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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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Relationship between degree of proliferation and concentration of kynurenine in coculture supernatants. (A) PHA-activated PBLs (PHA blasts) were cultured alone, in the presence of monocytes at the ratios indicated, in the presence of MCSF-treated macrophages at the ratios indicated, or in the presence of the MCSF solution (200 U/ml) used with macrophages. Cell culture was stopped after 96 h from PHA activation, and proliferation was evaluated by measuring 3[H] thymidine incorporation. m ± 1CD, n = 3. (B) PHA-activated PBLs were cultured in the presence of MCSF-treated macrophages at ratios of 4:1 (♦), 16:1 (▴), 64:1(▪), each ratio being performed in triplicate. Supernatants were collected from cocultures 72 h after the addition of PBLs, and levels of kynurenine were measured, as described in Materials and Methods section. Cell culture was stopped after 24 more hours, and proliferation was evaluated by measuring 3[H]thymidine incorporation. The amount of kynurenine in each supernatant was related to the level of proliferation in the well. The regression line is indicated. Star indicates the mean of thymidine incorporation of three wells with PHA-activated PBLs alone.
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fig7: Relationship between degree of proliferation and concentration of kynurenine in coculture supernatants. (A) PHA-activated PBLs (PHA blasts) were cultured alone, in the presence of monocytes at the ratios indicated, in the presence of MCSF-treated macrophages at the ratios indicated, or in the presence of the MCSF solution (200 U/ml) used with macrophages. Cell culture was stopped after 96 h from PHA activation, and proliferation was evaluated by measuring 3[H] thymidine incorporation. m ± 1CD, n = 3. (B) PHA-activated PBLs were cultured in the presence of MCSF-treated macrophages at ratios of 4:1 (♦), 16:1 (▴), 64:1(▪), each ratio being performed in triplicate. Supernatants were collected from cocultures 72 h after the addition of PBLs, and levels of kynurenine were measured, as described in Materials and Methods section. Cell culture was stopped after 24 more hours, and proliferation was evaluated by measuring 3[H]thymidine incorporation. The amount of kynurenine in each supernatant was related to the level of proliferation in the well. The regression line is indicated. Star indicates the mean of thymidine incorporation of three wells with PHA-activated PBLs alone.

Mentions: The next step was aimed at comparing the effect of exogenously added l-kynurenine with the effect displayed by kynurenine produced as consequence of IDO expression in MCSF-treated macrophages upon incubation with activated PBLs. Cocultures of the two cell populations were performed, and kynurenine was measured in supernatants. Accordingly to previous data (10), different degrees of inhibition of PBL proliferation could be observed, depending on the macrophages to PBLs ratio (Fig. 7 A). Instead, untreated monocytes did not affect PBL proliferation. Afterward, the amount of kynurenine in the supernatant of cocultures at different macrophages to PBLs ratios was measured, and related to the degree of cell proliferation. A clear inverse relationship was found (Fig. 7 B). In this model substantial inhibition of proliferation was achieved with concentrations of kynurenine at 72 h above 10 μM.


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)

Relationship between degree of proliferation and concentration of kynurenine in coculture supernatants. (A) PHA-activated PBLs (PHA blasts) were cultured alone, in the presence of monocytes at the ratios indicated, in the presence of MCSF-treated macrophages at the ratios indicated, or in the presence of the MCSF solution (200 U/ml) used with macrophages. Cell culture was stopped after 96 h from PHA activation, and proliferation was evaluated by measuring 3[H] thymidine incorporation. m ± 1CD, n = 3. (B) PHA-activated PBLs were cultured in the presence of MCSF-treated macrophages at ratios of 4:1 (♦), 16:1 (▴), 64:1(▪), each ratio being performed in triplicate. Supernatants were collected from cocultures 72 h after the addition of PBLs, and levels of kynurenine were measured, as described in Materials and Methods section. Cell culture was stopped after 24 more hours, and proliferation was evaluated by measuring 3[H]thymidine incorporation. The amount of kynurenine in each supernatant was related to the level of proliferation in the well. The regression line is indicated. Star indicates the mean of thymidine incorporation of three wells with PHA-activated PBLs alone.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196046&req=5

fig7: Relationship between degree of proliferation and concentration of kynurenine in coculture supernatants. (A) PHA-activated PBLs (PHA blasts) were cultured alone, in the presence of monocytes at the ratios indicated, in the presence of MCSF-treated macrophages at the ratios indicated, or in the presence of the MCSF solution (200 U/ml) used with macrophages. Cell culture was stopped after 96 h from PHA activation, and proliferation was evaluated by measuring 3[H] thymidine incorporation. m ± 1CD, n = 3. (B) PHA-activated PBLs were cultured in the presence of MCSF-treated macrophages at ratios of 4:1 (♦), 16:1 (▴), 64:1(▪), each ratio being performed in triplicate. Supernatants were collected from cocultures 72 h after the addition of PBLs, and levels of kynurenine were measured, as described in Materials and Methods section. Cell culture was stopped after 24 more hours, and proliferation was evaluated by measuring 3[H]thymidine incorporation. The amount of kynurenine in each supernatant was related to the level of proliferation in the well. The regression line is indicated. Star indicates the mean of thymidine incorporation of three wells with PHA-activated PBLs alone.
Mentions: The next step was aimed at comparing the effect of exogenously added l-kynurenine with the effect displayed by kynurenine produced as consequence of IDO expression in MCSF-treated macrophages upon incubation with activated PBLs. Cocultures of the two cell populations were performed, and kynurenine was measured in supernatants. Accordingly to previous data (10), different degrees of inhibition of PBL proliferation could be observed, depending on the macrophages to PBLs ratio (Fig. 7 A). Instead, untreated monocytes did not affect PBL proliferation. Afterward, the amount of kynurenine in the supernatant of cocultures at different macrophages to PBLs ratios was measured, and related to the degree of cell proliferation. A clear inverse relationship was found (Fig. 7 B). In this model substantial inhibition of proliferation was achieved with concentrations of kynurenine at 72 h above 10 μM.

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