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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 concentration on tryptophan catabolites-dependent inhibition of T cell proliferation. (A) Increasing concentrations of l-tryptophan were added to PBLs activated and cultured in a medium devoid of tryptophan. Cell culture was stopped after 96 h from PHA activation, and proliferation was evaluated by measuring 3[H]thymidine incorporation. (B–D) Increasing concentrations of l-kynurenine (B), picolinic acid (C), and quinolinic acid (D) were added to PBLs activated and cultured in tryptophan-free medium (▪——▪), or in the tryptophan-free medium supplemented with 26 μM l-tryptophan (▴——▴). Controls were represented by untreated PHA-activated PBLs grown in tryptophan-free medium and in the same medium supplemented with l-tryptophan, respectively. Cell culture was stopped after 96 h from PHA activation, and proliferation was evaluated by measuring 3[H]thymidine incorporation. To permit comparison, proliferation has been normalized to the control cultures (PHA-activated PBLs grown in tryptophan-free medium: 19,134 ± 1,109 cpm; PHA-activated PBLs grown in tryptophan-free medium supplemented with l-tryptophan: 20,021 ± 1,776 cpm). m ± 1 SD, n = 3.
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fig6: Effect of tryptophan concentration on tryptophan catabolites-dependent inhibition of T cell proliferation. (A) Increasing concentrations of l-tryptophan were added to PBLs activated and cultured in a medium devoid of tryptophan. Cell culture was stopped after 96 h from PHA activation, and proliferation was evaluated by measuring 3[H]thymidine incorporation. (B–D) Increasing concentrations of l-kynurenine (B), picolinic acid (C), and quinolinic acid (D) were added to PBLs activated and cultured in tryptophan-free medium (▪——▪), or in the tryptophan-free medium supplemented with 26 μM l-tryptophan (▴——▴). Controls were represented by untreated PHA-activated PBLs grown in tryptophan-free medium and in the same medium supplemented with l-tryptophan, respectively. Cell culture was stopped after 96 h from PHA activation, and proliferation was evaluated by measuring 3[H]thymidine incorporation. To permit comparison, proliferation has been normalized to the control cultures (PHA-activated PBLs grown in tryptophan-free medium: 19,134 ± 1,109 cpm; PHA-activated PBLs grown in tryptophan-free medium supplemented with l-tryptophan: 20,021 ± 1,776 cpm). m ± 1 SD, n = 3.

Mentions: To evaluate the relevance of the tryptophan levels in the extracellular environment, we used a culture medium devoid of tryptophan. As shown in Fig. 6 A, PHA-activated PBLs retained the capacity to proliferate in this medium. Furthermore, proliferation was not increased by adding increasing concentrations of l-tryptophan at the beginning of the test. In particular, no increase in proliferation was observed after adding 30 μM l-tryptophan, that is above the concentration of tryptophan in the normal culture medium. Afterward, the degree of inhibition of PBL proliferation was evaluated for each of the three catabolites both in tryptophan-free medium and in tryptophan-free medium supplemented with 26 μM l-tryptophan, that is the concentration of tryptophan in normal complete medium (Fig. 6, B–D). In the absence of tryptophan, l-kynurenine and, to a lesser extent, picolinic acid were endowed with inhibitory activity at concentrations that were below the concentrations that inhibited PBL proliferation in the presence of the amino acid (Fig. 6, B and C). Quinolinic acid, that was ineffective in the presence of l-tryptophan, in the absence of tryptophan showed a dose–response relationship for inhibition of proliferation (Fig. 6 D).


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 concentration on tryptophan catabolites-dependent inhibition of T cell proliferation. (A) Increasing concentrations of l-tryptophan were added to PBLs activated and cultured in a medium devoid of tryptophan. Cell culture was stopped after 96 h from PHA activation, and proliferation was evaluated by measuring 3[H]thymidine incorporation. (B–D) Increasing concentrations of l-kynurenine (B), picolinic acid (C), and quinolinic acid (D) were added to PBLs activated and cultured in tryptophan-free medium (▪——▪), or in the tryptophan-free medium supplemented with 26 μM l-tryptophan (▴——▴). Controls were represented by untreated PHA-activated PBLs grown in tryptophan-free medium and in the same medium supplemented with l-tryptophan, respectively. Cell culture was stopped after 96 h from PHA activation, and proliferation was evaluated by measuring 3[H]thymidine incorporation. To permit comparison, proliferation has been normalized to the control cultures (PHA-activated PBLs grown in tryptophan-free medium: 19,134 ± 1,109 cpm; PHA-activated PBLs grown in tryptophan-free medium supplemented with l-tryptophan: 20,021 ± 1,776 cpm). m ± 1 SD, n = 3.
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fig6: Effect of tryptophan concentration on tryptophan catabolites-dependent inhibition of T cell proliferation. (A) Increasing concentrations of l-tryptophan were added to PBLs activated and cultured in a medium devoid of tryptophan. Cell culture was stopped after 96 h from PHA activation, and proliferation was evaluated by measuring 3[H]thymidine incorporation. (B–D) Increasing concentrations of l-kynurenine (B), picolinic acid (C), and quinolinic acid (D) were added to PBLs activated and cultured in tryptophan-free medium (▪——▪), or in the tryptophan-free medium supplemented with 26 μM l-tryptophan (▴——▴). Controls were represented by untreated PHA-activated PBLs grown in tryptophan-free medium and in the same medium supplemented with l-tryptophan, respectively. Cell culture was stopped after 96 h from PHA activation, and proliferation was evaluated by measuring 3[H]thymidine incorporation. To permit comparison, proliferation has been normalized to the control cultures (PHA-activated PBLs grown in tryptophan-free medium: 19,134 ± 1,109 cpm; PHA-activated PBLs grown in tryptophan-free medium supplemented with l-tryptophan: 20,021 ± 1,776 cpm). m ± 1 SD, n = 3.
Mentions: To evaluate the relevance of the tryptophan levels in the extracellular environment, we used a culture medium devoid of tryptophan. As shown in Fig. 6 A, PHA-activated PBLs retained the capacity to proliferate in this medium. Furthermore, proliferation was not increased by adding increasing concentrations of l-tryptophan at the beginning of the test. In particular, no increase in proliferation was observed after adding 30 μM l-tryptophan, that is above the concentration of tryptophan in the normal culture medium. Afterward, the degree of inhibition of PBL proliferation was evaluated for each of the three catabolites both in tryptophan-free medium and in tryptophan-free medium supplemented with 26 μM l-tryptophan, that is the concentration of tryptophan in normal complete medium (Fig. 6, B–D). In the absence of tryptophan, l-kynurenine and, to a lesser extent, picolinic acid were endowed with inhibitory activity at concentrations that were below the concentrations that inhibited PBL proliferation in the presence of the amino acid (Fig. 6, B and C). Quinolinic acid, that was ineffective in the presence of l-tryptophan, in the absence of tryptophan showed a dose–response relationship for inhibition of proliferation (Fig. 6 D).

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