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Decreased tryptophan catabolism by placental indoleamine 2,3-dioxygenase in preeclampsia
Decreased tryptophan catabolism by placental indoleamine 2,3-dioxygenase in preeclampsia Yoshiki Kudo, MD,a C. A. R. Boyd, MD,a Ian L. Sargent, PhD,b and Christopher W. G. Redman, MDb Oxford, United Kingdom OBJECTIVE: Tryptophan degradation and depletion resulting from activation of indoleamine 2,3-dioxygenase is characteristic of inflammatory reactions and may control their intensity. Normal third-trimester pregnancy is characterized by a maternal systemic inflammatory response, which is more intense in preeclampsia. Therefore, we studied tryptophan metabolism in pregnant women, with or without preeclampsia, as well as expression and function of placental indoleamine 2,3-dioxygenase. STUDY DESIGN: Plasma concentrations of tryptophan and kynurenine in women with preeclampsia, appropriately matched women with normal pregnancy, and healthy nonpregnant women were measured. Placental enzymatic activity and messenger RNA (mRNA) expression level of indoleamine 2,3-dioxygenase were determined from the same placental material. Peripheral blood mononuclear cell proliferation was determined in medium conditioned by prior culture with villous tissue. RESULTS: The plasma ratio of kynurenine to tryptophan, an in vivo index of enzyme activity, was significantly increased compared with nonpregnant controls in normal pregnancy but not in preeclampsia. The activity and mRNA expression level of indoleamine 2,3-dioxygenase in term placentas were significantly lower in preeclampsia. Medium conditioned by culture of villous tissue explants of preeclampsia was less effective in inhibiting peripheral blood mononuclear cell proliferation compared with that of normal pregnancy. CONCLUSION: These observations suggest that in preeclampsia, reduced placental indoleamine 2,3-dioxygenase activity (and relatively elevated plasma tryptophan) could cause dysregulation of the inflammatory response that is intrinsic to normal pregnancy. This may contribute to the pathogenesis of the maternal syndrome of preeclampsia. (Am J Obstet Gynecol 2003;188:719-26.)
Key words: Indoleamine 2,3-dioxygenase, tryptophan, preeclampsia, lymphocyte, placenta
Indoleamine 2,3-dioxygenase is a widely distributed enzyme that catabolizes tryptophan. It is characterized best in monocytes and macrophages but can be induced in many cell types, such as endothelial cells,1 epithelial cells,2 fibroblasts,3 or T cells.4 It is primarily induced by interferon gamma (IFN-γ), but other proinflammatory stimulants are also effective.5 By reducing the availability of tryptophan, indoleamine 2,3-dioxygenase is thought to inhibit intracellular and other infections,6 the growth of malignant cells,7 and immune cell function.8,9 Indoleamine 2,3-dioxygenase is also expressed in the murine and human placentas.10,11 In the pregnant mouse, it is thought to have a crucial role in inhibiting
From the Department of Human Anatomy and Geneticsa and the Nuffield Department of Obstetrics and Gynaecology,b University of Oxford. Supported by Action Research. Y. K. was supported by Oxford Kobe Scholarship. Received for publication June 17, 2002; revised August 23, 2002; accepted October 11, 2002. Reprints not available from the authors. © 2003 Mosby, Inc. All rights reserved. 0002-9378/2003 $30.00 + 0 doi:10.1067/mob.2003.156
the activation of maternal T cells that are specific for the fetus.10,12 It is not known whether comparable mechanisms, which might control events in early pregnancy, are applicable in women. Nor is it known whether placental indoleamine 2,3-dioxygenase has a role in late (established) human pregnancy or contributes to pregnancy disorders such as preeclampsia. Preeclampsia is a dangerous, common, unpredictable, and highly variable complication of the second half of pregnancy, labor, or the early puerperium. The presence of a placenta is necessary to cause the disorder. The placental problem appears to be a relative failure of the uteroplacental circulation owing to spiral artery disease, with consequent oxidative stress and diminished placental function.13 The diversity of the maternal syndrome results from the circulatory and other disturbances, resulting from diffuse maternal endothelial cell dysfunction.14 We have more recently shown that the endothelial dysfunction is a single aspect of a generalized systemic maternal inflammatory response15,16 that also affects circulating leukocytes. Moreover, this inflammatory response is detected in normal pregnancy. It is not qualitatively different from that in preeclampsia but is less intense. We have proposed that preeclampsia devel719
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Table I. Characteristics of the women studied
Age (y) Nulliparas (%) Gestational ages (wk) Mean arterial pressure (mm Hg)* No. with proteinuria
Normal pregnancy (n = 12)
Preeclampsia (n = 12)
Nonpregnant (n = 9)
29.7 ± 3.5 75 35.5 ± 2.8 84.4 ± 5.7 0
26.9 ± 3.9 75 34.6 ± 3.2 113.7 ± 8.4† 12
28.1 ± 4.3 — — — —
Values are given as mean ± SD. *Calculated as diastolic pressure + pulse pressure/3. †P < .001 compared with normal pregnancy (Wilcoxon test).
ops when the systemic inflammatory process causes one or other maternal systems to decompensate. In medical conditions such as systemic lupus erythematosus17 or HIV infection,18 characterized by a systemic inflammatory response, tryptophan degradation appears to be enhanced, causing an increase in the ratio of plasma kynurenine (a degradation product of tryptophan) to tryptophan. The plasma concentration of tryptophan declines progressively as a function of gestational age.19 This has not previously been related to concurrent plasma kynurenine concentrations. An increase in the kynurenine/tryptophan ratio would be consistent with the systemic inflammatory response of normal pregnancy. If true, then in preeclampsia with its greater systemic inflammatory response, it would be predicted that the plasma kynurenine/tryptophan ratio would be further increased. However, if placental indoleamine 2,3dioxygenase function were the dominant influence, then the greater inflammatory response of preeclampsia (associated with poorer placental function) could be associated with a lower plasma kynurenine/tryptophan ratio and lower placental indoleamine 2,3-dioxygenase activity. Then, it could be hypothesized that the excessive systemic inflammatory response of preeclampsia represented a failure of a placental mechanism (indoleamine 2,3-dioxygenase depletion of tryptophan) that would be expected to down-regulate immune cell functions. To address these issues we studied placental indoleamine 2,3-dioxygenase activity and messenger RNA (mRNA) expression as well as indices of tryptophan catabolism in pregnant women with or without preeclampsia. Material and methods Plasma samples. Samples from 12 women with preeclampsia, 12 appropriately matched women with normal pregnancy, and 9 healthy nonpregnant women, not taking any medication, were studied with ethical committee permission. Preeclampsia was defined as new proteinuric hypertension of pregnancy (diastolic blood pressure raised above 90 mm Hg on at least two occasions 6 hours apart, combined with new proteinuria of at least 2+ on dipstick testing in the proved absence of a urinary tract
infection), both changes regressing remotely after delivery. The normal pregnant and preeclamptic women were well matched (Table I). Samples of heparinized blood were centrifuged immediately after collection. Plasma was vortexed with a one-tenth volume of ice-cold 2.4 mol/L perchloric acid. The mixture was chilled on ice for 15 minutes and centrifuged at 10000g for 3 minutes. The clear protein-free supernatant was used for high-performance liquid chromatography (HPLC) analysis. HPLC analysis of L-tryptophan and L-kynurenine. Concentrations of L-tryptophan and L-kynurenine were analyzed by HPLC. The system consisted of a Kontron 420 pump, a Kontron 460 autosampler, and a Kontron 432 variable wavelength detector (Watford, UK) with the Spherisorb S5-ODS1 column, 4.6 150 mm (Waters, Milford, Mass). The mobile phase consisted of 40 mmol/L citrate buffer (pH 2.25) and 50% methanol and 0.4 mmol/L sodium dodecylsulfate, which were used after filtration through a 0.45-µm membrane filter and degassed by a vacuum aspirator. A 20-µL volume of protein-free extract was injected on the column, chromatographed at a flow rate of 2.0 mL/min–1, and detected at 365 nm for Lkynurenine and at 280 nm for L-tryptophan. The minimum amount of L-kynurenine and L-tryptophan reproducibly detected was 20 pmol and calibration was linear up to 10 nmol of L-kynurenine or of L-tryptophan. Culture of villous tissue. Placentas were obtained (with ethical committee approval) from 8 normal and from 14 pregnancies associated with preeclampsia. Not all placentas were from the same donors as the plasma samples. Preeclampsia was defined as described previously. In this group age (years), gestational age (weeks) and mean arterial pressure (mm Hg) were 29.2 ± 5.7, 36.8 ± 1.3, and 111.9 ± 9.7 (mean ± SD), respectively. Placentas were collected within 15 minutes 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 solution (PBS) containing 100 U/mL–1 penicillin and 100 U/mL–1 streptomycin. The chorionic villi were then dissected into small pieces (a single piece was approximately 5 mg). All these procedures were carried out at less than 4°C. Three pieces of chorionic villi were placed on a polyester mesh
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Table II. Tryptophan and kynurenine concentrations and the ratio of kynurenine to tryptophan in plasma from women with preeclampsia or normal pregnancies and from nonpregnant women Normal pregnancy (n = 12) Tryptophan (µmol/L) Kynurenine (µmol/L) Kynurenine/tryptophan
32.7 ± 4.8 1.12 ± 0.17 0.034 ± 0.004
Preeclampsia (n = 12)
Nonpregnant (n = 9)
42.8 ± 6.9* 1.02 ± 0.22 0.024 ± 0.005*
53.0 ± 9.8†‡ 1.17 ± 0.28 0.022 ± 0.004‡
Concentrations of tryptophan and kynurenine in plasma were analyzed by HPLC as described in Methods. Values are given as mean ± SD. *P < .001 compared with normal pregnancy (Wilcoxon test). †P < .001 compared with preeclampsia (Mann-Whitney U test). ‡P < .001 compared with normal pregnancy (Mann-Whitney U test).
