Characteristics of interferon induced tryptophan metabolism in human cells in vitro

Biochimica et Biophysica Acta, 1012 (1989) 140-147 Elsevier 140 BBAMCR 12485 Characteristics of interferon induced tryptophan metabolism in human cells in vitro Gabriele W e r n e r - F e l m a y e r , Ernst R o b e r t W e r n e r , D i e t m a r Fuchs, A r n o H a u s e n , Gilbert R e i b n e g g e r a n d H e l m u t W a c h t e r Institute for Medical Chemistry and Biochemistry, University of lnnsbruck, Innsbruck (Austria) (Received 30 January 1989) Key words: Tryptophan metabolism; Tumor cell line; Indoleamine; Interferon; (Human cell culture) interferon-~/-induced tryptophan metabolism of human macrophages was compared to ten human neoplastic cell lines of various tissue origin and to normal dermal huma.-~ fibroblasts. Tryptophan and metabolites were determined in supernatants of cultures, after incubation for 48 h, by high-performance liquid chromatography with ultraviolet and fluorescence detection. With the exception of two cell lines (Hep G 2, hepatoma and CaCo 2, colon adenocarcinoma) in all of the ten other cells and cell lines tryptophan degradation was induced by interferon-7. Five of these ten formed 0nly kynurenlne (SK-N-SH, neuroblastoma; T 24, J 82, bladder carcinoma; A 431, epidermoid carcinoma; normal dermal fibroblasts), three formed kynuranine and anthranilic acid (U 138 MG, glioblastoma; SK-HEP-I, hepatoma; A 549, lung carcinoma). Only one line, A 498 (kidney carcinoma) showed the same pattern of metabolites as macrophages (kynurenine, anthranilie acid and 3-hydroxyanthranilic acid). Interferon-¥ regulated only the activity of indoleamine 2,3.dioxyganase. All other enzyme activities detected were independent of interferon-y, as shown by the capacity of the cells to metabolize L-kynurenine or N-formyI-L-kynurenine. Increasing the extraceilular L-tryptophan concentration resulted in a marked induction of tryptophan degradation by maerophages. Contrarily, a significant decrease of the tryptophan degrading activity was observed when the extracellular L-tryptophan concentration was increased ?,-fold with SK-N-SH, T 24 and J 82, 4-fold with A 431 and A 549 and 10-fold with U 138 MG and SK.HEP-L The activity was unaffected by extraceilular L-tryptophan with dermal fibroblasts and A 498. Though interferon-,/ was the most potent inducer of tryptophan metabolism, interferon-a a n d / o r -fl showed small but distinct action on some of the cells. |n all cells which reacted to interferon-¥ by enhanced expression of class i a n d / o r class !I major histocompatibility complex antigens tryptophan degradation was also inducible. These results demonstrate that induction of indoleamine 2,3-dioxygenase is a common feature of interferon-¥ action, that the extent of this induction is influenced by extracellular l.-tryptophan concentrations and that indoleamine 2,3-dioxygenase is the only enzyme in the formation nf 3-hydroxyanthranilie acid from tryptophan which is regulated by interferon.,/. Introduction Indoleamine 2,3-dioxygenase (IDO, EC 1.13.11.17) initiates the degradation of L-tryptophan via the Abbreviations: IDO, indoleamine 2,3-dioxygenase (EC 1.13.11.17): IFN, interferon; MHC, major histocompatibility complex; HLA, human leukocyte antigen; MEM, Eagles minimum essential medium; DMEM, Dulbecco's minimum essential medium; RPMI, Roswell Park Memorial Institute tissue culture medium; PBS, Dulbecco's phosphate-buffered saline; FCS, fetal calf serum; IgG (H + L), immunoglobulin G heavy and fight chain; FACS, fluorescence-activated cell sorter; FITC, fluorescein isothiocyanate Correspondence: H. Wachter, Institute for Medical Chemistry and Biochemistry, University of lrmsbruck, Fritz-PregI-Strasse 3, A-6020 innsbruck, Austria. kynurenine pathway in various extrahepatic tissues of mammals [1-3]. The activity of this enzyme can be markedly increased by interferon as was first demonstrated in mome lung slices [4]. The activation of IDO was suggested to be involved in host cell defense ~echanisms directed against intracellular parasites [5,6] and in the antipro!iferative effect of interferon-3, on human tumour cell lines [7,8]. It is unclear whether these phenomena are mediated via deprivation of L-tryptophan, via supply of nicotinic acid for pyridine nucleotide coenzyme biosynthesis or via formation of toxic metabolites. It has been demonstrated that human macrophages activated by interferon-y produce high amounts of 3hydroxyanthranilic acid from L-tryptophan which