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BOR - Papers in Press, published online ahead of print November 30, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.042333
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BIOLOGY OF REPRODUCTION 74, 552–559 (2006)
DOI: 10.1095/biolreprod.105.042333
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Indoleamine 2,3-Dioxygenase Participates in the Interferon-gamma-Induced Cell Death Process in Cultured Bovine Luteal Cells1

Matthew J. Cannon , and Joy L. Pate 2 

Department of Animal Sciences, The Ohio State University/Ohio Agricultural Research and Development Center, Wooster, Ohio 44691

ABSTRACT

Interferon-gamma (IFNG) induces apoptotic cell death in bovine luteal cells, but the pathway(s) involved in this process are not well defined. Evidence supporting the involvement of an IFNG-inducible enzymatic pathway that degrades tryptophan in IFNG-induced death of bovine luteal cells is presented in this study. The IFNG-inducible enzyme indoleamine 2,3-dioxygenase (INDO) catalyzes the first step in a metabolic pathway that degrades tryptophan. In the first experiment, RT-PCR revealed the presence of INDO mRNA in luteal cells treated with IFNG, but not in untreated cells. To determine whether INDO participates in IFNG-induced death of bovine luteal cells, an experiment was performed to test the effect of 1-methyl-D-tryptophan (1-MT), an inhibitor of INDO, on IFNG-induced DNA fragmentation in luteal cells. Single-cell gel electrophoresis and microscopic image analysis revealed that 1-MT inhibited DNA fragmentation induced by IFNG. To determine whether supplementation of cell cultures with additional tryptophan could also protect luteal cells from IFNG-induced DNA fragmentation, luteal cells were cultured in the presence of IFNG, and L-tryptophan was added to cultures to achieve final concentrations that were 5-, 10-, or 25-fold higher than the concentration of L-tryptophan found in nonsupplemented culture medium. Supplementation of IFNG-treated luteal cell cultures with elevated concentrations of tryptophan also prevented IFNG-induced DNA fragmentation. We conclude that INDO participates in IFNG-induced death of bovine luteal cells, through a mechanism that involves degradation of tryptophan, thereby reducing tryptophan concentrations to a point insufficient to meet luteal cells needs.

apoptosis, corpus luteum, cytokines, ovary

INTRODUCTION

The inflammatory cytokine interferon gamma (IFNG) has been implicated as a mediator of luteal regression. The presence of mRNA encoding IFNG has been demonstrated in the corpus luteum (CL) of several species, including the cow [1–5], and an inhibitory effect of IFNG on progesterone production by cultured luteal cells has been demonstrated [6–11]. In addition, multiple studies have described the detrimental effect of IFNG on viability of cultured luteal cells [6, 7, 11–16]. Fairchild and Pate (1991) first demonstrated that treatment of cultured bovine luteal cells with IFNG causes a reduction in the number of cells present in cultures [6]. Subsequently, these authors demonstrated that the proinflammatory cytokine tumor necrosis factor alpha (TNF), which is also believed to be involved in modulation of luteal function (reviewed in [17, 18]), enhances the cytotoxic effect of IFNG on luteal cells [7], an observation later confirmed by others [12–15]. However, studies conducted to determine the mechanism(s) by which IFNG mediates cytotoxic actions in luteal cells have yielded inconclusive results. Induction of luteal cell death by TNF and IFNG occurs in a manner independent of nitric oxide production, because the nitric oxide synthase inhibitor NG-monomethyl-L-arginine did not inhibit death of murine or bovine luteal cells induced by treatment with these cytokines in combination [13, 14]. Despite the ability of IFNG to increase prostaglandin synthesis in cultured bovine luteal cells [6], the cyclooxygenase inhibitor indomethacin was without effect on IFNG-induced luteal cell death [6, 14]. Moreover, inhibitors of phospholipase A2 and lipoxygenase, as well as the antioxidant vitamin C, displayed only negligible (if any) ability to protect bovine luteal cells from death induced by the combination of these cytokines [14]. However, the combination of superoxide dismutase and catalase in high concentrations, or recombinant bovine interferon-alpha, was able to partially inhibit bovine luteal cell death induced by IFNG and TNF [14]. How these agents prevent cytokine-induced luteal cell death is not known.

Indoleamine 2,3-dioxygenase (INDO) is an IFNG-inducible enzyme that catalyzes the degradation of tryptophan to N-formyl-L-kynurenine, the first step in degradation of tryptophan via the kynurenine pathway [19]. The involvement of tryptophan degradation in the antiproliferative and cytotoxic effects of IFNG has been demonstrated [20–23], and some mutant cell types that do not express INDO in response to IFNG are also resistant to IFNG-mediated cytotoxicity [20, 24, 25]. The induction of gene expression by IFNG is mediated via the janus activated kinases and the signal transducers and activators of transcription (STATs). Upon activation, STAT proteins translocate to the nucleus, where they induce transcription of IFNG-responsive genes [26]. Treatment of bovine luteal cells with IFNG causes elevation in nuclear amounts of STAT-1 and phosphorylated STAT-3 [11], as well as induction of cell surface class II MHC molecules [27], the latter being a well-documented effect of IFNG [28, 29]. Because IFNG regulates expression of INDO in other cell types, we hypothesized that IFNG treatment would induce expression of INDO in luteal cells, and that expression of INDO might participate in the cytotoxic effects of IFNG. The objectives of the present study were to determine whether IFNG causes an increase in steady-state amounts of INDO mRNA in bovine luteal cells, and to determine whether and how INDO participates in IFNG-induced bovine luteal cell death.

