation was evaluated by measuring 3[H]thymidine incorporation. (B) PHA-activated PBLs were incubated with the indicated combinations of 250 μM l-kynurenine, picolinic acid, or quinolinic acid. Control is represented by cells incubated with medium alone. Cell culture was stopped after 96 h and proliferation was evaluated by measuring 3[H]thymidine incorporation. (C) PHA-activated PBLs were incubated with 250 μM of l-kynurenine, picolinic acid, or quinolinic acid, respectively. Control is represented by untreated PHA-blasts. Cell culture was stopped after 96 h and the cells in the cell cycle were counted. m ± 1 SD, n = 3.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196046&req=5

fig4: Effect of tryptophan catabolites on T cell proliferation. (A) Increasing concentrations of l-kynurenine (▪––▪), picolinic acid (•- - -•), or quinolinic acid (▴…▴) were added to PHA-activated PBLs. Cell culture was stopped after 96 h and proliferation was evaluated by measuring 3[H]thymidine incorporation. (B) PHA-activated PBLs were incubated with the indicated combinations of 250 μM l-kynurenine, picolinic acid, or quinolinic acid. Control is represented by cells incubated with medium alone. Cell culture was stopped after 96 h and proliferation was evaluated by measuring 3[H]thymidine incorporation. (C) PHA-activated PBLs were incubated with 250 μM of l-kynurenine, picolinic acid, or quinolinic acid, respectively. Control is represented by untreated PHA-blasts. Cell culture was stopped after 96 h and the cells in the cell cycle were counted. m ± 1 SD, n = 3.
Mentions: Tryptophan is converted by IDO to N-formylkynurenine; this substance is further catabolized to kynurenine and then to the terminal metabolites picolinic acid or quinolinic acid. To understand the mechanisms of the inhibitory effect of the enzyme on PBL proliferation, it is mandatory to elucidate the effects that reduction of tryptophan and/or increase of downstream catabolites in the medium have on cell proliferation. Therefore, we started to investigate the effects exerted on PBL proliferation by three tryptophan-derived catabolites: l-kynurenine, picolinic acid, and quinolinic acid. The test was performed in complete culture medium, that contains 26 μM tryptophan, and the substances were added at the beginning of the test (Fig. 4 A). At concentrations above 1 mM a toxic effect was detected for all the three substances. Below this threshold, a dose-dependent inhibition of proliferation was observed with l-kynurenine and, to a lesser extent, with picolinic acid; instead, quinolinic acid was ineffective. Upon coincubation, l-kynurenine and picolinic acid showed a clear synergistic effect (Fig. 4 B). The two substances exerted their effect by blocking cell cycle in the mid-G1 phase (Fig. 4 C).

Bottom Line: Macrophages exposed to macrophage colony-stimulating factor acquire the capacity to suppress T cell proliferation; this effect is associated with de novo expression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO).Inhibition of cell proliferation induced by the tryptophan catabolites resulting from IDO activity was selective, applying only to cells undergoing activation.We suggest that IDO exerts its effect on cell proliferation by (i) starting the cascade of biochemical reactions that produce the three catabolites and by (ii) enhancing their inhibitory potential by depriving the extracellular microenvironment of tryptophan.

View Article: PubMed Central - PubMed

Affiliation: Immunogenetics Laboratory, National Cancer Research Institute, University of Genoa, Largo Rosanna Benzi 10, 16132 Genoa, Italy. guido.frumento@istge.it

ABSTRACT
Macrophages exposed to macrophage colony-stimulating factor acquire the capacity to suppress T cell proliferation; this effect is associated with de novo expression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). We have purified IDO and tested its activity in in vitro models of T cell activation. IDO was able to inhibit proliferation of CD4(+) T lymphocytes, CD8(+) T lymphocytes, and natural killer (NK) cells; proliferation of B lymphocytes was not affected. The inhibitory role of tryptophan and of its catabolites was then tested. In the presence of tryptophan, only L-kynurenine and picolinic acid inhibit cell proliferation. In a tryptophan-free medium cell proliferation was not affected. In the absence of tryptophan inhibition induced by L-kynurenine and picolinic acid was observed at concentrations below the lowest concentration that was effective in the presence of tryptophan, and quinolinic acid acquired some inhibitory capacity. Inhibition of cell proliferation induced by the tryptophan catabolites resulting from IDO activity was selective, applying only to cells undergoing activation. Resting cells were not affected and could subsequently activate normally. We suggest that IDO exerts its effect on cell proliferation by (i) starting the cascade of biochemical reactions that produce the three catabolites and by (ii) enhancing their inhibitory potential by depriving the extracellular microenvironment of tryptophan.

Show MeSH
Related in: MedlinePlus