could possibly enhance their cytotoxic potential [9]. With other human cells, kynurenine was the only detectable de- 0167-4889/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division) 141 gradation product [10]. We therefore checked whether there were any essential differences of interferon-induced tryptophan degradation in macrophages in comparison with a panel of other human cells originating from various tissues. This was done by studying (i) the pattern of tryptophan metabolites produced upon interferon-y stimulation, (ii) the action of interferon-)' on enzyme activities of the kynurenine pathway, (iii) the re~ativity of cells upon other interferons, (iv) the influence of increasing the extracellular tryptophan concentration and (v) the expression of major histocompatibility complex (MHC) antigens as an independent additional parameter of interferon-), action. Materials and Methods Materials 4,6-Diamidino-2-phenylindole was obtained from Sigma (Munich, F.R.G.). Eagle's minimum essential medium (MEM) with Earle's sails base and Dulbecco's modified Eagle's medium (DMEM) with 4.5 g/1 glucose were purchased from Serva (Heidelberg, F.R.G.), fetal calf serum (FCS) was from Biochrom (Berlin, F.R.G.) and all other cell culture medium supplements were from Sigma. Roswell Park Memorial Institute tissue culture medium (RPMI) 1640 (endotoxin < 50 pmol/1) was from Biochrom. RPMI 1640 Select Amine Kit was from Gibco (Grand Island, NY, U.S.A.). Ficoll-Paque and Percoll were from Pharmacia (Uppsala, Sweden). Cell culture dishes and 96-well microtiter plates were from Falcon (Beckton Dickinson, Lincoln Park, N J, U.S.A.). Human recombinant interferon-a2b (IFN-a) from Escherichia coli with a specific activity of 2. l0 s U / m g protein was a product of Sehering Corp. (Kenilworth, N J, U.S.A.). Recombinant and natural human interferon-fl (IFN-fl) and -y (IFN-)') were a generous gift from Bioferon GmbH (Laupheim, F.R.G.). Recombinant interferon-B was from CHO-cells and had a specific activity of 3. l0 s U / m g protein. Recombinant human interferon-)' had an activity of 2-10 7 U / m g protein. Natural interferon-fl-1, from FS-4 cells (3. l0 s U / m g protein) and natural interferon-)' (2.10 7 U/nag) protein) were compared to recombinant interferons. LTryptophan was purchased from Calbiochem (Los Angeles, CA, U.S.A.), 3-hydroxy-DL-kynurenine and Lkynurenine were from Sigma, anthranilic acid was from Merck. N-Formyl-L-kynurenine was a gift from Professor R.R. Brown (Madison, WI, U.S.A.). 3-Hydroxyanthranilic acid, N-formylanthranilic acid and cinnabarinic acid were obtained from Professor W. Pfieiderer (Konstanz, F.R.G.). Quinolinic acid, 2-picolinic acid, kynurenic acid and xanthurenie acid were products of Fluka (Buchs, Switzerland). Pure bovine serum albumin was from Serva. The protein assay dye reagent was purchased from Bio-Rad (Richmond, CA, U.S.A.). Sources of antibodies were: Seralab (Sussex, U.K.) providing BI-3D3 (M_AS 114) and the anti-mouse IgG (H + L)-FITC conjugate (produced in sheep, affinity purified) and Beckton Dickinson (Mountain View, CA, U.S.A.) providing L 243 (anti HLA-DR). Ceils were obtained either directly from the American Type Culture Collection (Rockville, MD, U.S.A.) or were kindly supplied by various institutions of the medical faculty of the University of Innsbruck. Dermal fibroblasts were generously pro,tided by Professor M. Schweiger (Innsbruck, Austria). Buffy coats of healthy blood donors and human serum (pooled) were generously supplied by the blood transfusion unit of the University Hospital of Innsbruck. Cell cultivation Cells used in the course of this study were the neuroblastoma line SK-N-SH [11], the bladder transitional-cell carcinoma lines T 24 [121 and J 82 [13], the hepatoma line Hop G 2 [14], the liver adenocarcinoma line SK-HEP-1 [15l, the colon adenocarcinoma line CaCo 2 [16], the lung carcinoma line A 549 [17], the epidermoid carcinoma line A 43I [18], the kidney carcinoma line A 498 [18], the glioblastoma line U 138 MG [19L normal human skin fibroblasts during the 16th and 23rd passage and macrophages isolated from blood of healthy donors (buffy co~.ts). The ceil lines were selected on the basis of the organ distribution of their origin and the reported absence of HeLa cell contamination [16]. All cell lines were mycoplasmanegative as determined by staining with 4,6-diamidino2-phenylindole [20]. T 24 was grown in MEM with Earle's salts base supplemented with 10% FCS, 2 mmol/l L-glutamine, 100 IE/ml penicillin