(Netwell 500 µm mesh, Costar, NY) and cultured in 35mm plastic culture dishes at 37°C in an atmosphere of 5% carbon dioxide and 95% air for 36 hours. RPMI medium 1640 was used as the culture medium, with added 5% fetal bovine serum, 100 U/mL–1 penicillin, 100 U/mL–1 streptomycin, and IFN-γ or vehicle. Cultures were conducted in triplicate. The number of placentas used in each experiment is mentioned in the figure and table legends. The conditioned media were then used for HPLC analysis after extraction with perchloric acid or for culture of peripheral blood mononuclear cells. Assay of indoleamine 2,3-dioxygenase. The fresh or cultured pieces of chorionic villi were washed three times, suspended in ice-cold PBS, and disrupted by sonication for 30 seconds in an ice bath at a power of 100 watts. The homogenate was centrifuged at 800g for 10 minutes at 4°C to remove unbroken fragments of the tissue. The supernatant was then centrifuged at 15000g for 15 minutes at 4°C. The resultant supernatant was used for the colorimetric assay of indoleamine 2,3-dioxygenase activity, as described previously.11 RNA extraction and reverse transcription–polymerase chain reaction (RT-PCR) analysis. Indoleamine 2,3-dioxygenase, tryptophanyl-tRNA synthetase and signal transducer and activator of transcriptional 1 (STAT1) gene expression were analyzed by semiquantitative RT-PCR by using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal standard. Total RNA was extracted from fresh or cultured chorionic villi with or without IFN-γ by using QuickPrep Total RNA Extraction Kit according to the manufacturer’s protocol. RNA samples were treated with deoxyriboneuclease I before RT-PCR to remove any contaminating DNA. The primers used in the subsequent RT-PCR were as follows: indoleamine 2,3dioxygenase, forward, 5´-TGCTAAACATCTGCCTGATC3´ and backward, 5´-GGAGCAATTGACTCAAATCA-3´; tryptophanyl-tRNA synthetase, forward, 5´-AGCTCAACTGCCCAGCGTGACC -3´ and backward, 5´-CAGTCAGCCTTGTAATCCTCCCCC-3´; STAT1, forward, 5´-AAGGTGGCAGGATGTCTCAGTG-3´ and backward, 5´-TGGTCTCGTGTTCTCTGTTCTG-3´; GAPDH, for-
ward, 5´-CGGGAAGCTTGTGATCAATGG-3´ and backward, 5´-GGCAGTGATGGCATGGACTG-3´. The expected size of the PCR products were 144 bp for indoleamine 2,3dioxygenase, 314 bp for tryptophanyl-tRNA synthetase, 564 bp for STAT1, and 358 bp for GAPDH. One microgram of RNA was reverse transcribed into complementary DNA (cDNA) with the use of oligo(dT)12-18 primer. The reverse transcription reaction, containing 500 µmol/L dNTP, 25 µg/mL-1 oligo(dT)12-18 primer, 10 U/µL-1 MMLV reverse transcriptase, 3 mmol/L magnesium chloride, 75 mmol/L potassium chloride, 10 mmol/L dithiothreitol, and 50 mmol/L TRIS (tris-[hydroxymethyl]-aminomethane)–hydrochloric acid (pH 8.3), was sequentially incubated at 25°C for 10 minutes, at 42°C for 50 minutes, and at 70°C for 15 minutes 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–1 Taq DNA polymerase, 1.5 mmol/L magnesium chloride, 50 mmol/L potassium chloride, and 20 mmol/L TRIS–hydrochloric acid (pH 8.4). The PCR conditions were 94°C for 3 minutes, 60°C for 1 minute, and 72°C for 2 minutes; then 25 cycles (for indoleamine 2,3-dioxygenase, tryptophanyl-tRNA synthetase and STAT1) and 22 cycles (for GAPDH) of 94°C for 1 minute, 60°C for 1 minute, and 72°C for 2 minutes; followed by a 10-minute 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 genes. PCR products were separated on a 2% agarose gel. Gels were stained with ethidium bromide. The intensity of either the target gene or GAPDH band for each sample was quantitated by using a gel documentation and analysis system (GDS8000, Ultra-Violet Products, Cambridge, UK) and the ratio of the target gene to GAPDH was used as a normalized measure of the target gene.
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Fig 2. Peripheral blood mononuclear cell proliferation in medium previously conditioned by culture with villous explants. Peripheral blood mononuclear cells were cultured for 72 hours either in nonconditioned medium or in medium previously conditioned by culture of villous explants from normal and preeclamptic placenta with 1000 U/mL–1 IFN-γ (open bars) or vehicle (closed bars) for 36 hours. [3H]Thymidine incorporation was then determined. Data represent mean ± SD of quadruplicate assays performed with 8 normal and 11 preeclamptic placentas. Asterisk, Significantly different from normal pregnancy. Fig 1. Indoleamine 2,3-dioxygenase, tryptophanyl-tRNA synthetase, and STAT1 mRNA expression level in placental villous tissue. Total RNA was extracted either from fresh (Fr) or cultured villous tissue with (Cul [+]) or without (Cul [–]) 1000 U/mL–1 IFN-γ for 36 hours. Levels of indoleamine 2,3-dioxygenase mRNA (a), tryptophanyl-tRNA synthetase (b), STAT1 (c), and GAPDH mRNA (d) were analyzed by RT-PCR. Results presented are from a single representative experiment.
