Neopterin Formation and Tryptophan Degradation by a Human Myelomonocytic Cell Line (THP-1) Upon Cytokine Treatment1

[CANCER RESEARCH 50. 2863-2867. May 15. 1990) Neopterin Formation and Tryptophan Degradation by a Human Myelomonocytic Cell Line (THP-1) upon Cytokine Treatment1

Gabriele Werner-Felmayer, Ernst Robert Werner, Dietmar Fuchs, Arno Hausen, Gilbert Reibnegger, and Helmut WÃ¤chter2 Institute for Medical Chemistry and Biochemistry. L'niversity of Innsbruck, Frit:-Pregl-Str. 3, 4-6020 Innsbruck, Austria

ABSTRACT stereoisomer of neopterin, was detectable only in macrophages (11). So far monocytes/macrophages are the only known cell Determination of neopterin lD-erythro-6-(l',2',3'-tnh\dro\)pro- type releasing substantial amounts of neopterin into the super pyl)pterin| in body fluids is a powerful diagnostic tool in a variety of natant. In all of the tested cells, 7-interferon-induced pteridine diseases in which activation of cellular immune mechanisms is involved, synthesis was paralleled by induction of IDO,3 an enzyme which such as certain malignancies, allograft rejection, and autoimmune and cleaves tryptophan to yV-formylkynurenine. In macrophages, infectious diseases. In vitro, neopterin is released into the supernatant by peripheral blood-derived monocytes/macrophages upon stimulation IDO activity and the amount of neopterin formed were aug with 7-interferon. In parallel, cleavage of tryptophan by indoleamine 2,3- mented by increasing the extracellular tryptophan concentra dioxygenase is induced. We report here that the human myelomonocytic tion, which is a special feature of this cell type (11). However, cell line THP-1 forms neopterin and degrades tryptophan upon treatment the biological function of 7-interferon-induced pteridine syn with 7-interferon. Like in macrophages a-interferon and /3-interferon thesis, its connection with IDO activity and the special position, induce these pathways only to a much smaller degree. The action of which macrophages are occupying in this regard, is not yet interferons is enhanced by cotreatment with tumor necrosis factor a, understood. lipopolysaccharide, or dexamethasone. 7-Interferon-induced neopterin For in-depth investigation of this issue, an in vitro system formation and indoleamine 2,3-dioxygenase activity are increased by more readily available than freshly purified monocytes/macro raising extracellular tryptophan concentrations. The pattern of intracel lulari) formed pteridines upon stimulation with 7-interferon shows the phages from peripheral blood would be advantageous. Among unique characteristics of human monocytes/macrophages. Neopterin, the established cell lines with monocytic characteristics only monapterin, and biopterin are produced in a 50:2:1 ratio. Thus, the THP- certain subclones of U 937 have been reported to secrete neop 1 cell line provides a permanent, easily accessible in vitro system for terin, but the parental cell line is negative for neopterin produc studying the induction and mechanism of neopterin formation. tion (12). Also the promyelomonocytic cell line HL-60 does not release neopterin upon 7-interferon treatment (13). Here we report that the monocytic cell line THP-1 releases INTRODUCTION neopterin upon stimulation with 7-interferon, which could not Neopterin [D-ery/7zro-6-(r,2',3'-trihydroxypropyl)pterin], a be shown for any other established cell line so far. Evidence is compound detectable in human body fluids, is released by presented that these cells have the same intracellular pteridine monocytes/macrophages specifically upon stimulation with 7- pattern as macrophages. Characteristics of 7-interferon-in interferon derived from activated T-cells (1). In vivo, elevated duced tryptophan metabolism and the influence of tryptophan neopterin levels were demonstrated for a panel of diseases in on neopterin synthesis and IDO activity were studied. Further, the effects of Â«-and 0-interferon, of LPS, of TNF-Â«, and of which activation of cellular immune mechanisms is involved, such as allograft rejection, viral infections, infections with in- dexamethasone on neopterin formation and IDO activity were tracellularly living bacteria and protozoa, autoimmune diseases, investigated and compared with results obtained in a previous study with macrophages, peripheral blood-derived mononuclear and certain tumors (reviewed in Refs. 2 to 4). For carcinoma of the cervix (5), ovarian cancer (6), and human immunodeficiency cells, and normal dermal fibroblasts (14). virus type 1 infection (7). neopterin has been shown to be a prognostic marker for disease progression. MATERIALS AND METHODS Biochemically, neopterin originates from the cleavage of GTP by GTP-cyclohydrolase I (EC 3.5.4.16). This reaction Cytokines, LPS, and Dexamethasone. Human recombinant Â«2b-interferon produced in Escherichia coli was from Schering Corp. (Kenil- yields 7,8-dihydroneopterin triphosphate (8), the joint precur worth, NJ) and had a specific activity of 2 x IO8 units/mg of protein. sor of dihydroneopterin and of tetrahydrobiopterin, a cofactor Human recombinant /3-interferon (specific activity, 3 x IO8 units/mg of aromatic amino acid monooxygenases (9) and of etherlip- of protein) from Chinese hamster ovary' cells and human recombinant idoxidase (10). The activity of GTP-cyclohydrolase I can be 7-interferon (specific activity, 2 x IO7 units/mg of protein) were a enhanced up to a hundredfold by stimulation with 7-interferon generous gift from Bioferon GmbH (Laupheim, FRG). Human recom not only in macrophages, but also in normal dermal fibroblasts binant TNF-a (2 x IO7 units/mg of protein as assayed on L-929 cells and a panel of human tumor cell lines from different tissue in the presence of actinomycin D by the manufacturer) was obtained origin, as was reported recently (11). Each of the tested cell from Genzyme (Boston, MA). LPS (phenolic extract) of E. coli 055:B5 types showed its characteristic pattern of intracellular pteridines was from Sigma (Munich, FRG). Dexamethasone (research grade) was upon 7-interferon treatment, the formed neopterin:biopterin purchased from Serva (Heidelberg, FRG). Cell Culture. THP-1, a human leukemic cell line with monocyte ratio ranging from 0.02 for T-24 bladder carcinoma cells to 73 characteristics (15), was obtained from the American Type Culture for peripheral blood-derived macrophages. Monapterin, the Collection (Rockville, MD) and was Mycoplasma negative as tested by staining with 4',6-diamidino-2-phenyl-indole (16). Cells were grown in Received 9/11/89; revised I/I 1/90. The costs of publication of this article were defrayed in part by the payment RPMI 1640 (endotoxin concentration <1 pg/ml; Biochrom, Berlin. of page charges. This article must therefore be hereby marked advertisement in FRG) supplemented with 10% heat-inactivated fetal calf serum (Bio- accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1Supported by the Austrian Research Funds "Zur FÃ¶rderungder wissenschaf 3The abbreviations used are: IDO. indoleamine 2,3-dioxygenase: TNF-n, tlichen Forschung." Project 6922. tumor necrosis factor Â«;LPS, lipopolysaccharide; HPLC, high-pressure liquid 2To whom requests for reprints should be addressed. chromatography; FRG. Federal Republic of Germany. 2863