and 0.1 ng/ml streptomycin. The same medium with a further addition of 1 mmol/l pyruvic acid was used for SK-N-SH, A 498 and U 138 MG. A 431, A 549, CaCo 2, Hop G 2 and fibroblasts were cultivated in DMEM, supplemented as described for T 24 cells. SK-HEP-1 was grown in DMEM with supplements, plus pyruvic acid and J 82, for which an addition of 15% FCS was used. Monocytes were purified from buffy coats by density gradients centrifugation over Ficoll-Paque and subsequently over a discontinuous Percoll gradient and a final step of adherence to plastic as described previously [9]. Cells were cultivated in RPMI 1640 (low endotoxin) with 10% heat-inactivated pooled human serum, 2 mmol/1 L-glutamine and antibiotics (see above) in 24well multidishes. The resulting cell population was more than 99% monocytes as judged by nonspecific esterase staining on day 3 of culture and referred to further as macrophages. All cells were cultured at 37°C in humidified air containing 5% CO 2. Treatment of cells with additives Cells were seeded at a density of (1-5). 105 per well in 24-well multidishes containing I ml of culture medium 142 per well. When monolayers had reached confluency the medium was replaced by 1 ml of fresh culture medium containing interferons in a range of 1-1000 U/ml. Macrophages (about 3-105/well) were stimulated on day 3 of culture after replacement of culture medium. Increasing concentrations (50-1000 ~mol/l) of Ltryptophan were added to cultures together with or without an appropriate dose of interferon-y for testing the effect of the extracellular tryptophan concentration. L-Kynurenine (50 j~mol/1), N-formyl-L-kynurenine (100 /~mol/1), 3-hydroxy-kynurenine (50 pmol/l) and anthranilic acid (20 /~mol/l) were added to the culture medium in order to distinguish between interferon-dependent and constitutive steps of the kynurenine pathway. For macrophages these experiments were also carded out in tryptophan-free RPMI 1640 (Select Amine Kit, Gibco). All cultures were incubated for 48 h at 37°C. Supernatants were collected for HPLC analysis and cells of individual wells were detached by 0.02% EDTA in Dulbecco's phosphate-buffered saline without Ca 2+ or Mg 2+ (PBS). After centrifugation (5 rain, 3000 X g), pellets were resuspended in 0.5-1 mi of 0.05 mol/l H3PO4 and stored at - 2 0 ° C for protein determination. As macrophages can be easily damaged by removing them from plastic surfaces, these cells were scraped from the plates with a rubber policeman in a small volume of PBS. Samples were collected an'! H3PO.: was added (final concentration 0.05 reel/l). Protein determination The protein concentration of cell lysates (see above) was determined according to Bradford [21], modified for 96-well flat bottom microtiter plates. The absorbance of samples was measured with a Titertek Multiskan MCC 340 microplate reader (Flow Laboratories, Irvine, U.K.) at 620 nm. Lyophilized pure bovine serum albumin was used as protein standard. M H C antigen expression on human tumour cell lines For estimation of MHC class I (HLA-ABC) antigen expression the IgG 2a (determined by Ouchtedony immunodiffusion) mouse monoclonal antibody from the clone BI-3D3 reacting with a nonpolymorphic epitope on HLA heavy chain was used. HLA-DR, as a representative for MHC class II antigens, was stained with the IgG 2a mouse monoclonal antibody L 243 [22] detecting a nonpolymorphic site. For isotype control, the mouse IgG 2a monoclonal antibody 14-4-4S [23] recognizing mouse I-E K determinants which was produced in HB 32 (American Type Culture Collection, Rockville, U.S.A.) was used. BI-3D3 and 14-4-4S were applied as undiluted supematants (100/~1 to 106 cells), L 243 as purified immunoglobulin (0.5 pg in 20/ll PBS containing gelatine and 0.1~ azide was applied to 106 cells in 50/~1 DMEM/2% FCS/0.1% azide). Confluent monolayers were incubated for 72 h with 250 U / m l of interferon-y. Cells were detached by 0.05% trypsin/ 0.02% EDTA in PBS, washed once with culture medium containing 10% FCS and once with DMEM/2% FCS/0.1% azide. Aliquots of 5- 10 s to 5- 106 cells with a viability of more than 90%, as determined by Trypan blue exclusion, were reacted with saturating concentrations of the appropriate antibody for 30 rain on ice. After washing with DMEM/2% FCS/0.1% azide cells were counter-stained with the FITC-conjugated antimouse IgG (H + L) serum, diluted 1 : 100 in DMEM/2% FCS/0.1% azide for 40 rain on ice. Cells were washed twice with PBS/0.1% azide and finally fixed with 1% paraformaldehyde