Peripheral blood mononuclear cell culture and proliferation assay. Human peripheral blood mononuclear cells were isolated from healthy, volunteer, nonpregnant donors by Ficoll-Hypaque gradient centrifugation. The cells (2 105 cells per well) were cultured in media conditioned by prior culture with villous tissue and stimulated with 5 µg/mL–1 phytohemagglutinin in 96-well flat-bottom plates at 37°C in an atmosphere of 5% carbon dioxide and 95% air for 72 hours. Peripheral blood mononuclear cell proliferation was determined by pulse labeling with tritiated thymidine (0.037 MBq/well) for 12 hours. Protein estimation. Protein concentration of placental homogenate was determined with the method of Lowry et al20 by using bovine serum albumin as a standard. Statistical analysis. The measurements with samples from preeclampsia and control normal pregnancy were compared by using the Wilcoxon test. Other comparisons used the Mann-Whitney U test. P < .01 was considered statistically highly significant; P < .05 was considered statistically significant. Chemicals. [Methyl-3H] thymidine (25.0 Ci mmol–1 or 925 GBq mmol–1) was purchased from Amersham Life Science (Amersham, UK). Human recombinant IFN-γ and phytohemagglutinin were obtained from SigmaAldrich Chemical (Poole, UK), and tissue culture supple-
ments were from Gibco BRL (Paisley, UK). QuickPrep Total RNA Extraction Kit was purchased from Amersham Pharmacia Biotech (Rainham, UK), Moloney murine leukemia virus (M-MLV) reverse transcriptase, oligo(dT)12-18 primer, deoxynucleotide 5´-triphosphate (dNTP), and Taq DNA polymerase were from Gibco BRL, and deoxyribonuclease I (DNase I) was from Promega (Southampton, UK). All other chemicals were of the highest purity commercially available. Results Tryptophan concentration in plasma. Table II summarizes the results of HPLC analysis of tryptophan and kynurenine concentrations in plasma. Tryptophan concentrations in plasma samples taken from both groups of pregnant women were significantly lower than those from women who were not pregnant. However, plasma samples from women with normal pregnancy also had significantly lower tryptophan concentrations than those from women with preeclampsia. Plasma kynurenine concentrations showed the converse patterns. Hence, the ratios of indoleamine 2,3-dioxygenase product (plasma kynurenine) to substrate (plasma tryptophan), an index of tryptophan catabolism, were significantly increased in normal pregnant women compared either with women who were not pregnant or with women with preeclampsia. The ratios of kynurenine to tryptophan for women with preeclampsia were not different from those for women who were not pregnant. Indoleamine 2,3-dioxygenase activity and mRNA expression level in placental villous tissue. The indoleamine 2,3-dioxygenase activity in fresh placental villous tissue
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Table III. Indoleamine 2,3-dioxygenase activity in placental villous tissue IDO activity (nmol/mg/min) Treatment Fresh Cultured Nil IFN-γ
Normal pregnancy (n = 8)
Preeclampsia (n = 14)
0.48 ± 0.06
0.29 ± 0.04*
0.47 ± 0.02 1.58 ± 0.04 (329.1 ± 43.3)
0.30 ± 0.04* 0.76 ± 0.14* (258.8 ± 48.3†)
Villous explants were cultured with or without 1000 U/mL–1 IFN-γ for 36 h. Indoleamine 2,3-dioxygenase (IDO) activity in the tissue extract of fresh or cultured villous explants was determined colorimetrically as described in Methods. Percentage stimulation is given in the parentheses. Values are mean ± SD of triplicate assay from indicated number of placentas. *Highly significantly different from normal pregnancy (P < .01). †Significantly different from normal pregnancy (P < .05).
Table IV. Relative quantitation of indoleamine 2,3-dioxygenase, tryptophanyl-tRNA synthetase, and STAT1 mRNA expression level in placental villous tissue mRNA expression level (% of control) Treatment Normal pregnancy Fresh Cultured Nil IFN-γ Preeclampsia Fresh Cultured Nil IFN-γ
IDO
WRS
STAT1
100
100
100
103.7 ± 6.9 212.3 ± 19.6
107.7 ± 4.3 207.5 ± 13.3
107.0 ± 5.3 302.3 ± 23.3
55.8 ± 5.3*
106.1 ± 10.6
109.5 ± 7.3
52.1 ± 5.3* 95.9 ± 8.3*
103.6 ± 9.8 216.1 ± 10.3
102.9 ± 6.3 322.9 ± 22.3
The intensity of both the each target gene and the GAPDH band for each sample was quantitated by using a gel documentation and analysis system of PCR products and the ratio of the two was used as a normalized expression value of each target gene. Data represent the mean ± SD of experiments performed with 8 normal and 8 preeclamptic placentas, expressed as percentage of control (ie, values for fresh villous tissue of normal placenta). IDO, Indoleamine 2,3-dioxygenase; WRS, tryptophanyl-tRNA synthetase. *Significantly different from normal pregnancy.
from preeclampsia was significantly lower than from normal pregnancy (Table III). When villous tissue explants were cultured for 36 hours with or without IFN-γ at 1000 U/mL–1 (maximally stimulating indoleamine 2,3-dioxygenase expression21), indoleamine 2,3-dioxygenase activity was still significantly lower; the percentage stimulation in villous tissue from preeclampsia was also significantly less than in normal pregnancy. Indoleamine 2,3-dioxygenase mRNA expression levels were studied either in fresh villous tissue or in cultured villous tissue with or without IFN-γ stimulation (Fig 1). Table IV shows the relative quantitation of gel analysis normalized by GAPDH. Compared with normal pregnancy, the level of indoleamine 2,3-dioxygenase mRNA expression was approximately half of that in fresh villous tissue from preeclampsia. Treatment with a maximally effective concentration of IFN-γ (1000 U/mL–1) induced a 2.1-fold increase of indoleamine 2,3-dioxgenase mRNA expression in villous tissue from normal pregnancy. However, in preeclampsia, IFN-γ had a smaller (1.7-fold) stimulatory effect on mRNA expression. To analyze the mechanisms involved,
expression of mRNA encoding tryptophanyl-tRNA synthetase and STAT1 either in fresh villous tissue or in cultured villous tissue with or without IFN-γ stimulation was determined (Fig 1). STAT1 is required for IFN-γ-dependent transcription22 and tryptophanyl-tRNA synthetase expression is induced by IFN-γ through the same pathway as indoleamine 2,3-dioxgenase.23 In contrast to indoleamine 2,3-dioxgenase mRNA expression, there were no differences in mRNA expression levels for STAT1 or tryptophanyl-tRNA synthetase between normal and preeclamptic placentas, either in fresh or in cultured villi, with or without IFN-γ stimulation (Table IV). Tryptophan catabolism by indoleamine 2,3-dioxygenase in placental villous tissue. Concentrations of tryptophan and kynurenine in the conditioned medium after 36 hours of in vitro culture of placental villous explants either from normal or from preeclamptic pregnancy were determined (Table V). As predicted from the results shown in Table III, the presence of IFN-γ in the culture medium stimulated tryptophan degradation, with a marked increase in kynurenine concentration both in
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Table V. Tryptophan catabolism by indoleamine 2,3dioxygenase in placental villous explants Treatment Nonconditioned Conditioned Normal pregnancy Nil IFN-γ Preeclampsia Nil IFN-γ
Fig 3. Schematic representation of the role of placental indoleamine 2,3-dioxygenase in human pregnancy.
