Molecular Human Reproduction, Vol. 6, No. 4, 369-374,
April 2000
© 2000 European Society of Human Reproduction and Embryology
Pregnancy |
Modulation of indoleamine 2,3-dioxygenase by interferon-
in human placental chorionic villi
1 Department of Human Anatomy and Genetics, University of Oxford, South Parks Road, Oxford OX1 3QX and 2 Nuffield Department of Obstetrics and Gynaecology, University of Oxford, John Radcliffe Hospital, Oxford OX3 9DU, UK
Abstract
The effect of interferon-
on indoleamine 2,3-dioxygenase, a tryptophan catabolizing enzyme, was studied in cultured human placental chorionic villi. The activity of indoleamine 2,3-dioxygenase was markedly stimulated by interferon- in a time- and concentration-dependent manner. Interferon-
and interferon-ß also showed a slight stimulatory effect on indoleamine 2,3-dioxygenase activity. The level of indoleamine 2,3-dioxygenase mRNA expression (determined by reverse transcription—polymerase chain reaction) was also enhanced by interferon-. Interleukin-4 showed a dose-dependent inhibitory effect on interferon--induced stimulation of indoleamine 2,3-dioxygenase activity and mRNA expression.
human placenta/indoleamine 2,3-dioxygenase/interferon/interleukin
Introduction
The enzyme indoleamine 2,3-dioxygenase is widely expressed in a variety of tissues of mammals such as rabbits (Hirata and Hayaishi, 1972
), rats (Cook et al., 1980), mice (Yoshida et al., 1980) and humans (Yamazaki et al., 1985). It catalyses the oxidative cleavage of the pyrrole ring of the indole nucleus of various indoleamines derivatives (e.g. tryptophan, 5-hydroxytryptophan, tryptamine and serotonin) upon the insertion of two oxygen atoms of molecular oxygen (Yoshida and Hayaishi, 1987). One tissue with particularly high activity is the human placenta (Yamazaki et al., 1985). Although the precise physiological role of indoleamine 2,3-dioxygenase is still unknown, the enzyme is induced under pathological conditions including virus infection (Yoshida et al., 1979), parasitic infestation (Dai et al., 1994) and tumour transplantation into allogenic animals (Yoshida et al., 1988; Takikawa et al., 1991), resulting in the rapid degradation of tryptophan to kynurenine in the infected cells and tumour cells. Interferon- (IFN-), which exerts potent immunomodulatory and antiproliferative effects, strongly induces the expression of genes coding for indoleamine 2,3-dioxygenase (Dai and Gupta, 1990). Therefore the antiproliferative effect of IFN- on tumour cells and its inhibitory effect on intracellular pathogens are, at least in part, attributable to the depletion of the essential amino acid, L-tryptophan, through the induction of indoleamine 2,3-dioxygenase. With regard to its role in normal pregnancy Munn et al. proposed the hypothesis that the expression of this enzyme in the placenta is crucial to prevent immunological rejection of the fetal allograft (Munn et al., 1998). They administered 1-methyl-tryptophan, a pharmacological inhibitor of intestinal indoleamine 2,3-dioxygenase (Cady and Sono, 1991), to pregnant mice and found a rapid T cell-induced rejection of allogenic concepti. They suggested that the mechanism of T cell inhibition involves indoleamine 2,3-dioxygenase-dependent localized depletion of L-tryptophan at the site of placentation. We have recently confirmed that human placental indoleamine 2,3-dioxygenase is a cytoplasmic enzyme of the syncytiotrophoblast and is competitively inhibited by 1-methyl-tryptophan (Kudo and Boyd, 2000). In order to clarify the physiological significance of human placental indoleamine 2,3-dioxygenase, we have investigated the regulation of its expression by IFN- using cultured human placental chorionic villi. We also studied the effect of interleukin-4 (IL-4) [a cytokine having inhibitory effect on indoleamine 2,3-dioxygenase expression in human monocytes (Musso et al., 1994) and the concentration of which is significantly elevated in the serum of pre-eclamptic women (Omu et al., 1995)], both singly and in combination with IFN-, on the expression of this enzyme.
Materials and methods
Materials
Human recombinant IFN- and IL-4 were purchased from Sigma Chemical (Poole, Dorset, UK), human recombinant IFN- and IFN-ß were from ICN Biomedicals (Thame, Oxfordshire, UK), tissue culture supplements were from Gibco BRL (Paisley, UK). QuickPrep Total RNA Extraction Kits were purchased from Amersham Pharmacia Biotech (Rainham, Essex, UK), moloney murine leukaemia virus (M-MLV) reverse transcriptase, oligo(dT)12–18 primer, deoxynucleotide 5'-triphosphate (dNTP) and Taq DNA polymerase were from Gibco BRL, deoxyribonuclease I (DNase I) was from Promega (Southampton, Hampshire, UK). All other chemicals were of the highest purity commercially available.