in PBS. For reagent control, the first incubation step was carried out with DMEM/2% FCS/0.1% azide. Human dermal fibroblasts, which are induced for enhanced class I and class II MHC aatigen expression by interferon-,t [24], were used as positive control. Fv~ analysis a fluorescence-activated cell sorter (FACS IIl, Beckton Dickinson) was used. 104 cells were analyzed fc,r each determination using identical settings. Data are e~pressed as the fluorescence intensity of 50% of the positively stained cells in relation to the isotype control and reagent control. Fluorescence intensities of the isotype control and the reagent control were identical in individual cell lines. Arbitrary fluorescence units were calculated according to 10 <n-"°)/52 (n ~chaL, nel number of 50~ of the positively stained cells of the specific staining, ~o = channel number of 50~ ot the positively stained cells of the isotype control; 52 channels represent a 10-fold difference in fluorescence intensity). H P L C methods The HPLC apparatus consisted of a liquid chromatograph 5500 (Varian, Paio Alto, CA, U.S.A.), a UV 200 variable wavelength detector (Varian), and an LS4 variable wavelength fluorescence detector (Perkin Elmer, Beaconsfield, U.K.) controlled by a data system 402 (Varian). Determination of tryptophan and metabolites in supernatants was carried out as described [9] with minor modifications. A 125 mm × 4 mm column protected with a 4 mm × 4 mm precolunm (LiChroCART, Merck, Darmstadt, F.R.G.) filled Ath reversed~phas¢ C-18 material (7 p m particle tze, LiChroSORB RP-18, Merck) was eluted with ,J.015 m o l / l potassium phosphate buffer (pH 6.4) containing 1.8% acetonitrile, at a flow rate of 0.8 ml/min. 10-50 pl crude supernatant were injected into the system. Tryptophan and metabolites were detected by ultraviolet (UV) or fluorescence (F1) detection as follows: tryptophan (UV 280 nm, FI 285/365 nm), N-formylanthranilic acid (FI 300/405 nm), kynurenine (UV 260 nm), 3-hydroxykynurenine (UV 235 nm), N-formylkynurenine (UV 260 nm), anthranilic acid (1=1300/405 nm), 3-hydroxyanthranilic acid (FI 323/414 nm), kynurenic acid (F1 325/405 nm), 143 xeu~thurenic acid (F1 350/460 nm). Detection Emits were 5 nmol/l for anthranilic acid and 3-hydroxyanthranilic acid, 10 nmol/1 for tryptophan, 50 nmol/1 for kynurenic and xanthurenic acid, 150 nmol/1 for N-formylanthranflic acid, 0.5/~mol/l for kynurenine, 1 /~mol/l for N-formylkynurenine and for 3-hydroxykynurenine. Determination of picolinic acid: A 250 m m x 4 mm SCX Partisil 10 column 0Vhatman, Clifton, N J, U.S.A.) was eluted with 0.01 M potassium phosphate buffer (pH 2.2) containing 5~ methanol at a flow rate of 0.8 ml/min. Picolinic acid was detected at UV 272 nm with a detection limit of 0.5/~mol/'l. Determination of quinolinic acid: a 250 nun x 4 mm SAX Partisil 10 column (Whatman) was eluted with 0.05 M potassium phosphate (pH 1.5) containing 10~ methanol at a flow rate of 2.0 ml/min. 3 ml of samples were concentrated on AASP-SAX cartridges (Anaiytithem, Harbor City, CA, U.S.A.), washed with 400 /~1 water and eluted with 200 #1 1 M H3PO4 (recovery of spiked quinolinic acid 94%). 50/~l of this concentrate was injected and detected by UV 272 nm with a detection limit of 0.5/zmol/l (related to the unconcentrated supernatant). Determination of cinnabarinic acid: a 250 mm x 4 mm SCX Partisil 10 column (Whatman) was eluted with 0.01 M potassium phosphate buffer (pH 2.2) with a 10 min gradient from 5-50~ methanol. Cinnabarinic acid was detected at UV 460 nm with a detection limit of 0.5 /~mol/1. Values are expressed as nmol of metabolites per mg of total cell protein of triplicate cultures + S.D. L-Tryptophan ,,,,-, ,. nO N-FormyI-L-Kynurenine ~> N-Formyl anthranilic acid L-Kynurenine ~ Anthranilic acid 3-Hydroxy-L-kynurenine ~ 3-Hydroxy-anthronilic acid Fig. 1. Schematic presentation of enzyme activities of Ihe kynurenine pathway found in the tested human cells and cell lines. 1, indoleamine 2,3-dioxygenase (EC 1.13.11.17); 2, kynurenine formamidase (EC 3.5.1.9); 3, kynurenine 3-monooxygenase (BC 1.14.13.9); 4, kynureninase (EC 3.7.1.3). the additional formation of anthranilic acid. Beside the macrophage, only A 498 also released 3-hydroxyanthranilic acid in addition to anthranilic acid and kynurenine. A 549 is peculiar in its accumulation of N-formylkynurenine and N-formylanthranilic acid in the supernatant, obviously due to a low formamidase activity. In all other cells and cell lines, N-formylkynurenine, the product of the I D a reaction, is not detectable but is quantitatively converted by formaafidase to kynurenine during the incubation period (Fig. 1). In A 549, the ratio of N-formylkynurenine to kynurenine changed frora approx. 