MATERIALS AND METHODS

Reagents

Powdered Ham's F-12 culture medium, gentamicin, calf serum, E. coli DH5{alpha} chemically competent cells, and TRIzol Reagent were all purchased from Life Technologies. The National Hormone and Pituitary Program provided bovine LH (NIAMMD-bLH-4). Insulin-transferrin-selenium (ITS) premix was obtained from BD Biosciences. Recombinant murine TNF was purchased from Life Technologies and R & D Systems. Recombinant bovine IFNG was provided by Ciba or was purchased from Serotec. L-tryptophan and 1-methyl-D-tryptophan were purchased from Aldrich Chemical Company. Tissue culture flasks and 24-well plates were from Corning. Type I collagenase was acquired from Worthington Biochemical Corp. Restriction enzymes, recombinant RNase inhibitor, Moloney murine leukemia virus reverse transcriptase, and TaqBead Hot Start Polymerase were purchased from Promega. All other chemicals were purchased from Sigma Chemical Co. or Fisher Scientific.

Animals

Corpora lutea were collected from normally cycling, multiparous, lactating dairy cows between 3 and 6 yr of age. Cows were housed indoors, had complete freedom of movement, and were fed a total mixed ration ad libitum. Corpora lutea were removed transvaginally on Day 10 or 11 following behavioral estrus, immediately placed in ice-cold Ham's F-12 culture medium, and transported to the laboratory for dissociation. Handling of animals and surgical procedures were carried out according to protocols approved by the Institutional Laboratory Animal Care and Use Committee of The Ohio State University.

Luteal Cell Dissociation and Culture

Dissociation of luteal cells was carried out according to procedures described previously [30]. Following dissociation, cells were resuspended in cell culture medium (Ham's F-12 medium containing 5 µg/ml insulin, 5 µg/ml transferrin, 5 ng/ml selenium, 20 µg/ml gentamicin, and 10 ng/ml LH) and cell viability was determined via Trypan Blue exclusion. The number of viable cells was counted using a hemacytometer. All cultures were carried out in a humidified atmosphere of 5% CO2 in air at 37°C. All experiments were repeated a total of four times using CL from different cows.

Experiment 1: Induction of INDO mRNA by IFNG

Dispersed luteal cells were placed in culture in 25-cm2 flasks (5 x 106 cells/flask) in a total of 5 ml of culture medium. Cells were allowed to adhere overnight, at which time medium was replaced and treatments were added. Treatments were arranged in a 2 x 4 factorial design, and all treatments were carried out in duplicate. Cells were treated with either 0 or 50 ng/ml murine TNF and 0, 10, 100, or 1000 international units (IU)/ml IFNG. It has been demonstrated previously that these concentrations of TNF and IFNG exert significant effects on bovine luteal cell viability and function in vitro [6, 7, 14]. Total RNA was extracted from cultured cells 48 h after initiation of treatments, because a previous study demonstrated a significant effect of TNF on steady-state concentrations of mRNA encoding IFNG-regulated genes in cultured bovine luteal cells at this time [31].

Steady-state concentrations of INDO mRNA were determined using semiquantitative RT-PCR (SQ-RT-PCR). SQ-RT-PCR was performed using a GeneAmp PCR System 9700 thermal cycler (Perkin Elmer). INDO primers were designed based on regions of highest homology between published human, mouse, and rat sequences. Sequences of forward and reverse primers were 5'-ATCACCATGGCGGATGTGTG-3' and 5'- CTGGAAGATGTTGCTCTGGG-3', respectively. Amplification using these primers resulted in a single 559-bp cDNA fragment, which was the size expected for the amplified INDO cDNA product. Amplification products were cloned and sequenced as described previously [32] to determine the identity of the cDNA inserts. The resulting partial bovine INDO cDNA sequence shares 81%, 79%, and 78% sequence identity, respectively, with homologous regions of the human, mouse, and rat INDO sequences. An 854-bp glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA fragment was amplified in parallel reactions carried out in separate tubes, using ovine-specific primers described previously [33], and was used as an external control as has been described previously [31]. The number of cycles in which the reactions entered the logarithmic phase of amplification was determined individually for each primer set. The number of cycles to be used for each primer set was determined by performing serial reactions with a pooled sample of RNA from IFNG-treated cells, incrementally increasing the number of cycles, and determining empirically the cycle number at which amplification was in the logarithmic phase. In these experiments, GAPDH amplification was in the logarithmic phase at 24 cycles, whereas INDO amplification was logarithmic at 31 cycles. Reverse transcription was performed on 200 ng of total RNA, followed by either 25 cycles (GAPDH) or 31 cycles (INDO) of PCR using the following conditions: denaturation, 94°C for 30 sec; annealing, 58°C for 30 sec; extension, 72°C for 60 sec. Following amplification, PCR products were separated on 1.5% agarose gels and visualized with ethidium bromide. The densitometric value (in arbitrary densitometric units) of the INDO band from each sample was standardized to the value of the corresponding GAPDH band.

Experiment 2: Effect of INDO Inhibition on IFNG-Induced DNA Fragmentation

Dispersed luteal cells were placed in culture in 24-well plates (2.5 x 105 cells/well) in a total of 1 ml of culture medium containing 10% calf serum. Cells were allowed to adhere overnight, at which time serum-containing medium was replaced with serum-free medium, and treatments were initiated. Treatments were arranged in a 2 x 2 factorial design, and all treatments were carried out in duplicate. Luteal cells were treated with 100 IU/ml IFNG in the absence or presence of 25 µg/ml of the INDO inhibitor 1-methyl-D-tryptophan. This compound has previously been shown to ameliorate various effects of tryptophan catabolism by the INDO enzyme [34–36]. Cells were cultured for 96 h following initiation of treatments, with replacement of medium and treatments after 48 h.

To determine the amount of DNA fragmentation in individual cell nuclei following culture, single-cell gel electrophoresis was performed as has been previously described [14, 37]. Briefly, cells were removed from the culture wells using a solution of 0.05% trypsin/0.53% EDTA, suspended in 1% (w/v) low-melting-temperature agarose, and layered onto slides. Slides were then submerged in lysis buffer (1% [v/v] Triton X-100, 2.5 M NaCl, 100 mM EDTA, 10 mM Tris, 10% [v/v] DMSO, pH 10), and subsequently placed in a horizontal electrophoresis box containing electrophoresis buffer (1 mM EDTA, 300 mM NaOH). Cells were allowed to equilibrate for 40 min in electrophoresis buffer, and electrophoresis was performed at 22 V/350 mA for 24 min. Following electrophoresis, cells were fixed with neutralization buffer (0.4 M Tris, pH 7.5), and stained with 20 µg/ml ethidium bromide in water.