Culture of small pieces of chorionic villi
Term placentae were obtained from women without any complications other than cephalopelvic disproportion requiring repeat Caesarean section at between 38 and 40 weeks of gestation within 15 min from delivery and chilled on ice. The placenta was cut into cotyledons and the decidual surface was removed. The tissue was washed three times with ice-cold phosphate-buffered saline (PBS) containing 100 U/ml penicillin and 100 U/ml streptomycin and chorionic villi was dissected into small pieces (a single piece was ~5 mg). All procedures were carried out below 4°C. Three pieces of chorionic villi were placed on a Millipore filter (0.65 µm) and cultured in 35 mm plastic culture dish at 37°C in an atmosphere of 5% CO2 and 95% air for the indicated times. Culture medium used was RPMI medium 1640 with addition of 5% fetal bovine serum, 100 U/ml penicillin, 100 U/ml streptomycin and IFN- or vehicle. Other additions are described in the table or figure legends. Cultures were conducted in triplicate to assess reproducibility. The number of placentae used in each study is mentioned in the table and figure legends.
Preparation of tissue extract
The cultured pieces of chorionic villi were washed twice, suspended in ice-cold PBS and disrupted by sonication for 30 s in an ice bath at a power of 100 W. The homogenate was centrifuged at 800 g for 10 min at 4°C to remove unbroken fragments of the tissue. The supernatant was then centrifuged at 15000 g for 15 min at 4°C. The resultant supernatant (tissue extract) was stored at –80°C before analysis.
Assay of indoleamine 2,3-dioxygenase
Indoleamine 2,3-dioxygenase activity was determined colorimetrically by a modified procedure based on a previous report (Takikawa et al., 1988). The reaction was initiated by adding 0.25 ml of the tissue extract to 0.25 ml of the incubation medium composed of 0.8 mmol/l L-tryptophan, 40 mmol/l ascorbic acid, 20 µmol/l methylene blue, 200 U/ml catalase and 100 mmol/l potassium phosphate buffer (pH 6.5). Both the tissue extract and the incubation medium were pre-incubated independently at 37°C for 5 min before mixing, followed by further incubation at 37°C for 30 min. The reaction was terminated by adding 0.1 ml of 30% (w/v) trichloroacetic acid and incubated at 50°C for 30 min to hydrolyse N-formylkynurenine produced by indoleamine 2,3-dioxygenase to kynurenine. The reaction mixture was then centrifuged for 20 min at 3000 g to remove sediment. To 0.5 ml of the supernatant, 0.5 ml of 1% (w/v) p-dimethylaminobenzaldehyde in acetic acid was added. The absorbance at 480 nm for the yellow colour derived from kynurenine was determined. All assays were conducted in triplicate.
RNA extraction and reverse transcription–polymerase chain reaction (RT–PCR) analysis
Indoleamine 2,3-dioxygenase gene expression was analysed by semi-quantitative RT–PCR using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal standard. Total RNA was extracted from cultured chorionic villi with QuickPrep Total RNA Extraction Kit according to the manufacturer's protocol. RNA samples were treated with DNase I before RT–PCR to remove any contaminating DNA. The primers used in the subsequent RT–PCR were as follows: indoleamine 2,3-dioxygenase (Kadoya et al., 1992), forward, 5'-TGCTAAACATCTGCCTGATC-3' and backward, 5'-GGAGCAATTGACTCAAATCA-3'; GAPDH (Tso et al., 1985), forward, 5'-CGGGAAGCTTGTGATCAATGG-3' and backward, 5'-GGCAGTGATGGCATGGACTG-3'. The expected size of the PCR products were 144 bp for indoleamine 2,3-dioxygenase and 358 bp for GAPDH. One microgram RNA was reverse transcribed into cDNA using oligo(dT)12–18 primer. The reverse transcription reaction, containing 500 µmol/l dNTP, 25 µg/ml oligo(dT)12—18 primer, 10 U/µl M-MLV reverse transcriptase, 3 mmol/l MgCl2, 75 mmol/l KCl, 10 mmol/l dithiothreitol and 50 mmol/l Tris—HCl (pH 8.3), was sequentially incubated at 25°C for 10 min, at 42°C for 50 min and at 70°C for 15 min and cooled on ice. To control for DNA contamination, reactions were run without RNA or with RNA in the absence of the reverse transcriptase and revealed no amplified product (data not shown). The synthesized cDNA (0.05 µg equivalent to RNA) was used for PCR amplification in a reaction mixture containing 200 µmol/l dNTP, 1 µmol/l forward and backward primers, 0.05 U/µl Taq DNA polymerase, 1.5 mmol/l MgCl2, 50 mmol/l KCl and 20 mmol/l Tris—HCl (pH 8.4). The PCR conditions were: 94°C for 3 min, 60°C for 1 min and 72°C for 2 min; then 25 cycles (for indoleamine 2,3-dioxygenase) and 22 cycles (for GAPDH) of 94°C for 1 min, 60°C for 1 min and 72°C for 2 min; followed by a 10 min final extension at 72°C. The amount of template cDNA and the number of cycles were determined experimentally so that quantitative comparison could be made during the exponential phase of the amplification process for both target and reference gene. PCR products were separated on a 2% agarose gel. Gels were stained with ethidium bromide. The intensity of either the indoleamine 2,3-dioxygenase or GAPDH band for each sample was quantified using a gel documentation and analysis system (GDS8000, Ultra-Violet Products, Cambridge, UK) and the ratio of the two was used as a normalized expression value of the indoleamine 2,3-dioxygenase gene. All assays were conducted in triplicate.