1 : 1 after 48 h to 1 : 6 after 72 h (not shown). N-Formylanthranilic acid, a metabolite thus far not described as occurring in human cells or tissues, was characterized by comparison of fluorescence spectra to synthetic material. Further evidence for its identity comes from its formation from Results TABLE l Tryptophan metabolites released by interferon-'/ treated human cells and cell lines The pattern of metabolites produced by macrophages upon interferon-,/ stimulation was compared to that found with a panel of eleven human cell lines of different tissue origin. A schematic presentation of the metabolic activities observed is given in Fig. 1. In two of the cell lines tested, Hep G 2 (hepatoma) and CaCo2 (colon adenocarcinoma), oxidative tryptophan metabolism could not be induced by interferon-,/ during an incubation period of 48 and 72 hours, respectively. Tryptophan metabolites formed by the other ten cells and cell lines investigated upon interferon-), treatment are shown in Table I. With five of these interferon-responsive cell lines (SK-N-SH, neuroblastoma; T 24, J 82, bladder carcinoma; A 431, epidermoid carcinoma; normal dermal fibroblasts) kynurenine was the only detectable tryptophan metabolite. A 549 (lung carcinoma), U 138 MG (glioblastoma) and SK-HEP-1 (hepatoma) displayed kynureninase activity resulting in Tryptophan metabolites in supernatants of interferon-v treated human cells and cell lines On day 3, confluent monolayers and macrophages were stimulated with 100 U / m l interferon-y for 48 h. Supernatants were analyzed for tryptophan metabolites by HPLC (mean + S.D. of triplicate cultures). NFK, N-formylkynurenine; NFAA, N-formylanthranilic acid; Kyn, kynurenine; AA, anthranilic acid; 3HAA, 3-hydroxyanthranilic acid; N.D. not detectable. Cell line Metabolites (% of degraded tryptophan) NFK Macrophages N.D. A 498 N.D. NFAA N.D. N.D. Kyn AA 3HAA 28.2+ 2.4 1.60+0.2 19.1+2.3 24.9+ 1.8 2.3 +(~.8 29.8+2.0 A 549 48.8+5.3 7.9+0.2 37.1+ 0.7 3.2 +0.1 N.D. SK-HEP-1 U 138 MG N.D. N.D. N.D. N.D. 80.04- 5.3 3.4 +1.1 N.D. 96.5±18.5 1.7 +0.2 N.D. SK-N-SH T 24 J 82 A 431 Fibroblasts N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 69.1+ 104.7+ 83.2+ 102.3+ 80.4+ 2.6 4.3 8.1 4.0 3.1 N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. N.D. 144 TABLE II Enzyme activities of the kynurenine pathway in human cells: influence of interferon. 7 O~. day 3, confluent monolayers and macrophages were treated with either L-kynurenine (50 /zmol/l), 3-hydroxykynurenine (50 /tmol/I), anthranllic acid (20/tmol/l) and N-formyl-L-kynurenine(100 ~tmol/l), or with interferon-y (U 138 MG, SK-HEP-1 with 1000 U/ml, A 498 with 100 U/ml; A 549 with 10 U/ml; macrophages with 250 U/ml). After 48 h, metabolites in supematants were determined (triplicate cultures 4-S.D.). No degradation of medium tryptophan was observed in cultures treated with various metabolites (not shown). Addition of anthranilic acid did not result in the formation of 3-hydroxyanthranilic acid (not shown). NFK, N-formylkynurenine; NFAA, N-formylanthranilic acid; Kyn, kynurenine; AA, anthranilic acid; 3HAA, 3-hydroxyanthranilic acid; 3HK, 3-hydroxy-kynurenine. N.D., not detectable. Cell line/ Metabolites (nmol/mg protein) additive NFK NFAA Kyn AA 3HAA Macrophages IFN-'r Kyn 3HK N.D, N.D, N.D. N,D. N,D. N,D. 137.2±13.2 832.0 4-64.0 a N.D. 8.54-1.0 5.9 + 1.1 N,D. 108.~+ 4.8 107.9 4. 35.4 27.24. 5.5 A 498 IFN-v Kyn 3HK N.D. N.D. N.D, N,D. N,D. N,D. 79.94- 5.9 80.9 + 14.8 a N.D. 7.44-2.6 6.3 + 1.4 N,D. 93.44- 6.7 122.0 4.14.8 33.5 4.10.3 SK-HEpol IFN-y Kyn N.D. N.D. N,D. N,D. 107.24- 3.5 86.64- 2.0 a 2.64-0.2 3.34-0.2 N.D. N.D. 157.24-17.3 N.D. 134.1 4. 33.5 a 25.54-0.9 N.D. 17.2 4-1.6 118.94- 2.4 150.0 ± 25.9 a 115.5 4- 4.7 10.45:0.4 31.7 4. 6.6 12.8 ± 0.5 N.D. N.D. N.D. N.D. N.D. N,D. N,D. 494.1 4. 