Microscopic image analysis was conducted using an Olympus MagnaFire digital camera coupled to an Olympus BX51 fluorescence microscope (Olympus America) and Image-Pro Plus software, version 4.5 (Media Cybernetics). Quantification of DNA fragmentation was performed by measuring the percentage of DNA remaining in each individual nucleus following single-cell gel electrophoresis. Micrographs of 20 randomly selected individual cell nuclei per treatment were taken at 400x magnification, and were subjected to quantitative image analysis as described previously [14]. In brief, to determine the percentage of DNA in the head of each individual comet, a thick band profile measuring total pixel intensity of the entire comet was taken. The amount of DNA in the comet head was then determined by encircling the head and excluding all regions outside of the head from the thick band measurement. This number was pixel intensity of the comet head, and was divided by the total pixel intensity to give the percentage of DNA remaining in the comet head (cell nucleus). This allows accurate determination of the extent of DNA fragmentation in individual cell nuclei, and also allows the number of cells with a specific amount of DNA fragmentation to be determined for each treatment.

Experiment 3: Effect of Tryptophan Supplementation on IFNG-Induced DNA Fragmentation

Dispersed luteal cells were cultured as described for Experiment 2. Treatments were arranged in a 2 x 4 factorial design, and all treatments were carried out in duplicate. Luteal cells were cultured in medium to which no supplemental tryptophan had been added, or in medium to which L-tryptophan had been added to achieve a final concentration that was 5-, 10-, or 25-fold greater than that found in nonsupplemented medium. Cells cultured in nonsupplemented and supplemented medium were treated with 0 or 100 IU/ml IFNG. Cells were cultured for 96 h following initiation of treatments, with replacement of medium and treatments after 48 h. The experiment was repeated a total of four times. At the end of the culture period, cells were subjected to single-cell gel electrophoresis and microscopic image analysis as described for Experiment 2.

Statistical Analysis

All statistical analyses were performed using Sigma Stat software (Jandel Corporation). One-way ANOVA followed by the Student-Newman-Keuls test was performed on densitometric values of INDO bands (standardized to corresponding GAPDH bands) to determine differences in steady-state concentrations of INDO mRNA between specific treatment means. Cells subjected to single-cell gel electrophoresis were assigned to one of four arbitrarily-determined categories based on the percentage of DNA remaining in the nucleus (80%–100%, 60%–79%, 40%–59%, or 20%–39%). Differences between treatments in the number of cells in each percentage group were determined by one-way ANOVA followed by the Student-Newman-Keuls test. All differences were considered statistically significant at P < 0.05.

RESULTS

Induction of INDO mRNA by IFNG

SQ-RT-PCR was used to determine relative steady-state concentrations of INDO mRNA in luteal cells treated with IFNG. INDO mRNA was not detectable in luteal cells in the absence of IFNG (Fig. 1A). IFNG induced expression of INDO in luteal cells, with greater (P < 0.05) concentrations of INDO mRNA present in cultures treated with 100 or 1000 IU/ml compared with 10 IU/ml IFNG (Fig. 1, A and B). Tumor necrosis factor-{alpha} had no effect on steady-state concentrations of INDO mRNA (Fig. 1, A and B).


Figure 1
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  		FIG. 1. A) Representative gel showing amplification products of RT-PCR using INDO-specific primers (top panel) and GAPDH-specific primers (bottom panel) in cultured luteal cell RNA. Cultures received either 0, 10, 100, or 1000 IIU/ml (IU/ml) IFNG in the absence or presence of 50ng/ml TNF. B) Effect of IFNG, in the absence or presence of TNF, on steady-state concentrations of INDO mRNA in cultured bovine luteal cells. Bars represent relative concentrations of INDO mRNA (mean ± SEM, expressed in arbitrary units), standardized to GAPDH mRNA. Different letters denote significant differences (P < 0.05; n = 4)

Effect of INDO Inhibition on IFNG-Induced DNA Fragmentation

Single-cell gel electrophoresis, also known as the comet assay, was used to measure the effect of INDO inhibition on IFNG-induced DNA fragmentation. In the present study, nuclei from control cells, in which little to no DNA fragmentation would be expected, retained greater than 85% of the DNA in the nucleus (head of the comet), whereas nuclei from IFNG-treated cells retained as little as 25% of the DNA. To determine whether INDO participates in IFNG-induced luteal cell death, an experiment was performed to test the effect of 1-methyl-D-tryptophan (1-MT), an inhibitor of INDO, on IFNG-induced DNA fragmentation in luteal cells. Single-cell gel electrophoresis and microscopic image analysis revealed that the majority of cells cultured in the absence of IFNG (control cells) had 80%–100% of the DNA remaining in the nucleus, with some cells having 60%–79% of the DNA remaining in the nucleus (Fig. 2a). The somewhat lower percentage of DNA in the nuclei of a few control cells is attributable to the extensive processing procedure used before fixation of cells in the assay. Treatment with 100 IU/ml IFNG, shown in the first experiment to induce maximum steady-state concentrations of INDO mRNA, induced significant DNA fragmentation (Fig. 2, A vs. B), lessening (P < 0.05) the number of cells with 80%–100% of the DNA in the nucleus when compared with controls, and also resulting in the appearance of cells with only 20%–39% or 40%–59% of the DNA remaining in the nucleus (Fig. 3). The INDO inhibitor 1-MT dramatically inhibited this effect (Fig. 2, D vs. B). Similarly to controls, cells treated with IFNG and 1-MT retained 80%–100% of the DNA in the nucleus following single-cell gel electrophoresis. Moreover, 1-MT completely inhibited the ability of IFNG to induce the presence of cells with 20%–39% or 40%–59% of the DNA remaining in the nucleus (Fig. 3).


