In this study, we have investigated the mechanisms of IDO-dependent inhibition of PBL proliferation. For this purpose we have set up a new protocol for IDO purification. Our approach allows the same recovery, but is both easier and faster than the ones described previously (7
Afterward, we tested the capacity of purified IDO to inhibit PBL proliferation in in vitro models. We have used PHA as mitogen, instead of anti-CD3 (6
), to avoid any possible interference via Fc receptors, that are highly expressed on macrophage surface (15
). The other model we used was based on alloreactive T cell lines. The two models gave equivalent results, but for brevity only results with PHA-activated PBLs are shown here.
In detail, we found that purified IDO at a concentration of 4,000 U/ml was able to reduce proliferation of PBLs to the half, when administered at the beginning of the test. However, because of the short half-life of the enzyme at 37°C in culture medium (unpublished data and reference 16
), purified IDO was more effective when administered repeatedly than in a single dose. Better results were obtained by coincubating the enzyme with methylene blue and l
-ascorbic acid: these cofactors optimize the efficiency of the enzyme, possibly by increasing production of O2−
). It is possible that cofactors exist in macrophage cytosol that perform the same function of the two above mentioned substances. We got a confirmation that the inhibition of PBL proliferation was due to the enzymatic activity of IDO by demonstrating that 1-methyl-DL-tryptophan, a competitive inhibitor of IDO activity (14
), was able to almost completely prevent the inhibition of proliferation induced by the enzyme.
In agreement with previous studies (6
), indicating that macrophage-dependent inhibition of T cell proliferation was related to a sustained arrest of the cell cycle, we found that also IDO treatment blocks cells in the mid-G1 phase.
These data, taken together, suggest that macrophage-dependent inhibition of PBL proliferation can result from the sole IDO enzymatic activity, and other antiproliferative factors released by macrophages might be not required.
We also found that, among the different subpopulations making up PBLs, only CD4+
T lymphocytes and NK cells were sensitive to IDO, proliferation of B lymphocytes not being affected. This finding, together with previous data indicating that proliferation of fibroblasts and of vascular endothelial cells is not affected by the presence of macrophages (10
), rules out the possibility that IDO might act as a nonspecific inhibitor for proliferating cells.
Instead, inhibition of cell proliferation induced by the tryptophan catabolites resulting from IDO activity is selective, applying to specific leukocyte subpopulations and only to cells undergoing activation. Resting cells are not affected and can subsequently activate normally.
Previous data show that both macrophages (6
) and dendritic cells (17
) can inhibit T cell proliferation via IDO-dependent mechanisms. Our results confirm the immunological role played by the enzyme.
In this regard, it has been hypothesized that IDO expression by APCs helps maintain peripheral tolerance by regulating autoreactive T cells that have escaped intrathymic deletion (6
). It has also been suggested that IDO production by APCs could be part of a mechanism self-limiting immune response (17
). As a matter of fact, IDO expression is induced in APCs by signals, namely IFN-γ and CD40 ligand, produced by T cells undergoing activation. Therefore, IDO might be part of a negative feedback loop.
To gain insights into the mechanisms of IDO-dependent inhibition of T and NK cell proliferation, we analyzed the effects of changes in the concentration of tryptophan and its catabolites in the extracellular microenvironment. We found that in the absence of tryptophan substantial levels of proliferation were retained. It is conceivable that cells could get the tryptophan that is required for proliferation by catabolizing proteins contained in the medium. The slight reduction in cell proliferation that was observed under these conditions could not be related to the lack of tryptophan, since addition of l-tryptophan did not increase proliferation. Instead, we think that part of factors sustaining proliferation, that are normally contained in FCS, were probably lost during the dialysis performed to remove unbound tryptophan.
The three tryptophan-derived catabolites we tested were found to be able, at various degree and under different conditions, to inhibit cell proliferation. In the presence of tryptophan, inhibition of proliferation was observed with l-kynurenine and, to a lesser extent, with picolinic acid; instead, quinolinic acid was ineffective. When the same test was performed in the absence of thryptophan, all the three catabolites were effective, at various degrees, in inducing a dose-dependent inhibition of proliferation. In the case of l-kynurenine and, to a lesser extent, of picolinic acid, inhibition was observed at concentrations below the lowest concentration that was effective in the presence of tryptophan, while in the case of quinolinic acid the inhibitory capacity was acquired de novo.