8.0 638.0 4-31.7 a 1.54.0.3 4.4 4-0.6 N.D. N.D. A 549 IFN-y Kyn NFK U 138 MG IFN-y Kyn a Non-metabolized remainder of added compound. supplied N-formylkynurenine by A 549 (see Table II). None of the other six metabolites tested (kynurenic, xanthurenic, cinnabarinic, picolinic and quinolinic acid and 3-hydroxykynurenine) was detectable in supernatants of interferon-~/-activated m~ erophages. Recovery of metabofites from d~graded tryptophan TABLE llI Potential of recombinant interferons to induce IDO in human cells and cell lines One day 3, confluent monolayers of cell lines and macrophages were incubated with various doses of interferons for 48 h. Tryptophan met~bolhes from tripli~te cultures were determined in supernatants by HPLC. Values are expressed as the mean sum of metabolites in nmol per mg cell protein±S.D. Metabolites were kynurenine for SK-N-SH, T24, J 82, A 431, fibroblasts; kynurenine, anthranilic acid for U 138 MG, SK-HEP-1; kynurenine, N-formylkynurenine, anthranilic acid, N-formylanthranilic acid for A 549; kynurenine, anthranilic acid, 3-liydroxyanthranilic acid for A 498 and macrophages. Tryptophan degradation was not inducible in Hep G 2 and CaCo 2. For details see Table I. N.D.., no tryptophan degrading activity was detectable. Cell line J 82 SK-HEP-I SK-N-SH U 138 MG A431 T24 A 549 Macrophages A498 Fibroblasts Control N.D. N.D. N,D, N.D, N.D, 200.04-36.0 N.D. N.D. 1.94- 0.4 N.D. IFN-a IFN-/3 IFN-y (1000 U/nil) (1000 U/ml) 10 U/ml N.D. N.D. N.D, N.D, N.D. 180.84-6.0 N.D. N.D. 5.5+1.5 30.8 + 7.0 N.D. N.D. N.D. N.D. N.D. 226.0+ 8.3 0.9+ 0.1 81.1 + 17.9 42.1+ 6.2 42.3 4- 6.5 a Medium tryptophan was totally degraded. 9.7 ± 0.1 N.D. 107.8 4-13.3 N.D. 18.8+ 2.6 908.3+ 8.8 310.9+21.1 117.1 4. 28.8 10.4+ 2.0 33.4 4- 2.9 100 U/ml 1000 U/ml 127,5 + 12.5 34.0± 2.4 394.9 4-18.6 154.64-25.7 > 500 a >1250 a > 350 a 195.2 4.15.0 > 300 a 941.0 ± 36.4 112.04-16.4 109.94- 3.8 427.5 4-26.0 > 500 a > 500 ~ >1250 a > 350 a > 330 a > 300 a 1072,0 4-26.5 145 was about 50~ for macrophages and A 498, 70~ for SK-N-SH and more than 80~ for the other cell lines (Table I). The lower recovery with A 498 and macrophages may be caused by the chemical lability of 3-hydroxyanthranilic acid under cultivating conditions. Enzyme activities of the kynurenine pathway: influence of interferon.`/ For investigating whether enzyme activities of the kynurenine pathway other than that of IDO were also regulated by interferon-`/, the capacity of cells to metabolize N-formyl-L-kynureninc, L-kynurenine, 3-hydroxy-DL-kynurenine and anthranilic acid was tested. The results were compared to that obtained with interferon-`/-activated cells (Table II). The addition of N-formyl.r.kyn~renine or l.-kynurenine to cells resulted in the formation of the other metabolites to a degree comparable to interferon-,/-treated cultures (Table If). No L-tryptophan was degraded in these unstimulated cultures. With macrophages, the same experiments were also performed using culture medium which was free of r-tryptophan. Degradation rates for L-kynurenine, 3-hydroxy-DL-kynurenine and anthranilic acid were exactly the same in interferon-,/-stimulated as those in unstimulated calls (not shown). Thus, only the first step of the sequence, i.e., the IDO reaction, is regulated by interferon-`/. The other enzymes are constitutively present in the cells. 3-Hydroxyanthranilic-acid was formed from the Lkynurenine supplied and, though less efficiently, from 3-hydroxy-DL-kynurcninc (Table II) but not from the anthranilic acid supplied (not shown). This indicates that macrophages and A 498 have a kynurenine 3monooxygenase activity, degrading tryptophan via the classical kynurenine pathway (Fig. 1) rather than an anthranilic acid 3-hydroxylase activity as reported for rat liver [25] and suggested previously for macrophages [261. 3-Hydroxykynurenine formed by the cells was ~=ot detectable in supematants. This may be ~,,e to the mitochondrial localization of kynurenine 3-monooxygenas e [271. Capacity of interferon species to induce IDO As shown in Table III, interferon-,/ was the most potent inducer of tryptophan metabolism when compared to the other interferons and in the majority of the cell lines tested it was the only effective one. This was confirmed with natural interferons (not shown). However, high doses of interferon-fl (1000 U / m l ) activated macrophages, fibroblasts and A 498 to a degree comparable to the action of low doses of interferon-~, (Table III). In A 549 104 U / n i l interferon-fl produced 17.0 + 3.2 nmol of metabolites per mg cell protein and prolon- gation of the treatment with 1000 U / n i l to 72 h led to the formation of 92.8 4- 3.1 nmol per mg protein (mean of triplicates _+ S.D.). Interferon-a was only effective in A 498 and fibroblasts. T 24 cells could also degrade L-tryptophan to a considerable degree when unstimulated. This was not found with the other cell lines tested except for a small activity of A 498 control cells (Table III). In relation to the protein content of cells, T 24 and dermal