Figure 2
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  		FIG. 2. Representative fluorescent micrographs displaying the results of single-cell gel electrophoresis of A) control cells, or cells cultured in the presence of B) 100 IU/ml IFNG, C) 25 µg/ml 1-MT, or D) 100 IU/ml IFNG + 25 µg/ml 1-MT. Original magnification x400


Figure 3
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  		FIG. 3. DNA fragmentation, as determined by single-cell gel electrophoresis and microscopic image analysis, in luteal cells cultured in the presence or absence of 100 IU/ml IFNG and/or 25 µg/ml of the INDO inhibitor 1-MT. Data are grouped arbitrarily according to the measurable percentage of DNA remaining in the nucleus (head region of each comet; X-axis). Bars represent the number of cells (mean ± SEM) in each percentage category per 20 cells measured for each treatment (frequency; Y-axis). Different letters indicate significant differences (P < 0.05; n = 4 cultures). Asterisks (*) denote the presence of cells from one treatment in a category in which no other treatment resulted in cells in that category

Effect of Tryptophan Supplementation on IFNG-Induced DNA Fragmentation

To determine if addition of supplemental tryptophan could also prevent IFNG-induced DNA fragmentation in luteal cells, L-tryptophan was added to luteal cell cultures to achieve 5-, 10-, or 25-fold greater concentrations of L-tryptophan than nonsupplemented medium. The majority of cells cultured in medium without additional tryptophan (control cells), and the majority of cells cultured in medium with 5-, 10-, or 25-fold greater concentrations of L-tryptophan, retained 80%–100% of the DNA in the nucleus, with the remaining cells having 60%–79% of the DNA remaining in the nucleus, following single-cell gel electrophoresis (Fig. 4, C, E, and G). As in the previous experiment, IFNG induced significant DNA fragmentation (Fig. 4, B vs. A). However, addition of L-tryptophan at all concentrations inhibited IFNG-induced DNA fragmentation (Fig. 4, D, F, and H vs. B). Supplementation of IFNG-treated cultures with 5-fold greater concentrations of tryptophan increased (P < 0.05) the number of cells with 80%–100% of the DNA remaining in the nucleus, reduced (P < 0.05) the number of cells with 40%–59% of the DNA remaining in the nucleus, and prevented the appearance of cells with only 20%–39% of the DNA remaining in the nucleus (Fig. 5). Compared with IFNG-treated cells cultured in the absence of supplemental tryptophan, addition of 10- or 25-fold greater concentrations of tryptophan to IFNG-treated cultures increased (P < 0.05) the number of cells with 80%–100% of the DNA remaining in the nucleus, reduced (P < 0.05) the number of cells with 60%–79% of the DNA remaining in the nucleus, and prevented the appearance of cells with only 40%–59% and 20%–39% of the DNA remaining in the nucleus (Fig. 5). There were no differences in DNA fragmentation between control cells and IFNG-treated cells cultured in the presence of 10- and 25-fold greater concentrations of tryptophan (Fig. 4, A, F, and H).


Figure 4
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  		FIG. 4. Representative fluorescent micrographs displaying the results of single-cell gel electrophoresis of cells cultured in the absence (A, C, E, and G) or presence (B, D, F, and H) of 100 IU/ml IFNG without supplemental tryptophan (A and B) or supplemented with 5- (C and D), 10- (E and F), or 25-fold (G and H) greater concentrations of L-tryptophan. Original magnification x400


Figure 5
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  		FIG. 5. DNA fragmentation, as determined by single-cell gel electrophoresis and microscopic image analysis, in luteal cells treated with 100 IU/ml IFNG in the absence of supplemental tryptophan, or in the presence of 5-, 10-, or 25-fold greater concentrations of supplemental tryptophan. Data are grouped arbitrarily according to the measurable percentage of DNA remaining in the nucleus (head region of each comet; X-axis). Bars represent the number of cells (mean ± SEM) in each percentage category per 20 cells measured for each treatment (frequency, Y-axis). Different letters indicate significant differences (P < 0.05, n = 4 cultures). Asterisks (*) denote the presence of cells from one treatment in a category in which no other treatment resulted in cells in that category

DISCUSSION

In the present study, we have demonstrated that IFNG induces expression of the INDO gene in bovine luteal cells, and that expression of INDO plays a significant role in IFNG-mediated death of bovine luteal cells. To the best of our knowledge, this is the first description of INDO expression by ovarian cells in any species. The presence of detectable amounts of INDO mRNA was induced by the lowest concentration of IFNG used in this study (10 IU/ml), and steady-state INDO mRNA concentrations were maximal when cells were treated with 100 IU/ml IFNG. We have also attempted to identify immunoreactive INDO protein in IFNG-treated luteal cells, without success. We have found that commercially available antibodies to human and murine INDO do not cross-react with the homologous bovine protein in a Western blot assay (data not shown), most likely because of the limited degree of homology (82% and 83% sequence identity between the bovine protein and homologous regions of the murine and human proteins, respectively). It is unfortunate that we were unable to demonstrate the presence of the protein using this technique, and this is one acknowledged limitation of the present study. It is of interest to note that TNF, which enhances other effects of IFNG on luteal cells [7, 12–15], did not stimulate further increases of INDO mRNA above that achieved with IFNG alone. Given that INDO is an IFNG-inducible enzyme, and that TNF was without effect on steady-state INDO mRNA concentrations, the latter cytokine was omitted from experiments examining the role of INDO in IFNG-mediated luteal cell death.