Protein estimation
Protein concentration of the tissue extract was determined by an established method (Lowry et al., 1951) using bovine serum albumin as a standard.
Statistical analysis
Differences between groups were analysed using Student's t-test and results were considered statistically significant at P < 0.05.
Results
Effect of IFN- on indoleamine 2,3-dioxygenase activity in cultured chorionic villi
The presence of IFN- in the culture medium markedly stimulated indoleamine 2,3-dioxygenase activity (Figure 1). An effect could be detected 12 h after the initiation of culture, peaked at 36 h of exposure and remained elevated thereafter. In the absence of IFN-, indoleamine 2,3-dioxygenase activity appeared to decline during the 48 h culture period, but this decrease was not statistically significant. If IFN- was removed after 3 h of treatment and indoleamine 2,3-dioxygenase activity was determined 9 h later, there was no stimulation of enzyme activity (control, 0.39 ± 0.03; 3 h treatment, 0.42 ± 0.05 nmol/min/mg protein). This suggests that prolonged exposure to IFN- is necessary for stimulation of indoleamine 2,3-dioxygenase activity. Different concentrations of IFN- over the range of 10 to 104 U/ml stimulated indoleamine 2,3-dioxygenase activity in a concentration-dependent manner (Figure 2). Significant and maximal stimulation were observed at 50 and 103 U/ml IFN- respectively. Therefore, chorionic villi were cultured for 36 h in the presence of 103 U/ml IFN- to estimate the IFN--induced stimulation of indoleamine 2,3-dioxygenase in subsequent experiments. Both IFN- and IFN-ß at a concentration of 103 U/ml also had a slight and a significant stimulatory effect on indoleamine 2,3-dioxygenase activity after 36 h of culture activity (P < 0.05) (control, 0.41 ± 0.01; IFN-, 0.71 ± 0.09; IFN-ß, 0.61 ± 0.04 nmol/min/mg protein).
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), vehicle (•). Data represent the mean ± SD of three separate experiments performed with three placentae.
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Effect of IFN-
on indoleamine 2,3-dioxygenase mRNA expression in cultured chorionic villiIn order to examine the role of IFN-
as a regulator of indoleamine 2,3-dioxygenase gene expression in chorionic villi, indoleamine 2,3-dioxygenase mRNA levels after treatment of IFN- were determined by RT–PCR. A single band of the expected size for indoleamine 2,3-dioxygenase (144 bp) was detected at each time point (Figure 3A). IFN- at 103 U/ml stimulated indoleamine 2,3-dioxygenase mRNA levels after 3 h with further increases after 6 and 12 h. Without treatment there was a constant expression level of indoleamine 2,3-dioxygenase mRNA during the 48 h of culture. Gel analysis revealed that IFN- increased indoleamine 2,3-dioxygenase mRNA by 1.4-fold after 3 h, by 1.7-fold after 6 h and by 2.2-fold after 12 h (Figure 3B). The density of the RT—PCR product obtained using primers for GAPDH was constant for each sample, indicating that equal quantities of mRNA from the housekeeping gene were present in each extracted RNA sample.
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Effect of IL-4 on IFN-
-induced stimulation of indoleamine 2,3-dioxygenase in cultured chorionic villiThe effect of IL-4 was studied to characterize further the action of IFN-
on induction of indoleamine 2,3-dioxygenase in chorionic villi. As shown in Figure 4, IL-4 produced a dose-dependent inhibition of IFN--induced indoleamine 2,3-dioxygenase activity. The half-maximal inhibition was observed at ~4 ng/ml and maximal inhibition at 10 ng/ml IL-4. However, in control culture, increasing concentrations of IL-4 had no significant inhibitory effect on indoleamine 2,3-dioxygenase activity. Indoleamine 2,3-dioxygenase mRNA levels after treatment with IL-4 and/or IFN- were determined by RT—PCR (where again GAPDH mRNA was constant for each treatment, Figure 5). Gel analysis revealed that treatment with maximally effective doses of IFN- (103 U/ml) induced 2.2-fold increase of indoleamine 2,3-dioxygenase mRNA expression above basal levels (Table I). IL-4 at 10 ng/ml inhibited IFN--induced stimulation of indoleamine 2,3-dioxygenase mRNA expression by 77.2%. In contrast, IL-4 alone had no effect.