normal or in preeclampsia villous tissue. However, in tissue from preeclamptic women, the degradation of tryptophan was significantly less with production of significantly less kynurenine, in the presence or absence of IFN-γ. Peripheral blood mononuclear cell proliferation in the medium conditioned by culture of placental villous tissue. To study the functional consequence of suppressed indoleamine 2,3-dioxygenase expression in preeclamptic placental villous tissue, peripheral blood mononuclear cell proliferation activity was determined after the culture in the same medium of which tryptophan concentration was measured in Table V. Thymidine incorporation into DNA is significantly inhibited by conditioned medium. With medium conditioned in the presence of IFN-γ inhibition is significantly increased. Thymidine incorporation is significantly greater in the conditioned medium of IFN-γ–treated explants from preeclampsia than from normal pregnancy. Thus, the inhibitory effect of the conditioning medium on peripheral blood mononuclear cell activation is significantly reduced in preeclampsia compared with normal villous tissue (Fig 2). Comment The increased plasma kynurenine-to-tryptophan ratio in normal pregnancy compared with nonpregnancy is consistent with the evidence that normal third-trimester pregnancy is associated with a systemic inflammatory response.16 Although there is a significantly more intense inflammatory response in preeclampsia,15 we found that the plasma kynurenine/tryptophan ratio is not increased but nearer to that of nonpregnant women. This is associated with a suppressed expression of placental indoleamine 2,3-dioxygenase in preeclampsia. Not only is functional enzyme activity suppressed in vitro (Table V), but the level of indoleamine 2,3-dioxygenase gene expression is also decreased. However, IFN-γ could still stim-
Tryptophan (µmol/L)
Kynurenine (µmol/L)
20.1 ± 0.3
Not detectable
11.7 ± 0.5 1.9 ± 0.2
5.3 ± 0.4 13.2 ± 0.6
16.2 ± 1.2* 4.6 ± 0.7*
2.9 ± 0.2* 9.9 ± 0.4*
Villous explants from normal and preeclamptic placenta were cultured with 1000 U/mL–1 IFN-γ or vehicle for 36 hours. Concentrations of tryptophan and L-kynurenine in the conditioned medium were analyzed by HPLC. Data represent the mean ± SD of triplicate assays performed with 8 normal and 11 preeclamptic placentas. *Significantly different from normal pregnancy.
ulate activity in vitro (Table III and Fig 1), albeit from a lower baseline. The expression of other IFN-γ inducible genes (ie, tryptophanyl-tRNA synthetase and STAT1) was normal and responded appropriately to exogenous IFN-γ (Fig 1). The enzyme activity could also be affected by posttranscriptional events (eg, message stability, protein synthesis) or by molecules, which can regulate enzyme activity. Although each of these steps remains to be studied, our unpublished work shows that the sequence of the indoleamine 2,3-dioxygenase gene, including the promoter region, in cord blood obtained from infants from preeclamptic pregnancies is normal. Both interleukin-421 and transforming growth factor24 β can down-regulate expression of indoleamine 2,3dioxygenase. However, it is not known whether the expression of either cytokine is increased in the preeclampsia placenta. In preeclampsia the placenta is characteristically subjected to oxidative25 and nitrosative stress.26 It is known that the superoxide anion acts as a cofactor for indoleamine 2,3-dioxygenase, which, together with its metabolic products, has antioxidant activity.27 However, these functions would not be predicted to decrease expression of indoleamine 2,3-dioxygenase. Hence, the cause of the reduced placental indoleamine 2,3-dioxygenase expression and activity is unknown at present. But the implication is that placental indoleamine 2,3-dioxygenase activity determines, at least in part, maternal systemic tryptophan concentrations. Depletion of tryptophan by indoleamine 2,3-dioxygenase is a general mechanism of inhibiting cell growth and thought to be how IFN-γ, produced by leukocytes, stimulates tumor rejection28 and inhibits intracellular microorganisms.6 Tryptophan depletion also inhibits lymphocyte proliferation, including that of T lymphocytes (this inhibition is due to arrest of cell cycle progression)8,29 and has the potential to down-regulate inflammatory responses.
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This mechanism has been shown to protect the allogeneic murine conceptus from T cell–mediated rejection.10,12 In nonpregnant women, increases in the systemic kynurenine/tryptophan ratio are a feature of inflammatory disorders and ascribable to systemic activation of indoleamine 2,3-dioxygenase in monocytes and possibly endothelium.17 In the peripheral blood of normal pregnant women in the third trimester, leukocyte (including lymphocyte) activation is known to be exaggerated compared with the findings in matched nonpregnant women.15,16 The increased kynurenine/tryptophan ratio found in normal pregnancy is similar to that of other disorders characterized by systemic inflammation where it can be ascribed to indoleamine 2,3-dioxygenase activity of circulating leukocytes. However, in preeclampsia the systemic inflammatory response is even more intense than in normal pregnancy,15 so it would have been predicted that the kynurenine/tryptophan ratio would have been increased further. Instead, the converse was observed (Table II). This paradox can be explained if the systemic kynurenine/tryptophan ratio in preeclamptic women relative to normal pregnant women depends on placental indoleamine 2,3-dioxygenase expression and activity, which are reduced (Tables III and IV). The conclusion supports the concept that, in pregnancy, placental indoleamine 2,3-dioxygenase activity, at least in part, regulates the systemic kynurenine/tryptophan ratio. These findings therefore provide evidence for a mechanism whereby placental indoleamine 2,3-dioxygenase may down-regulate the maternal systemic inflammatory response intrinsic to normal pregnancy, a process which, our evidence suggests, becomes disrupted in preeclampsia. A general proposal for the role of placental indoleamine 2,3-dioxygenase in suppressing the maternal systemic inflammatory response during human pregnancy and in the pathogenesis of preeclampsia is illustrated in Fig 3. The systemic inflammatory response stimulated by normal pregnancy would be excessive without the physiologic suppression caused by placental indoleamine 2,3dioxygenase. Even so, in the third trimester, it is more intense than in nonpregnancy.15 In preeclampsia, the response is released because of decreased placental indoleamine 2,3-dioxygenase activity. It is thought that the features of the preeclampsia syndrome arise from this excessive systemic inflammatory response.13 The results in this article raise the possibility that novel therapeutic intervention in preeclampsia might usefully be focused on manipulation of plasma tryptophan concentration. We thank Research Midwives Hazel Coburn and Carol Simms for their tireless help in obtaining clinical samples. REFERENCES
1. Hansen AM, Driussi C, Turner V, Takikawa O, Hunt NH. Tissue distribution of indoleamine 2,3-dioxygenase in normal and malaria-infected tissue. Redox Rep 2000;5:112-5.