Indoleamine 2,3-dioxygenase catalyzes the conversion of L-tryptophan to L-kynurenine, which is the first stable metabolite in the degradation of tryptophan through the kynurenine pathway [38]. The specificity of INDO for tryptophan (and to a lesser extent other indoleamine derivatives such as serotonin and melatonin) as substrate has been described and characterized in numerous biochemical studies (reviewed in [39]). The IFNG-inducibility of this enzyme was first demonstrated in a murine model in which IFNG was found to be the agent responsible for induction of INDO in response to intraperitoneal administration of bacterial endotoxin [40]. The ability of IFNG to induce INDO has since been demonstrated in an array of diverse cell types and systems (reviewed in [19]). More recently, the methylated derivative of tryptophan, 1-methyl-D-tryptophan (1-MT), which is a pharmacologic competitive inhibitor of INDO, has been used in various studies to inhibit tryptophan depletion by INDO [34, 41–44].

Degradation of tryptophan by INDO depletes the cellular microenvironment of available tryptophan, an effect that has been shown to have both antiproliferative and cytotoxic effects on various cell types [20–23, 45–47]. Induction of INDO is an IFNG-inducible event [19]. Given the previously observed detrimental effects of IFNG on bovine luteal cell viability [6, 7, 11, 14] and because some mutant cell types that are incapable of upregulating INDO in response to IFNG are resistant to IFNG-mediated cell death [20, 24, 25], we hypothesized that INDO may play a role in IFNG-mediated death of bovine luteal cells. In the present study, IFNG induced significant DNA fragmentation in cultured luteal cells, and the INDO inhibitor 1-MT inhibited this effect.

It was hypothesized that two possible mechanisms may account for the INDO-mediated effect of IFNG. First, induction of INDO by IFNG could catalyze the degradation of tryptophan, thereby reducing available free L-tryptophan to concentrations insufficient to support luteal cell survival, an effect that induces apoptosis in other cell types [22, 45–47]. Alternatively, induction of INDO by IFNG may have caused accumulation of metabolites of tryptophan, including kynurenine and quinolinic acid, which can induce apoptosis in a variety of cell types [23, 48–50]. The former possibility assumes that a sufficient concentration of tryptophan is necessary for luteal cell survival, and that therefore depletion of available tryptophan from cultures by INDO results in cell death. The latter possibility assumes that luteal cells are sensitive to metabolites produced via INDO-dependent degradation of tryptophan through the kynurenine pathway, and that accumulation of these metabolites in culture medium results in cell death. Following the above rationale, we supplemented cultures of luteal cells with high concentrations of L-tryptophan, expecting one of two outcomes. If IFNG-induced cell death was mediated by depletion of available tryptophan, then supplementation with additional tryptophan would protect luteal cells from IFNG-induced cell death. Alternatively, if IFNG induced luteal cell death by promoting the buildup of toxic metabolites of tryptophan, then addition of supplemental tryptophan would augment IFNG-induced cell death, because there would be more substrate present to convert to cytotoxic metabolites.

To determine which of these possibilities was the case, the culture medium of IFNG-treated luteal cells was supplemented with either 5-, 10-, or 25-fold greater concentrations of L-tryptophan than found in nonsupplemented medium. Because elevation of L-tryptophan concentrations in culture medium inhibited IFNG-induced DNA fragmentation, it appears that the mechanism of action by which IFNG induces death of cultured bovine luteal cells involves depletion of this amino acid to concentrations that are insufficient to meet luteal cell needs for survival. To the best of our knowledge, there are no data concerning concentrations of tryptophan in luteal tissue of any species, and it is unknown whether concentrations of this amino acid fluctuate in the tissue over the estrous cycle. Therefore, we can only speculate as to the minimal tryptophan requirements for survival of luteal cells and how a reduction in concentration of this amino acid might occur in vivo. The most plausible mechanism would seem to be that induction of INDO in luteal cells would deplete available tryptophan from the cellular microenvironment, as has been demonstrated in other studies [20, 21, 34, 51, 52], which would then lead to apoptosis.

The duration of luteal cell cultures in the present study has been used previously to demonstrate the effects of IFNG on luteal cell viability and function [6, 7, 14]. At first glance, this appears to be a large amount of time compared to the time required for luteolysis in vivo. However, a significant distinction must be made with regard to the meaning of the term "luteolysis" in vivo. The decline in circulating concentrations of progesterone, which typically characterizes the onset of luteal regression, occurs relatively rapidly in response to the luteolytic stimulus. However, the rapid decline in circulating progesterone concentration does not reflect the time it takes for the cells that make up the CL to subsequently undergo cell death. We therefore believe that the experimental conditions reflect, to a limited degree, the in vivo situation, namely prolonged exposure to luteolytic agents that ultimately results in the structural demise of the CL.

One downstream event in the apoptotic process in luteal cells appears to be the activation of caspase-3 [53, 54]. Tumor necrosis factor-{alpha} and IFNG both upregulate expression of Fas [54–56], and the action of TNF and Fas both upregulate caspase-3, TNF presumably indirectly via upregulation of Fas, and Fas directly through its signaling activity [57–59]. Using caspase-3 deficient mice, Carambula et al. [59, 60] demonstrated the fundamental importance of the caspase-3 system in the apoptotic process in luteal cells, and to luteal regression in general. Because IFNG can upregulate caspase-3 activity via upregulation of Fas, the contribution of IFNG to the caspase-3-dependent apoptotic cell death process in luteal cells is apparent. How the induction of INDO by IFNG and INDO-mediated tryptophan degradation is related to luteal cell death in this context is uncertain. However, one possible mechanism by which tryptophan may protect luteal cells from IFNG-induced cell death may involve an antioxidant effect. Tryptophan is known to possess antioxidant and free radical scavenger activities, and free L-tryptophan has been identified as a primary antioxidant in human placental extracts [61]. It is possible that the elevated concentrations of tryptophan used in the culture medium in the third experiment of the present study prevented cell death through actions as an antioxidant. This possibility is intriguing, given the observation from a previous study that high concentrations of superoxide dismutase and catalase were also able to inhibit (at least partially) IFNG-induced luteal cell death [14]. Therefore, it seems plausible that IFNG induces multiple pathways in luteal cells that mediate apoptotic cell death, and that the IFNG-mediated upregulation of INDO functions as one pathway.