fibroblasts exhibited the highest IDO activities. The enzyme inducibility in SK-N-SH, J 82 and dermal fibroblasts was saturated in the range 100-1000 U / m l interferon-,/. For the other cell lines and for macrophages, no plateau of the interferon-`/ effect was observ~:d within the investigated concentration range. Hep G 2 and CaCo 2, in which no IDO activity was inducible by interferon-`/, did not react upon treatment with the other interferons. Protein levels of monolayers were not diminished by incubation with interferon-,/ at all doses tested during 48 or 72 h as long as the nutritional provision was sufficient (not shown), thus indicating that under these conditions no direct growth inhibitory effect of interferon-,/occurred. Influence of extracellular L-tryptophan concentration on tryptophan.degrading activity Depending on the cell line, the addition of increasing L-tryptophan concentrations to the culture medium at the time of stimulation with interferon-'t resulted in (i) significantly decreased, (ii) several-fold increased or (iii) unaffected tryptophan-metabolizing activity (Fig. 2). With seven (J 82, SK-HEP-1, SK-N-SH, U 138 MG, A 431, T 24, A 549) out of nine inducible cell lines, an inhibitory effect of additional L-tryptophan on the degrading activity was observed. The sensitivity to tryptophan was different for individual cell lines. Addition of 50/zmol/1 L-tryptophan led to a significant (Student's t-test) reduction of tryptophan metabolism in SK-N-SH ( P < 0.02, at 100 U / m l IFN-,/), T 24 (P < 0.01, at 10 U / m l IFN-,/) and J 82 ( P < 0.01, at 100 U / m l IFNq,. The activity was significantly reduced after supplementation with 150 # m o l / l L-tryptophan in A 431 (P < 0.001, at 100 U / m l IFN-),) and A 549 (P < 0.01, at 10 U / m l IFN-,/). Addition of 500 # m o l / l L-tryptophan was necessary to obtain significant inhibition with U 138 MG ( P < 0.02, at 250 U / m l IFN-~/) and SK-HEP-1 (P < 0.05, at 100 U/rnl IFN-`/). Contrarily, interferon-induced IDO activity in macrophages was increased several times by the addition of L-tryptophan and remained constant over the concentration range tested with fibroblasts and A 498 (Fig. 2). In all cases, the added L-tryptophan quantities did not influence cell viability (data not shown). 146 1~0- -120 100- -100 "CO -CO ~ 40- -40 E 20- -20 a 120- ~..~..~ .'e°° B 0 " i 40< fore checked whether expression of MHC-antigens in the cell lines tested in the course of this study changed upon interferon-7 treatment. All cell lines in which IDO activity was inducible showed enhanced expression of either HLA-ABC (SK-N-SH, T 24, A 549), HLA-DR (J 82, A 431) or both antigens (U 138 MG, SK-HEP-1, A 498) upon interferon-,/ treatment (Table IV). The expression of the tested HLA-antigens remained unchanged with Hep G 2 and CaCo 2. E ~ -2oo 20s 50 , 150 , 500 i 1000 50 ) 150 i 500 i 1000 L-Tryptophun added to c u l t u r e m e d i u m ( u m o l / I ) Fig. 2. Dependence of interferon-y-induced indoleamine 2,3-di- oxygenaseactivityon extracellularL-tryptophanconcentration.Confluent monolayers and macrophages (3.10S/nd) were treated on day 3 with increasingconcentrationsof L-tryptophanand with interferon-'r for 48 h. Data are expressed as the amount of tryptophan metabolites released into the supernatant (triplicate culture+S.D.) in relation to results obtained with L-tryptophan concentrations of culture medium (-100~). (A) and (B) are cell lines showing inhibition: (e) U 138 MG, 250 U/m] IFN-¥; (o) A 549, 10 U/ml IFN-7; (~) SK-N-SH, 100 U/nd IFN-~; (It) T 24, 10 U/ml IFN-7; (1[]) SK-HEP-1, 100 U/ml IFN-¥; (<>) A 431, 100 U/ml IFN-,/; (v) J 82, 100 U / m l IFN-~,. (C) No influence: (*) fibroblasts, 25 U/ml IFN-7; (,,) A 498, 100 U/ml IFN-7. (D) Stimulation: ( ~ ) macrophages, 100 U / m l IFN-~,. Interferon-y-induced alterations of MHC antigen expression Interferon-`/ enhances the expression of HLA-ABC and HLA-DR antigens on monocytes [28], fibroblasts [24] and a number of tumour cell lines [29]. We thereTABLE IV Influence of interferon.y on the expression of HLA.ABC and HLA.DR antigens on human tumonr cell lines wi~i, inducible IDO. Monolayers were treated with IFN-~, (250 U/ml) for 72 h. Cells were reacted with saturating amounts of BI-3D3, L 243 or a control IgG 2a mouse myeloma antibody and were counter-stained with FITC-conjuga:ed sheep anti-mouse lgG (H+L). Control cells were treated in the same way but without interferon. Data are expressed as arbitrary units (see Materials and Methods) of fluorescence intensity of 505$ of the positively stained cells relative to a nonreactive mouse myeloma protein. (Fluorescence intensity ffi1.00). Cell line Discussion -100 HLA-ABC HLA-DR control IFN-'r control IFN-7 SK-N-SH T 24 A 549 1.