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2. Bodaghi B, Goureau O, Zipeto D, Laurent L, Virelizier JL, Michelson S. Role of IFN-gamma–induced indoleamine 2,3 dioxygenase and inducible nitric oxide synthase in the replication of human cytomegalovirus in retinal pigment epithelial cells. J Immunol 1999;162:957-64. 3. Chaves AC, Ceravolo IP, Gomes JA, Zani CL, Romanha AJ, Gazzinelli RT. IL-4 and IL-13 regulate the induction of indoleamine 2,3-dioxygenase activity and the control of Toxoplasma gondii replication in human fibroblasts activated with IFN-gamma. Eur J Immunol 2001;31:333-44. 4. Curreli S, Romerio F, Mirandola P, Barion P, Bemis K, Zella D. Human primary CD4+ T cells activated in the presence of IFNalpha 2b express functional indoleamine 2,3-dioxygenase. J Interferon Cytokine Res 2001;21:431-7. 5. Taylor MW, Feng GS. Relationship between interferon-gamma, indoleamine 2,3-dioxygenase, and tryptophan catabolism. FASEB J 1991;5:2516-22. 6. Daubener W, MacKenzie CR. IFN-gamma activated indoleamine 2,3-dioxygenase activity in human cells is an antiparasitic and an antibacterial effector mechanism. Adv Exp Med Biol 1999; 467:517-24. 7. Burke F, Knowles RG, East N, Balkwill FR. The role of indoleamine 2,3-dioxygenase in the anti-tumour activity of human interferon-gamma in vivo. Int J Cancer 1995;60:115-22. 8. Munn DH, Shafizadeh E, Attwood JT, Bondarev I, Pashine A, Mellor AL. Inhibition of T cell proliferation by macrophage tryptophan catabolism. J Exp Med 1999;189:1363-72. 9. 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;164:3596-9. 10. Munn DH, Zhou M, Attwood JT, Bondarev I, Conway SJ, Marshall B, et al. Prevention of allogeneic fetal rejection by tryptophan catabolism. Science 1998;281:1191-3. 11. Kudo Y, Boyd CAR. Human placental indoleamine 2,3-dioxygenase: cellular localization and characterization of an enzyme preventing fetal rejection. Biochim Biophys Acta 2000;1500:119-24. 12. Mellor AL, Sivakumar J, Chandler P, Smith K, Molina H, Mao D, et al. Prevention of T cell-driven complement activation and inflammation by tryptophan catabolism during pregnancy. Nat Immunol 2001;2:64-8. 13. Redman CWG, Sargent IL. Placental debris, oxidative stress and pre-eclampsia. Placenta 2000;21:597-602. 14. Roberts JM, Taylor RN, Musci TJ, Rodgers GM, Hubel CA, McLaughlin MK. Preeclampsia: an endothelial cell disorder. Am J Obstet Gynecol 1989;161:1200-4. 15. Sacks GP, Studena K, Sargent K, Redman CW. Normal pregnancy and preeclampsia both produce inflammatory changes in peripheral blood leukocytes akin to those of sepsis. Am J Obstet Gynecol 1998;179:80-6. 16. Redman CWG, Sacks GP, Sargent IL. Preeclampsia: an excessive maternal inflammatory response to pregnancy. Am J Obstet Gynecol 1999;180:499-506. 17. Widner B, Sepp N, Kowald E, Ortner U, Wirleitner B, Fritsch P, et al. Enhanced tryptophan degradation in systemic lupus erythematosus. Immunobiology 2000;201:621-30. 18. Huengsberg M, Winer JB, Gompels M, Round R, Ross J, Shahmanesh M. Serum kynurenine-to-tryptophan ratio increases with progressive disease in HIV-infected patients. Clin Chem 1998;44:858-62. 19. Schrocksnadel H, Baier Bitterlich G, Dapunt O, Wachter H, Fuchs D. Decreased plasma tryptophan in pregnancy. Obstet Gynecol 1996;88:47-50. 20. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurements with the Folin phenol reagent. J Biol Chem 1951;193:265-75.
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21. Kudo Y, Boyd CAR, Sargent IL, Redman CWG. Modulation of indoleamine 2,3-dioxygenase by interferon-γ in human placental chorionic villi. Mol Hum Reprod 2000;6:369-74. 22. Chon SY, Hassanain HH, Gupta SL. Cooperative role of interferon regulatory factor 1 and p91 (STAT1) response elements in interferon-gamma-inducible expression of human indoleamine 2,3-dioxygenase gene. J Biol Chem 1996;271:17247-52. 23. Tolstrup AB, Bejder A, Fleckner J, Justesen J. Transcriptional regulation of the interferon-gamma-inducible tryptophanyltRNA synthetase includes alternative splicing. J Biol Chem 1995;270:397-403. 24. Yuan W, Collado Hidalgo A, Yufit T, Taylor M, Varga J. Modulation of cellular tryptophan metabolism in human fibroblasts by transforming growth factor-beta: selective inhibition of indoleamine 2,3-dioxygenase and tryptophanyl-tRNA synthetase gene expression. J Cell Physiol 1998;177:174-86.
O
25. Sikkema JM, van Rijn BB, Franx A, Bruinse HW, de Roos R, Stroes ES, et al. Placental superoxide is increased in pre-eclampsia. Placenta 2001;22:304-8. 26. Myatt L, Rosenfield RB, Eis AL, Brockman DE, Greer I, Lyall F. Nitrotyrosine residues in placenta: evidence of peroxynitrite formation and action. Hypertension 1996;28:488-93. 27. 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. 28. Takikawa O, Habara Ohkubo A, Yoshida R. IFN-gamma is the inducer of indoleamine 2,3-dioxygenase in allografted tumor cells undergoing rejection. J Immunol 1990;145:1246-50. 29. Kudo Y, Boyd CAR, Sargent IL, Redman CWG. Tryptophan degradation by human placental indoleamine 2,3-dioxygenase regulates lymphocyte proliferation. J Physiol 2001;535:207-15.
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Key words: Indoleamine 2,3-dioxygenase, tryptophan, preeclampsia, lymphocyte, placenta
Indoleamine 2,3-dioxygenase is a widely distributed enzyme that catabolizes tryptophan. It is characterized best in monocytes and macrophages but can be induced in many cell types, such as endothelial cells,1 epithelial cells,2 fibroblasts,3 or T cells.4 It is primarily induced by interferon gamma (IFN-γ), but other proinflammatory stimulants are also effective.5 By reducing the availability of tryptophan, indoleamine 2,3-dioxygenase is thought to inhibit intracellular and other infections,6 the growth of malignant cells,7 and immune cell function.8,9 Indoleamine 2,3-dioxygenase is also expressed in the murine and human placentas.10,11 In the pregnant mouse, it is thought to have a crucial role in inhibiting
From the Department of Human Anatomy and Geneticsa and the Nuffield Department of Obstetrics and Gynaecology,b University of Oxford. Supported by Action Research. Y. K. was supported by Oxford Kobe Scholarship. Received for publication June 17, 2002; revised August 23, 2002; accepted October 11, 2002. Reprints not available from the authors. © 2003 Mosby, Inc. All rights reserved. 0002-9378/2003 $30.00 + 0 doi:10.1067/mob.2003.156
the activation of maternal T cells that are specific for the fetus.10,12 It is not known whether comparable mechanisms, which might control events in early pregnancy, are applicable in women. Nor is it known whether placental indoleamine 2,3-dioxygenase has a role in late (established) human pregnancy or contributes to pregnancy disorders such as preeclampsia. Preeclampsia is a dangerous, common, unpredictable, and highly variable complication of the second half of pregnancy, labor, or the early puerperium. The presence of a placenta is necessary to cause the disorder. The placental problem appears to be a relative failure of the uteroplacental circulation owing to spiral artery disease, with consequent oxidative stress and diminished placental function.13 The diversity of the maternal syndrome results from the circulatory and other disturbances, resulting from diffuse maternal endothelial cell dysfunction.14 We have more recently shown that the endothelial dysfunction is a single aspect of a generalized systemic maternal inflammatory response15,16 that also affects circulating leukocytes. Moreover, this inflammatory response is detected in normal pregnancy. It is not qualitatively different from that in preeclampsia but is less intense. We have proposed that preeclampsia devel719
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Table I. Characteristics of the women studied
Age (y) Nulliparas (%) Gestational ages (wk) Mean arterial pressure (mm Hg)* No. with proteinuria
Normal pregnancy (n = 12)
Preeclampsia (n = 12)
Nonpregnant (n = 9)
29.7 ± 3.5 75 35.5 ± 2.8 84.4 ± 5.7 0
26.9 ± 3.9 75 34.6 ± 3.2 113.7 ± 8.4† 12
28.1 ± 4.3 — — — —
Values are given as mean ± SD. *Calculated as diastolic pressure + pulse pressure/3. †P < .001 compared with normal pregnancy (Wilcoxon test).