In conclusion, we have described in the present study the induction of INDO in bovine luteal cells in response to IFNG. This is the first report to describe INDO expression in ovarian cells of any species. Moreover, we have also demonstrated a role for INDO in IFNG-induced bovine luteal cell death. IFNG-induced DNA fragmentation was inhibited by addition of the INDO inhibitor 1-MT to luteal cell cultures, and addition of supplemental tryptophan also protected cells from IFNG-induced DNA fragmentation. From these results it can be inferred that tryptophan degradation by INDO plays a significant role in IFNG-induced apoptosis in bovine luteal cells. From the protective effect of additional tryptophan in this study, we conclude that the mechanism by which IFNG-induced INDO mediates luteal cell death is tryptophan depletion, rather than production of cytotoxic metabolites from tryptophan. Collectively, we conclude from these results that IFNG-dependent, INDO-mediated tryptophan degradation plays a significant role in IFNG-induced death of cultured bovine luteal cells.

ACKNOWLEDGMENTS

The authors would like to thank Jodi Winkler for excellent technical assistance.

FOOTNOTES

2 Correspondence: Joy L. Pate, Department of Animal Sciences, The Ohio State University/Ohio Agricultural Research and Development Center, 1680 Madison Ave., Wooster, OH 44691. FAX: 330 263 3949; pate.1{at}osu.edu Back

1 Supported by NIH grant HD37550 to JLP. Salaries and research support also provided by state and federal funds appropriated. Back

Received: 28 March 2005.

First decision: 22 April 2005.

Accepted: 23 November 2005.