Confirming their role as major cause of IDO effect, tryptophan-derived catabolites were found to exert their inhibitory effect by blocking the cell cycle progression.
It has to be noted that at concentrations of l
-kynurenine up to 500 μM the inhibition of PBL proliferation occurs only when the substance is added within 36 h from cell activation and inhibition can be overcome by removing the substance from the medium within the same period. This indicates that the target mechanisms of l
-kynurenine-dependent inhibition are operative only at the early stages of cell activation. This finding is in agreement with data from (10
), indicating that macrophage-dependent suppression of T cell proliferation specifically affects the initial transition from quiescence to proliferation.
Some considerations can also be done on the results of the attempt to compare the effect of exogenously added l-kynurenine with the effect displayed by kynurenine resulting from IDO expression by MCSF-treated macrophages in the presence of activated PBLs. Cocultures were performed, and kynurenine levels in the supernatants were related to the degree of inhibition observed. Confirming the role of kynurenine in IDO-dependent inhibition of cell proliferation, the amount of kynurenine in the supernatant was inversely related to the degree of cell proliferation ( B). We are well aware of the fact that we do not know the kinetics of release and consumption of kynurenine in cocultures of MCSF-treated macrophages and activated PBLs, therefore a precise comparison of the activity of kynurenine in the two systems cannot be done. However, it is noteworthy that the range of concentration of kynurenine that in this system is related with reduced cell proliferation is close to the range of concentration of exogenous l-kynurenine that is active in tryptophan-free medium in the absence of MCSF-treated macrophages. As a matter of fact, we found that in cocultures of MCSF-treated macrophages and activated PBLs, proliferation >50% was achieved with concentrations of l-kynurenine at 72 h below 10 μM. A degree of proliferation in the range of 50% of control was achieved in tryptophan-free medium in the absence of MCSF-treated macrophages by exogenously adding l-kynurenine at concentrations between 200 and 40 μM at the beginning of the test. Since l-kynurenine is quickly degraded in the supernatant of PBL cultures, it is conceivable that the concentration that is effective during the test is well below the concentration at the beginning of the test.
We have to take into account also the probable occurrence of synergic effect of different tryptophan-derived catabolites, as in the case of l-kynurenine and picolinic acid.
These considerations prompt us to suggest that l-kynurenine and, possibly, also other tryptophan-derived catabolites are the substances responsible for the inhibitory effect displayed by the enzyme on proliferation of IDO-sensitive cells. Our data indicate in tryptophan-derived catabolites a group of substances endowed with immunosuppressive activity.
These conclusions are in contrast with previous findings from D.H. Munn and coworkers, indicating that the inhibitory effect exerted by IDO on T cell proliferation can be due to the sole tryptophan starvation (6
). The authors reach their conclusions on the basis of the fact that conditioned medium from cocultures of MCSF-treated macrophages and activated T cells was unable to support T cell proliferation unless tryptophan was added. However, in the light of our results, the data displayed by Munn and coworkers do not rule out the possibility of tryptophan-derived immunosuppressive catabolites. As a matter of fact, we have found kynurenine in the supernatant of cocultures performed as described by Munn and coworkers. Therefore, we suggest that this substance, and presumably other tryptophan-derived catabolites in the conditioned medium, was responsible of the inhibitory effect on T cell proliferation displayed by conditioned medium in (6
) Also, the capacity of the same conditioned medium to support T cell proliferation after the addition of tryptophan could be explained on the basis of our results. To exert the same inhibitory effect, l
-kynurenine has to be one order of magnitude more concentrate in the presence of tryptophan than in the absence of the amino acid. As it regards the experiment described in (6
), we suggest that after tryptophan was added the concentration of kynurenine in the conditioned medium was not able anymore to inhibit T cell proliferation.
Instead, we speculate that IDO expressed by macrophages upon interaction with activated T cells exerts its effect on T and NK cell proliferation via two different mechanisms. On one side it starts the cascade of biochemical reactions that leads to the production of tryptophan-derived catabolites, themselves endowed with the capacity to inhibit cell proliferation. On the other side it depletes the medium of tryptophan, allowing at least three catabolites to put in action their inhibitory potential at concentrations that probably can be achieved in the extracellular microenvironment.