ops when the systemic inflammatory process causes one or other maternal systems to decompensate. In medical conditions such as systemic lupus erythematosus17 or HIV infection,18 characterized by a systemic inflammatory response, tryptophan degradation appears to be enhanced, causing an increase in the ratio of plasma kynurenine (a degradation product of tryptophan) to tryptophan. The plasma concentration of tryptophan declines progressively as a function of gestational age.19 This has not previously been related to concurrent plasma kynurenine concentrations. An increase in the kynurenine/tryptophan ratio would be consistent with the systemic inflammatory response of normal pregnancy. If true, then in preeclampsia with its greater systemic inflammatory response, it would be predicted that the plasma kynurenine/tryptophan ratio would be further increased. However, if placental indoleamine 2,3dioxygenase function were the dominant influence, then the greater inflammatory response of preeclampsia (associated with poorer placental function) could be associated with a lower plasma kynurenine/tryptophan ratio and lower placental indoleamine 2,3-dioxygenase activity. Then, it could be hypothesized that the excessive systemic inflammatory response of preeclampsia represented a failure of a placental mechanism (indoleamine 2,3-dioxygenase depletion of tryptophan) that would be expected to down-regulate immune cell functions. To address these issues we studied placental indoleamine 2,3-dioxygenase activity and messenger RNA (mRNA) expression as well as indices of tryptophan catabolism in pregnant women with or without preeclampsia. Material and methods Plasma samples. Samples from 12 women with preeclampsia, 12 appropriately matched women with normal pregnancy, and 9 healthy nonpregnant women, not taking any medication, were studied with ethical committee permission. Preeclampsia was defined as new proteinuric hypertension of pregnancy (diastolic blood pressure raised above 90 mm Hg on at least two occasions 6 hours apart, combined with new proteinuria of at least 2+ on dipstick testing in the proved absence of a urinary tract
infection), both changes regressing remotely after delivery. The normal pregnant and preeclamptic women were well matched (Table I). Samples of heparinized blood were centrifuged immediately after collection. Plasma was vortexed with a one-tenth volume of ice-cold 2.4 mol/L perchloric acid. The mixture was chilled on ice for 15 minutes and centrifuged at 10000g for 3 minutes. The clear protein-free supernatant was used for high-performance liquid chromatography (HPLC) analysis. HPLC analysis of L-tryptophan and L-kynurenine. Concentrations of L-tryptophan and L-kynurenine were analyzed by HPLC. The system consisted of a Kontron 420 pump, a Kontron 460 autosampler, and a Kontron 432 variable wavelength detector (Watford, UK) with the Spherisorb S5-ODS1 column, 4.6 150 mm (Waters, Milford, Mass). The mobile phase consisted of 40 mmol/L citrate buffer (pH 2.25) and 50% methanol and 0.4 mmol/L sodium dodecylsulfate, which were used after filtration through a 0.45-µm membrane filter and degassed by a vacuum aspirator. A 20-µL volume of protein-free extract was injected on the column, chromatographed at a flow rate of 2.0 mL/min–1, and detected at 365 nm for Lkynurenine and at 280 nm for L-tryptophan. The minimum amount of L-kynurenine and L-tryptophan reproducibly detected was 20 pmol and calibration was linear up to 10 nmol of L-kynurenine or of L-tryptophan. Culture of villous tissue. Placentas were obtained (with ethical committee approval) from 8 normal and from 14 pregnancies associated with preeclampsia. Not all placentas were from the same donors as the plasma samples. Preeclampsia was defined as described previously. In this group age (years), gestational age (weeks) and mean arterial pressure (mm Hg) were 29.2 ± 5.7, 36.8 ± 1.3, and 111.9 ± 9.7 (mean ± SD), respectively. Placentas were collected within 15 minutes 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 solution (PBS) containing 100 U/mL–1 penicillin and 100 U/mL–1 streptomycin. The chorionic villi were then dissected into small pieces (a single piece was approximately 5 mg). All these procedures were carried out at less than 4°C. Three pieces of chorionic villi were placed on a polyester mesh
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Table II. Tryptophan and kynurenine concentrations and the ratio of kynurenine to tryptophan in plasma from women with preeclampsia or normal pregnancies and from nonpregnant women Normal pregnancy (n = 12) Tryptophan (µmol/L) Kynurenine (µmol/L) Kynurenine/tryptophan
32.7 ± 4.8 1.12 ± 0.17 0.034 ± 0.004
Preeclampsia (n = 12)
Nonpregnant (n = 9)
42.8 ± 6.9* 1.02 ± 0.22 0.024 ± 0.005*
53.0 ± 9.8†‡ 1.17 ± 0.28 0.022 ± 0.004‡
Concentrations of tryptophan and kynurenine in plasma were analyzed by HPLC as described in Methods. Values are given as mean ± SD. *P < .001 compared with normal pregnancy (Wilcoxon test). †P < .001 compared with preeclampsia (Mann-Whitney U test). ‡P < .001 compared with normal pregnancy (Mann-Whitney U test).
(Netwell 500 µm mesh, Costar, NY) and cultured in 35mm plastic culture dishes at 37°C in an atmosphere of 5% carbon dioxide and 95% air for 36 hours. RPMI medium 1640 was used as the culture medium, with added 5% fetal bovine serum, 100 U/mL–1 penicillin, 100 U/mL–1 streptomycin, and IFN-γ or vehicle. Cultures were conducted in triplicate. The number of placentas used in each experiment is mentioned in the figure and table legends. The conditioned media were then used for HPLC analysis after extraction with perchloric acid or for culture of peripheral blood mononuclear cells. Assay of indoleamine 2,3-dioxygenase. The fresh or cultured pieces of chorionic villi were washed three times, suspended in ice-cold PBS, and disrupted by sonication for 30 seconds in an ice bath at a power of 100 watts. The homogenate was centrifuged at 800g for 10 minutes at 4°C to remove unbroken fragments of the tissue. The supernatant was then centrifuged at 15000g for 15 minutes at 4°C. The resultant supernatant was used for the colorimetric assay of indoleamine 2,3-dioxygenase activity, as described previously.11 RNA extraction and reverse transcription–polymerase chain reaction (RT-PCR) analysis. Indoleamine 2,3-dioxygenase, tryptophanyl-tRNA synthetase and signal transducer and activator of transcriptional 1 (STAT1) gene expression were analyzed by semiquantitative RT-PCR by using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as an internal standard. Total RNA was extracted from fresh or cultured chorionic villi with or without IFN-γ by using QuickPrep Total RNA Extraction Kit according to the manufacturer’s protocol. RNA samples were treated with deoxyriboneuclease I before RT-PCR to remove any contaminating DNA. The primers used in the subsequent RT-PCR were as follows: indoleamine 2,3dioxygenase, forward, 5´-TGCTAAACATCTGCCTGATC3´ and backward, 5´-GGAGCAATTGACTCAAATCA-3´; tryptophanyl-tRNA synthetase, forward, 5´-AGCTCAACTGCCCAGCGTGACC -3´ and backward, 5´-CAGTCAGCCTTGTAATCCTCCCCC-3´; STAT1, forward, 5´-AAGGTGGCAGGATGTCTCAGTG-3´ and backward, 5´-TGGTCTCGTGTTCTCTGTTCTG-3´; GAPDH, for-
ward, 5´-CGGGAAGCTTGTGATCAATGG-3´ and backward, 5´-GGCAGTGATGGCATGGACTG-3´. The expected size of the PCR products were 144 bp for indoleamine 2,3dioxygenase, 314 bp for tryptophanyl-tRNA synthetase, 564 bp for STAT1, and 358 bp for GAPDH. One microgram of RNA was reverse transcribed into complementary DNA (cDNA) with the use of oligo(dT)12-18 primer. The reverse transcription reaction, containing 500 µmol/L dNTP, 25 µg/mL-1 oligo(dT)12-18 primer, 10 U/µL-1 MMLV reverse transcriptase, 3 mmol/L magnesium chloride, 75 mmol/L potassium chloride, 10 mmol/L dithiothreitol, and 50 mmol/L TRIS (tris-[hydroxymethyl]-aminomethane)–hydrochloric acid (pH 8.3), was sequentially incubated at 25°C for 10 minutes, at 42°C for 50 minutes, and at 70°C for 15 minutes 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–1 Taq DNA polymerase, 1.5 mmol/L magnesium chloride, 50 mmol/L potassium chloride, and 20 mmol/L TRIS–hydrochloric acid (pH 8.4). The PCR conditions were 94°C for 3 minutes, 60°C for 1 minute, and 72°C for 2 minutes; then 25 cycles (for indoleamine 2,3-dioxygenase, tryptophanyl-tRNA synthetase and STAT1) and 22 cycles (for GAPDH) of 94°C for 1 minute, 60°C for 1 minute, and 72°C for 2 minutes; followed by a 10-minute 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 genes. PCR products were separated on a 2% agarose gel. Gels were stained with ethidium bromide. The intensity of either the target gene or GAPDH band for each sample was quantitated by using a gel documentation and analysis system (GDS8000, Ultra-Violet Products, Cambridge, UK) and the ratio of the target gene to GAPDH was used as a normalized measure of the target gene.