(30 3.60 1.85 3.02 14.80 5.87 1.00 1.00 1.00 1.00 1.00 1.00 J 82 A 431 4.30 8.01 4.50 8.01 1.00 1.00 3.70 1.77 U 138 MG SK-HEP-1 A 498 2.31 6.42 1.42 3.61 7.33 2.42 1.00 1.62 1.00 2.42 3.77 1.94 The present study demonstrates that in various human cell types, neoplastic and normal, originating from different tissues oxidative tryptophan metabolism is inducible using interferon-,/. Although interferon--/ was the most potent inducer of IDO, interferon-a a n d / o r -fl showed small but distinct action on some of the cells. These results considerably extend previous work on this topic [8-10,30]. The biological significance of IDO induction remains a matter of speculation. IDO has been suggested to be involved in the antiproliferative action of interferon-7 on tumour cells [7] and on intracellular parasites [5,31]. In vivo, the excretion of tryptophan metabolites of the kynurenine pathway is enhanced in various disease states [32], including malignant neoplasms and viral infections, which are both connected with T-cell activation and interferon-`/production. Interferon-y is known to activate human macrophages specifically to produce H 2 0 a and kill intracellular parasites such as Toxoplasma gondii [33]. It is further known that the expression of HLA-DR, which is essentially involved in antigen presentation, is increased in macrophages [28]. One of the multiple effects of interferon-`/-in macrophages is the induction of IDO. In human fibroblasts, which provided a model for studying the antiproliferative action of interferon-`/ on Toxoplasma gondii [5], IDO activity as well as HLA-DR expression was inducible [24]. We therefore were interested to determine whether other cells with inducible IDO activity reacted with enhanced HLA-DR expression. This was found to be the case in only five of the reactive cell lines, three of which increased only the expression of HLA-ABC antigen. Thus, no specific correlation to the expression of the HLA-DR locus was observed. In all cell lines responsive to interferon-`/by enhanced MHC antigen expression, IDO was ind~cible. We therefore conclude that activation of IDO may be a general feature of interferon-,/action. No homogenous metabolite pattern was found for the cell lines tested. In contrast to previous reports [10], however, the results clearly demonstrate that several cells contain kynurenine-metabolizing enzymes. Further, we show that formation of 3-hydroxyanthranilic acid is rare, but is not found exclusively in macro- 147 phages. With the exception of IDO, all enzyme activities of the kynurenine pathway detected here were also present without interferon-~, stimulation. Thus, only the first enzyme of the pathway is regulated by interferon-~,. These results suggest a cooperation of cells of different tissues in vivo: the interferon signal may cause many cells to produce kynurenine which is then further metabolized only by specialized cells like macrophages or kidney cells (A 498). Induction of IDO is not only influenced by the dose of interferon-,/but also by the extracellular tryptophan concentration. With some of the tumor cell lines, a significant inhibition of the tryptophan-degrading actiw ity was observed. This results from decreased enzyme induction rather than fiom substrate inhibition of IDO as demonstrated previously [34]. The effective L-tryptophan concentrations were in the range 50-1000 /~mol/l. It has been shown by other investigators [7] that addition of 25-100/~g/ml of L-tryptophan to the culture medium rescued human tumour cells, among them A 549 and T 24, from the antiproliferative effect of interferon-~,. These concentrations are equivalent to 125-500 #mol/1 L-tryptophan which lies within the IDO inhibitory range. Thus, addition of L-tryptophan not only restores this essential amino acid, but also decreases the IDO reaction in these cells. This decrease of the IDO reaction itself may cause two effects, namely a less pronounced tryptophan deprivation and reduced production of toxic metabolites. From these results il cannot be determined whether this inhibitory effect is restricted to tumour cells. However, fibroblasts were not influe_n,;ed and increased IDO activity was induced in macrophages by higher L-tryptophan concentrations. 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