REFERENCES

  1. Petroff MG, Petroff BK, Pate JL, Expression of cytokine messenger ribonucleic acids in the bovine corpus luteum. Endocrinology 1999 140:1018-1021[Abstract/Free Full Text]
  2. Penny LA, Armstrong D, Bramley TA, Webb R, Collins RA, Watson ED, Immune cells and cytokine production in the bovine corpus luteum throughout the oestrous cycle and after induced luteolysis. J Reprod Fertil 1999 115:87-96[Abstract/Free Full Text]
  3. Krusche CA, Vloet TD, Herrler A, Black S, Beier HM, Functional and structural regression of the rabbit corpus luteum is associated with altered luteal immune cell phenotypes and cytokine expression patterns. Histochem Cell Biol 2002 118:479-489[Medline]
  4. Komatsu K, Manabe N, Kiso M, Shimabe M, Miyamoto H, Changes in localization of immune cells and cytokines in corpora lutea during luteolysis in murine ovaries. J Exp Zool 2003 296: A 152-159
  5. Neuvians TP, Schams D, Berisha B, Pfaffl MW, Involvement of pro-inflammatory cytokines, mediators of inflammation, and basic fibroblast growth factor in prostaglandin F2{alpha}-induced luteolysis in bovine corpus luteum. Biol Reprod 2004 70:473-480[Abstract/Free Full Text]
  6. Fairchild DL, Pate JL, Modulation of bovine luteal cell synthetic capacity by interferon-gamma. Biol Reprod 1991 44:357-363[Abstract]
  7. Benyo DF, Pate JL, 1992. Tumor necrosis factor-{alpha} alters bovine luteal cell synthetic capacity and viability. Endocrinology 1992 130:854-860[Abstract]
  8. Wang HZ, Lu SH, Han XJ, Zhou W, Sheng WX, Sun ZD, Gong YT, Inhibitory effect of interferon and tumor necrosis factor on human luteal function in vitro. Fertil Steril 1992 58:941-945[Medline]
  9. Fukuoka M, Yasuda K, Fujiwara H, Kanzaki H, Mori T, Interactions between interferon gamma, tumour necrosis factor alpha, and interleukin-1 in modulating progesterone and oestradiol production by human luteinized granulosa cells in culture. Hum Reprod 1992 7:1361-1364[Abstract/Free Full Text]
  10. Best CL, Griffin PM, Hill JA, Interferon gamma inhibits luteinized human granulosa cell steroid production in vitro. Am J Obstet Gynecol 1995 172:1505-1510[CrossRef][Medline]
  11. Suter J, Hendry IR, Ndjountche L, Obholz K, Pru JK, Davis JS, Rueda BR, Mediators of interferon {gamma}-initiated signaling in bovine luteal cells. Biol Reprod 2001 64:1481-1486[Abstract/Free Full Text]
  12. Jo T, Tomiyama T, Ohashi K, Saji F, Tanizawa O, Ozaki M, Yamamoto R, Yamamoto T, Nishizawa Y, Terada N, Apoptosis of cultured mouse luteal cells induced by tumor necrosis factor-alpha and interferon-gamma. Anat Rec 1995 241:70-76[CrossRef][Medline]
  13. Jo T, Terada N, Takauchi Y, Saji F, Nishizawa Y, Tanaka S, Kosaka H, Cytotoxic actions of cytokines on cultured mouse luteal cells are independent of nitric oxide. J Steroid Biochem Mol Biol 1995 55:291-296[CrossRef][Medline]
  14. Petroff MG, Petroff BK, Pate JL, Mechanisms of cytokine-induced death of cultured bovine luteal cells. Reproduction 2001 121:753-760[Abstract]
  15. Komatsu K, Manabe N, Kiso M, Shimabe M, Miyamoto H, Soluble Fas (FasB) regulates luteal cell apoptosis during luteolysis in murine ovaries. Mol Reprod Devel 2003 65:345-352[CrossRef][Medline]
  16. Pru JK, Lynch MP, Davis JS, Rueda BR, Signaling mechanisms in tumor necrosis factor alpha-induced death of microvascular endothelial cells of the corpus luteum. Reprod Biol Endocrinol 2003 1:17[CrossRef][Medline]
  17. Terranova PF, Potential roles of tumor necrosis factor-{alpha} in follicular development, ovulation, and the life span of the corpus luteum. Domest Anim Endocrinol 1997 14:1-15[CrossRef][Medline]
  18. Pate JL, Keyes PL, Immune cells in the corpus luteum: friends or foes?. Reproduction 2001 122:665-676[Abstract]
  19. Taylor MW, Feng GS, Relationship between interferon-{gamma}, indoleamine 2,3-dioxygenase, and tryptophan catabolism. FASEB J 1991 5:2516-2522[Abstract]
  20. Ozaki Y, Edelstein MP, Duch DS, Induction of indoleamine 2,3-dioxygenase: a mechanism of the antitumor activity of interferon {gamma}. Proc Natl Acad Sci U S A 1988 85:1242-1246[Abstract/Free Full Text]
  21. Aune TM, Pogue SL, Inhibition of tumor cell growth by interferon-{gamma} is mediated by two distinct mechanisms dependent upon oxygen tension: induction of tryptophan degradation and depletion of intracellular nicotinamide adenine dinucleotide. J Clin Invest 1989 84:863-875[Medline]
  22. Taylor MW, Konan VK, Yu D, Tryptophan starvation is involved in human interferon-gamma mediated apoptosis. Adv Exp Med Biol 1996 398:155-159[Medline]
  23. Fallarino F, Grohmann U, Vacca C, Bianchi R, Orabona C, Spreca A, Fioretti MC, Puccetti P, T cell apoptosis by tryptophan catabolism. Cell Death Differ 2002 9:1069-1077[CrossRef][Medline]
  24. Feng GS, Taylor MW, Interferon {gamma}-resistant mutants are defective in the induction of indoleamine 2,3-dioxygenase. Proc Natl Acad Sci U S A 1989 86:7144-7148[Abstract/Free Full Text]
  25. Ozes ON, Taylow MW, Reversal of interferon-{gamma}-resistant phenotype by poly(I:C): possible involvement of ISGF2 (IFR1) in interferon-{gamma}-mediated induction of the IDO gene. J Interferon Res 1994 14:25-32[Medline]
  26. Heim MH, The Jak-STAT pathway: cytokine signalling from the receptor to the nucleus. J Recept Signal Transduct Res 1999 19:75-120[Medline]
  27. Fairchild DL, Pate JL, Interferon-{gamma} induction of major histocompatibility complex antigens on cultured bovine luteal cells. Biol Reprod 1989 40:453-457[Abstract]
  28. Boss JM, Regulation of transcription of MHC class II genes. Curr Opin Immunol 1997 9:107-113[CrossRef][Medline]
  29. LeibundGut-Landmann S, Waldburger JM, Krawczyk M, Otten LA, Suter T, Fontana A, Acha-Orbea H, Reith W, Specificity and expression of CIITA, the master regulator of MHC class II genes. Eur J Immunol 2004 34:1513-1525[CrossRef][Medline]
  30. Pate JL, Condon WA, Effects of serum and lipoproteins on steroidogenesis in cultured bovine luteal cells. Mol Cell Endocrinol 1982 28:551-562[CrossRef][Medline]
  31. Cannon MJ, Pate JL, Expression and regulation of interferon-{gamma}-inducible proteasomal subunits LMP7 and LMP10 in the bovine corpus luteum. Biol Reprod 2003 68:1447-1454[Abstract/Free Full Text]
  32. Bove SE, Petroff MG, Nishibori M, Pate JL, Macrophage migration inhibitory factor in the bovine corpus luteum: characterization of steady-state messenger ribonucleic acid and immunohistochemical localization. Biol Reprod 2000 62:879-885[Abstract/Free Full Text]