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Fig 2. Peripheral blood mononuclear cell proliferation in medium previously conditioned by culture with villous explants. Peripheral blood mononuclear cells were cultured for 72 hours either in nonconditioned medium or in medium previously conditioned by culture of villous explants from normal and preeclamptic placenta with 1000 U/mL–1 IFN-γ (open bars) or vehicle (closed bars) for 36 hours. [3H]Thymidine incorporation was then determined. Data represent mean ± SD of quadruplicate assays performed with 8 normal and 11 preeclamptic placentas. Asterisk, Significantly different from normal pregnancy. Fig 1. Indoleamine 2,3-dioxygenase, tryptophanyl-tRNA synthetase, and STAT1 mRNA expression level in placental villous tissue. Total RNA was extracted either from fresh (Fr) or cultured villous tissue with (Cul [+]) or without (Cul [–]) 1000 U/mL–1 IFN-γ for 36 hours. Levels of indoleamine 2,3-dioxygenase mRNA (a), tryptophanyl-tRNA synthetase (b), STAT1 (c), and GAPDH mRNA (d) were analyzed by RT-PCR. Results presented are from a single representative experiment.
Peripheral blood mononuclear cell culture and proliferation assay. Human peripheral blood mononuclear cells were isolated from healthy, volunteer, nonpregnant donors by Ficoll-Hypaque gradient centrifugation. The cells (2 105 cells per well) were cultured in media conditioned by prior culture with villous tissue and stimulated with 5 µg/mL–1 phytohemagglutinin in 96-well flat-bottom plates at 37°C in an atmosphere of 5% carbon dioxide and 95% air for 72 hours. Peripheral blood mononuclear cell proliferation was determined by pulse labeling with tritiated thymidine (0.037 MBq/well) for 12 hours. Protein estimation. Protein concentration of placental homogenate was determined with the method of Lowry et al20 by using bovine serum albumin as a standard. Statistical analysis. The measurements with samples from preeclampsia and control normal pregnancy were compared by using the Wilcoxon test. Other comparisons used the Mann-Whitney U test. P < .01 was considered statistically highly significant; P < .05 was considered statistically significant. Chemicals. [Methyl-3H] thymidine (25.0 Ci mmol–1 or 925 GBq mmol–1) was purchased from Amersham Life Science (Amersham, UK). Human recombinant IFN-γ and phytohemagglutinin were obtained from SigmaAldrich Chemical (Poole, UK), and tissue culture supple-
ments were from Gibco BRL (Paisley, UK). QuickPrep Total RNA Extraction Kit was purchased from Amersham Pharmacia Biotech (Rainham, UK), Moloney murine leukemia virus (M-MLV) reverse transcriptase, oligo(dT)12-18 primer, deoxynucleotide 5´-triphosphate (dNTP), and Taq DNA polymerase were from Gibco BRL, and deoxyribonuclease I (DNase I) was from Promega (Southampton, UK). All other chemicals were of the highest purity commercially available. Results Tryptophan concentration in plasma. Table II summarizes the results of HPLC analysis of tryptophan and kynurenine concentrations in plasma. Tryptophan concentrations in plasma samples taken from both groups of pregnant women were significantly lower than those from women who were not pregnant. However, plasma samples from women with normal pregnancy also had significantly lower tryptophan concentrations than those from women with preeclampsia. Plasma kynurenine concentrations showed the converse patterns. Hence, the ratios of indoleamine 2,3-dioxygenase product (plasma kynurenine) to substrate (plasma tryptophan), an index of tryptophan catabolism, were significantly increased in normal pregnant women compared either with women who were not pregnant or with women with preeclampsia. The ratios of kynurenine to tryptophan for women with preeclampsia were not different from those for women who were not pregnant. Indoleamine 2,3-dioxygenase activity and mRNA expression level in placental villous tissue. The indoleamine 2,3-dioxygenase activity in fresh placental villous tissue
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Table III. Indoleamine 2,3-dioxygenase activity in placental villous tissue IDO activity (nmol/mg/min) Treatment Fresh Cultured Nil IFN-γ
Normal pregnancy (n = 8)
Preeclampsia (n = 14)
0.48 ± 0.06
0.29 ± 0.04*
0.47 ± 0.02 1.58 ± 0.04 (329.1 ± 43.3)
0.30 ± 0.04* 0.76 ± 0.14* (258.8 ± 48.3†)
Villous explants were cultured with or without 1000 U/mL–1 IFN-γ for 36 h. Indoleamine 2,3-dioxygenase (IDO) activity in the tissue extract of fresh or cultured villous explants was determined colorimetrically as described in Methods. Percentage stimulation is given in the parentheses. Values are mean ± SD of triplicate assay from indicated number of placentas. *Highly significantly different from normal pregnancy (P < .01). †Significantly different from normal pregnancy (P < .05).
Table IV. Relative quantitation of indoleamine 2,3-dioxygenase, tryptophanyl-tRNA synthetase, and STAT1 mRNA expression level in placental villous tissue mRNA expression level (% of control) Treatment Normal pregnancy Fresh Cultured Nil IFN-γ Preeclampsia Fresh Cultured Nil IFN-γ
IDO
WRS
STAT1
100
100
100
103.7 ± 6.9 212.3 ± 19.6
107.7 ± 4.3 207.5 ± 13.3
107.0 ± 5.3 302.3 ± 23.3
55.8 ± 5.3*
106.1 ± 10.6
109.5 ± 7.3
52.1 ± 5.3* 95.9 ± 8.3*
103.6 ± 9.8 216.1 ± 10.3
102.9 ± 6.3 322.9 ± 22.3
The intensity of both the each target gene and the GAPDH band for each sample was quantitated by using a gel documentation and analysis system of PCR products and the ratio of the two was used as a normalized expression value of each target gene. Data represent the mean ± SD of experiments performed with 8 normal and 8 preeclamptic placentas, expressed as percentage of control (ie, values for fresh villous tissue of normal placenta). IDO, Indoleamine 2,3-dioxygenase; WRS, tryptophanyl-tRNA synthetase. *Significantly different from normal pregnancy.