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Discussion
The survival of the allogeneic fetus in the mother is an immune paradox. A unique scientific observation has been made concerning this issue (Munn et al., 1998): the placenta is protected from immune rejection by maternal T cells by means of localized, indoleamine 2,3-dioxygenase-dependent depletion of L-tryptophan. It is not known if this mechanism (discovered in mouse) is applicable to the human, and the extent to which defective activation of this process could contribute to disorders of pregnancy such as recurrent miscarriage and pre-eclampsia. These considerations prompted us to characterize further human placental indoleamine 2,3-dioxygenase.
In certain tissues indoleamine 2,3-dioxygenase activity is modulated by certain cytokines. Thus IFN- stimulates its expression in macrophages and monocytes (Takikawa et al., 1988, 1990); other cytokines (eg IL-4) inhibit indoleamine 2,3-dioxygenase expression (Musso et al., 1994). It was of interest to explore whether either expression or activity of human placental indoleamine 2,3-dioxygenase was similarly controlled. Human placental chorionic villi exposed to IFN- showed an increased indoleamine 2,3-dioxygenase activity in a time- (Figure 1
) and concentration-dependent (Figure 2) manner. The significant stimulatory effect of IFN- on indoleamine 2,3-dioxygenase activity could be detected after 12 h of exposure of chorionic villi and was fully expressed 36 h later, while the enhancement of mRNA expression began after 3 h stimulation and reached a maximum at 12 h (Figure 3). IFN--stimulated indoleamine 2,3-dioxygenase activity was inhibited in a dose-dependent way by increasing concentrations of IL-4 (Figure 4), as was expression of mRNA for IL-4 (Figure 5). Since IL-4 alone did not inhibit either mRNA level or enzyme activity, this effect may be due to the suppression of the increased transcription induced by IFN-. These data suggest that IL-4 may have a role in modulating indoleamine 2,3-dioxygenase availability in the human placenta.
It has been shown that in early pregnancy there is a particularly dense infiltrate of natural killer cells in the decidua having a different phenotype from peripheral blood natural killer cells (King et al., 1998) and that decidual natural killer cells also produce a wide variety of cytokines (Jokhi et al., 1994). It is possible to speculate that cytokines produced by these cells are regulating placental indoleamine 2,3-dioxygenase expression, thus forming a potential cytokine network at the site of placentation. Local tryptophan concentration (which may be controlled by the extent of indoleamine 2,3-dioxygenase expression) may thus control the differentiation and function of natural killer cells. This is obviously of relevance to the possible immunoregulatory role of indoleamine 2,3-dioxygenase at the maternal–fetal interface (Bonney and Matzinger, 1998; Mellor and Munn, 1999).
The maternal syndrome of pre-eclampsia is characterized by a systemic inflammatory response causing maternal endothelial cell activation and dysfunction (Redman et al., 1999). Although the aetiology of pre-eclampsia remains unknown, there is substantial evidence that this may be secondary to abnormalities of placentation and increased oxidative stress. The circulatory concentrations of tumour necrosis factor-, IL-4 and IL-6 are significantly elevated in women with pre-eclampsia (Omu et al., 1995; Vince et al., 1995) and there is indirect evidence for increased IFN- production (Saito et al., 1998). Placental content of tumour necrosis factor- is also increased (Wang and Walsh, 1996) but there is no information about placental IFN- production in pre-eclampsia. Preliminary evidence suggests that normal placental tissue can produce IFN- but at relatively low levels (Paradowska et al., 1997). Cytokine-induced changes in the activity or levels of indoleamine 2,3-dioxygenase in the placenta may be involved in the pathogenesis of pre-eclampsia. It is possible to test these speculations in human pregnancy by measuring the concentration of L-tryptophan or its metabolite, kynurenine, catalysed by indoleamine 2,3-dioxygenase in plasma or urine of women with pre-eclampsia and comparing them with those of normal pregnancy. Our findings also have implications for patients with recurrent miscarriage where indoleamine 2,3-dioxygenase activity can be tested by assessing levels of L-tryptophan and kynurenine. Our studies may thus provide additional understanding of human pregnancy and may open up novel potential targets for therapy of disorders of placental function.
Acknowledgments
We thank staff at the John Radcliffe Hospital, Oxford for assistance with obtaining placentae and Action Research for financial support.
Notes
3 To whom correspondence should be addressed 
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Submitted on November 5, 1999; accepted on January 13, 2000.
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