  33. Tsai S-J, Wiltbank MC, Bodensteiner KJ, Distinct mechanisms regulate induction of messenger ribonucleic acid for prostaglandin (PG) G/H synthase-2, PGE (EP3) receptor, and PGF2{alpha} receptor in bovine preovulatory follicles. Endocrinology 1996 137:3348-3355[Abstract]
  34. Munn DH, Zhou M, Attwood JT, Bondarev I, Conway SJ, Marshall B, Brown C, Mellor AL, Prevention of allogeneic fetal rejection by tryptophan catabolism. Science 1998 281:1191-1193[Abstract/Free Full Text]
  35. Hwu P, Du MX, Lapointe R, Do M, Taylor MW, Young HA, Indoleamine 2,3-dioxygenase production by human dendritic cells results in the inhibition of T cell proliferation. J Immunol 2000 164:3596-3599[Abstract/Free Full Text]
  36. Mellor AL, Sivakumar J, Chandler P, Smith K, Molina H, Mao D, Munn DH, Prevention of T cell-driven complement activation and inflammation by tryptophan catabolism during pregnancy. Nat Immunol 2001 2:64-68[CrossRef][Medline]
  37. Green MH, Lowe JE, Delaney CA, Green IC, Comet assay to detect nitric oxide-dependent DNA damage in mammalian cells. Methods Enzymol 1996 269:243-266[Medline]
  38. Fujiwara M, Shibata M, Watanabe Y, Nukiwa T, Hirata F, Mizuno N, Hayaishi O, Indoleamine 2,3-dioxygenase. Formation of L-kynurenine from L-tryptophan in cultured rabbit pineal gland. J Biol Chem 1978 253:6081-6085[Abstract/Free Full Text]
  39. Thomas SR, Stocker R, Redox reactions related to indoleamine 2,3-dioxygenase and tryptophan metabolism along the kynurenine pathway. Redox Rep 1999 4:199-220[CrossRef][Medline]
  40. Yoshida R, Imanishi J, Oku T, Kishida T, Hayaishi O, Induction of pulmonary indoleamine 2,3-dioxygenase by interferon. Proc Natl Acad Sci U S A 1981 78:129-132[Abstract/Free Full Text]
  41. Kudo Y, Boyd CA, Sargent IL, Redman CW, Tryptophan degradation by human placental indoleamine 2,3-dioxygenase regulates lymphocyte proliferation. J Physiol 2001 53:5207-215
  42. Logan GJ, Smyth CMF, Earl JW, Zaikina I, Rowe PB, Smythe JA, Alexander IE, HeLa cells cocultured with peripheral blood lymphocytes acquire an immuno-inhibitory phenotype through up-regulation of indoleamine 2,3-dioxygenase activity. Immunology 2002 105:478-487[CrossRef][Medline]
  43. Xiao BG, Liu X, Link H, Antigen-specific T cell functions are suppressed over the estrogen-dendritic cell-indoleamine 2,3-dioxygenase axis. Steroids 2004 69:653-659[CrossRef][Medline]
  44. Kudo Yoshiki, Boyd CAR, Spyropoulou I, Redman CWG, Takikawa O, Katsuki T, Hara T, Ohama K, Sargent IL, Indoleamine 2,3-dioxygenase: distribution and function in the developing human placenta. J Reprod Immunol 2004 61:87-98[CrossRef][Medline]
  45. Lee GK, Park HJ, Macleod M, Chandler P, Munn DH, Mellor AL, Tryptophan deprivation sensitizes activated T cells to apoptosis prior to cell division. Immunology 2002 107:452-460[CrossRef][Medline]
  46. Yen CE, Mar M, Craciunescu CN, Edwards LJ, Zeisel SH, Deficiency in methionine, tryptophan, isoleucine, or choline induces apoptosis in cultured cells. J Nutr 2002 132:1840-1847[Abstract/Free Full Text]
  47. Li Y, Tredget EE, Kilani RT, Iwashina T, Karami A, Lin X, Ghahary A, Expression of indoleamine 2,3-dioxygenase in dermal fibroblasts functions as a local immunosuppressive factor. J Invest Dermatol 2004 122:953-964[CrossRef][Medline]
  48. Okuda S, Nishiyama N, Saito H, Katsuki H, 3-hydroxykynurenine, an endogenous oxidative stress generator, causes neuronal cell death with apoptotic features and region selectivity. J Neurochem 1998 70:299-307[Medline]
  49. Martinsons A, Rudzite V, Bratslavska O, Saulite V, The influence of kynurenine, neopterin, and norepinephrine on tubular epithelial cells and alveolar fibroblasts. Adv Exp Med Biol 1999 467:347-352[Medline]
  50. Terness P, Bauer TM, Röse L, Dufter C, Watzlik A, Simon H, Opelz G, Inhibition of allogenic T cell proliferation by indoleamine 2,3-dioxygenase-expressing dendritic cells: mediation of suppression by tryptophan metabolites. J Exp Med 2002 196:447-457[Abstract/Free Full Text]
  51. Uyttenhove C, Pilotte L, Theate I, Stroobant V, Colau D, Parmentier N, Boon T, Van den Eynde BJ, Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase. Nat Med 2003 9:1269-1274[CrossRef][Medline]
  52. Wolf AM, Wolf D, Rumpold H, Moschen AR, Kaser A, Obrist P, Fuchs D, Brandacher G, Winkler C, Geboes K, Rutgeerts P, Tilg H, Overexpression of indoleamine 2,3-dioxygenase in human inflammatory bowel disease. Clin Immunol 2004 113:47-55[CrossRef][Medline]
  53. Rueda BR, Hendry IR, Tilly JL, Hamernik DL, Accumulation of caspase-3 messenger ribonucleic acid and induction of caspase activity in the ovine corpus luteum following prostaglandin F2{alpha} treatment in vivo. Biol Reprod 1999 60:1087-1092[Abstract/Free Full Text]
  54. Quirk SM, Harman RM, Huber SC, Cowan RG, Responsiveness of mouse corpora luteal cells to Fas antigen (CD95)-mediated apoptosis. Biol Reprod 2002 63:49-56
  55. Taniguchi H, Yokomizo Y, Okuda K, Fas-Fas ligand system mediates luteal cell death in bovine corpus luteum. Biol Reprod 2002 66:754-759[Abstract/Free Full Text]
  56. Komatsu K, Manabe N, Kiso M, Shimabe M, Miyamoto H, Soluble Fas (FasB) regulates luteal cell apoptosis during luteolysis in murine ovaries. Mol Reprod Devel 2003 65:345-352
  57. Abdo MA, Richards A, Atiya N, Singh B, Parkinson S, Hisheh S, Dharmarajan AM, Inhibitors of caspase homologues suppress an apoptotic phenotype in cultured rabbit corpora lutea. Reprod Fertil Dev 2001 13:395-403[CrossRef][Medline]
  58. Abdo M, Hisheh S, Dharmarajan A, Role of tumor necrosis factor-alpha and the modulating effect of the caspases in rat corpus luteum apoptosis. Biol Reprod 2003 68:1241-1248[Abstract/Free Full Text]
  59. Carambula SF, Pru JK, Lynch MP, Matikainen T, Goncalves PBD, Flavell RA, Tilly JL, Rueda BR, Prostaglandin F2alpha- and FAS-activating antibody-induced regression of the corpus luteum involves caspase-8 and is defective in caspase-3 deficient mice. Reprod Biol Endocrinol 2003 1:15[CrossRef][Medline]
  60. Carambula SF, Matikainen T, Lynch MP, Flavell RA, Goncalves PBD, Tilly JL, Rueda BR, Caspase-3 is a pivotal mediator of apoptosis during regression of the ovarian corpus luteum. Endocrinology 2002 143:1495-1501[Abstract/Free Full Text]
  61. Watanabe S, Togashi SI, Takahashi N, Fukui T, L-tryptophan as an antioxidant in human placenta extract. J Nutr Sci Vitaminol 2002 48:36-39




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