from preeclampsia was significantly lower than from normal pregnancy (Table III). When villous tissue explants were cultured for 36 hours with or without IFN-γ at 1000 U/mL–1 (maximally stimulating indoleamine 2,3-dioxygenase expression21), indoleamine 2,3-dioxygenase activity was still significantly lower; the percentage stimulation in villous tissue from preeclampsia was also significantly less than in normal pregnancy. Indoleamine 2,3-dioxygenase mRNA expression levels were studied either in fresh villous tissue or in cultured villous tissue with or without IFN-γ stimulation (Fig 1). Table IV shows the relative quantitation of gel analysis normalized by GAPDH. Compared with normal pregnancy, the level of indoleamine 2,3-dioxygenase mRNA expression was approximately half of that in fresh villous tissue from preeclampsia. Treatment with a maximally effective concentration of IFN-γ (1000 U/mL–1) induced a 2.1-fold increase of indoleamine 2,3-dioxgenase mRNA expression in villous tissue from normal pregnancy. However, in preeclampsia, IFN-γ had a smaller (1.7-fold) stimulatory effect on mRNA expression. To analyze the mechanisms involved,
expression of mRNA encoding tryptophanyl-tRNA synthetase and STAT1 either in fresh villous tissue or in cultured villous tissue with or without IFN-γ stimulation was determined (Fig 1). STAT1 is required for IFN-γ-dependent transcription22 and tryptophanyl-tRNA synthetase expression is induced by IFN-γ through the same pathway as indoleamine 2,3-dioxgenase.23 In contrast to indoleamine 2,3-dioxgenase mRNA expression, there were no differences in mRNA expression levels for STAT1 or tryptophanyl-tRNA synthetase between normal and preeclamptic placentas, either in fresh or in cultured villi, with or without IFN-γ stimulation (Table IV). Tryptophan catabolism by indoleamine 2,3-dioxygenase in placental villous tissue. Concentrations of tryptophan and kynurenine in the conditioned medium after 36 hours of in vitro culture of placental villous explants either from normal or from preeclamptic pregnancy were determined (Table V). As predicted from the results shown in Table III, the presence of IFN-γ in the culture medium stimulated tryptophan degradation, with a marked increase in kynurenine concentration both in
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Table V. Tryptophan catabolism by indoleamine 2,3dioxygenase in placental villous explants Treatment Nonconditioned Conditioned Normal pregnancy Nil IFN-γ Preeclampsia Nil IFN-γ
Fig 3. Schematic representation of the role of placental indoleamine 2,3-dioxygenase in human pregnancy.
normal or in preeclampsia villous tissue. However, in tissue from preeclamptic women, the degradation of tryptophan was significantly less with production of significantly less kynurenine, in the presence or absence of IFN-γ. Peripheral blood mononuclear cell proliferation in the medium conditioned by culture of placental villous tissue. To study the functional consequence of suppressed indoleamine 2,3-dioxygenase expression in preeclamptic placental villous tissue, peripheral blood mononuclear cell proliferation activity was determined after the culture in the same medium of which tryptophan concentration was measured in Table V. Thymidine incorporation into DNA is significantly inhibited by conditioned medium. With medium conditioned in the presence of IFN-γ inhibition is significantly increased. Thymidine incorporation is significantly greater in the conditioned medium of IFN-γ–treated explants from preeclampsia than from normal pregnancy. Thus, the inhibitory effect of the conditioning medium on peripheral blood mononuclear cell activation is significantly reduced in preeclampsia compared with normal villous tissue (Fig 2). Comment The increased plasma kynurenine-to-tryptophan ratio in normal pregnancy compared with nonpregnancy is consistent with the evidence that normal third-trimester pregnancy is associated with a systemic inflammatory response.16 Although there is a significantly more intense inflammatory response in preeclampsia,15 we found that the plasma kynurenine/tryptophan ratio is not increased but nearer to that of nonpregnant women. This is associated with a suppressed expression of placental indoleamine 2,3-dioxygenase in preeclampsia. Not only is functional enzyme activity suppressed in vitro (Table V), but the level of indoleamine 2,3-dioxygenase gene expression is also decreased. However, IFN-γ could still stim-
Tryptophan (µmol/L)
Kynurenine (µmol/L)
20.1 ± 0.3
Not detectable
11.7 ± 0.5 1.9 ± 0.2
5.3 ± 0.4 13.2 ± 0.6
16.2 ± 1.2* 4.6 ± 0.7*
2.9 ± 0.2* 9.9 ± 0.4*
Villous explants from normal and preeclamptic placenta were cultured with 1000 U/mL–1 IFN-γ or vehicle for 36 hours. Concentrations of tryptophan and L-kynurenine in the conditioned medium were analyzed by HPLC. Data represent the mean ± SD of triplicate assays performed with 8 normal and 11 preeclamptic placentas. *Significantly different from normal pregnancy.
ulate activity in vitro (Table III and Fig 1), albeit from a lower baseline. The expression of other IFN-γ inducible genes (ie, tryptophanyl-tRNA synthetase and STAT1) was normal and responded appropriately to exogenous IFN-γ (Fig 1). The enzyme activity could also be affected by posttranscriptional events (eg, message stability, protein synthesis) or by molecules, which can regulate enzyme activity. Although each of these steps remains to be studied, our unpublished work shows that the sequence of the indoleamine 2,3-dioxygenase gene, including the promoter region, in cord blood obtained from infants from preeclamptic pregnancies is normal. Both interleukin-421 and transforming growth factor24 β can down-regulate expression of indoleamine 2,3dioxygenase. However, it is not known whether the expression of either cytokine is increased in the preeclampsia placenta. In preeclampsia the placenta is characteristically subjected to oxidative25 and nitrosative stress.26 It is known that the superoxide anion acts as a cofactor for indoleamine 2,3-dioxygenase, which, together with its metabolic products, has antioxidant activity.27 However, these functions would not be predicted to decrease expression of indoleamine 2,3-dioxygenase. Hence, the cause of the reduced placental indoleamine 2,3-dioxygenase expression and activity is unknown at present. But the implication is that placental indoleamine 2,3-dioxygenase activity determines, at least in part, maternal systemic tryptophan concentrations. Depletion of tryptophan by indoleamine 2,3-dioxygenase is a general mechanism of inhibiting cell growth and thought to be how IFN-γ, produced by leukocytes, stimulates tumor rejection28 and inhibits intracellular microorganisms.6 Tryptophan depletion also inhibits lymphocyte proliferation, including that of T lymphocytes (this inhibition is due to arrest of cell cycle progression)8,29 and has the potential to down-regulate inflammatory responses.
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This mechanism has been shown to protect the allogeneic murine conceptus from T cell–mediated rejection.10,12 In nonpregnant women, increases in the systemic kynurenine/tryptophan ratio are a feature of inflammatory disorders and ascribable to systemic activation of indoleamine 2,3-dioxygenase in monocytes and possibly endothelium.17 In the peripheral blood of normal pregnant women in the third trimester, leukocyte (including lymphocyte) activation is known to be exaggerated compared with the findings in matched nonpregnant women.15,16 The increased kynurenine/tryptophan ratio found in normal pregnancy is similar to that of other disorders characterized by systemic inflammation where it can be ascribed to indoleamine 2,3-dioxygenase activity of circulating leukocytes. However, in preeclampsia the systemic inflammatory response is even more intense than in normal pregnancy,15 so it would have been predicted that the kynurenine/tryptophan ratio would have been increased further. Instead, the converse was observed (Table II). This paradox can be explained if the systemic kynurenine/tryptophan ratio in preeclamptic women relative to normal pregnant women depends on placental indoleamine 2,3-dioxygenase expression and activity, which are reduced (Tables III and IV). The conclusion supports the concept that, in pregnancy, placental indoleamine 2,3-dioxygenase activity, at least in part, regulates the systemic kynurenine/tryptophan ratio. These findings therefore provide evidence for a mechanism whereby placental indoleamine 2,3-dioxygenase may down-regulate the maternal systemic inflammatory response intrinsic to normal pregnancy, a process which, our evidence suggests, becomes disrupted in preeclampsia. A general proposal for the role of placental indoleamine 2,3-dioxygenase in suppressing the maternal systemic inflammatory response during human pregnancy and in the pathogenesis of preeclampsia is illustrated in Fig 3. The systemic inflammatory response stimulated by normal pregnancy would be excessive without the physiologic suppression caused by placental indoleamine 2,3dioxygenase. Even so, in the third trimester, it is more intense than in nonpregnancy.15 In preeclampsia, the response is released because of decreased placental indoleamine 2,3-dioxygenase activity. It is thought that the features of the preeclampsia syndrome arise from this excessive systemic inflammatory response.13 The results in this article raise the possibility that novel therapeutic intervention in preeclampsia might usefully be focused on manipulation of plasma tryptophan concentration. We thank Research Midwives Hazel Coburn and Carol Simms for their tireless help in obtaining clinical samples. REFERENCES
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