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1990 American Association for Cancer Research. NEOPTERIN FORMATION BY A HUMAN MYELOMONOCVTIC CELL LINE (THP-1) chrom. Berlin, FRG), 2 HIM L-glutamine, 100 units/ml of penicillin, are 5 nM for anthranilic acid and 3-hydroxyanthraniIic acid, 10 nM for 0.1 ng/ml of streptomycin, and 20 MM 2-mercaptoethanol (Serva, tryptophan and 0.5 MMfor kynurenine. IDO activity is expressed as the Heidelberg, FRG) in a humidified atmosphere containing 5% CO2. For amount of tryptophan metabolites detected in supernatants in nmol per maintenance, cells were seeded at a density of 105/ml with addition of mg of cell protein after 72 h. fresh medium on Day 3 and passaged once weekly. For experiments, cells were seeded at 6 x 105/ml in 24-well plates (Falcon, Beckton Dickinson, Lincoln Park, NJ) and treated with stimuli for 72 h. RESULTS Interferons were applied at doses which yielded significant effects in macrophages (14). Dexamethasone (50 nmol), when used, was added Action of Interferons, TNF-a, LPS, and Dexamethasone. As to cultures l h before other stimuli. In some experiments cells were shown in Table 1 IDO activity and neopterin formation and cultured in tryptophan-free RPMI 1640 (Select Amin Kit; Gibco, Grand release in THP-1 cells could be induced in parallel by all three Island, NY) supplemented with fetal calf serum, glutamine, penicillin, interferons. Tryptophan metabolites and neopterin accumu streptomycin (as described above), and various concentrations of L- lated in the supernatant in a time- and dose-dependent manner tryptophan (Calbiochem, Los Angeles, CA) or D-tryptophan (Sigma, (Table 1). The most pronounced effect was achieved by 7- Munich, FRG). Supernatants were harvested by centrifugation. Cell interferon. pellets were washed once with phosphate buffer, containing 130 mM TNF-Â«applied as a single stimulus was only a weak inducer NaCl, 2 HIMKC1, 6 mM Na2HPO4, and 1 mM KH2PO4 (supplied as phosphate-buffered saline from Serva, Heidelberg, FRG), and lysed in of the two pathways: 1000 units/ml yielded an IDO activity of 0.4 ml of 0.05 M H3PO4. The protein concentration of cell lysates was 3.68 Â±8.08 nmol/mg and neopterin levels of 57.40 Â±22.40 determined according to the method of Bradford ( 17), modified for 96- pmol/mg (n = 6 Â±SD, 72 h); 100 units/ml for 72 h resulted in well flat-bottomed microtiter plates using the protein assay dye reagent formation of 37.50 Â±8.00 pmol/mg of neopterin and unde- from Bio-Rad (Richmond. CA) and pure bovine serum albumin (Serva, tectable IDO activity. When used in combination, 100 units/ Heidelberg, FRG) as protein standard. The protein content of individ ml of TNF-Â« significantly enhanced the effect of interferons ual wells after the incubation period was 142.2 Â±17.1 Mgfor controls, concerning neopterin formation (Fig. I) and IDO activity (Fig. 137.1 Â±29.8 Mgfor cells treated with 1000 units/ml of Â«-interferon, 2) by 30 to 50%. 129.7 Â±26.4 Mgfor cells treated with 1000 units/ml of /S-interferon, 129.8 Â±19.3 Mgfor cells treated with 250 units/ml of -y-interferon, LPS (10 Mg/ml) applied as a single stimulus was a potent 130.7 Â±16.9 Mgfor cells treated with 1000 units/ml of TNF-a, and inducer of neopterin formation (Fig. 1) and IDO activity (Fig. 128.1 Â±16.0 Mgfor cells treated with 10 Mg/ml of LPS (n = 9 Â±SD). 2). As a costimulus it drastically enhanced the effect of inter Dexamethasone (50 n.M)did not influence protein levels. 7-Interferon ferons (Figs. 1 and 2). As shown in Fig. 3 the action of a given in combination with LPS (0.1 to 10 Mg/ml) alone and with further dose of 7-interferon was increased by LPS in a dose-dependent addition of dexamethasone, respectively, decreased protein concentra manner. Even 10 pg/ml of LPS, which caused no detectable tions by 33.4 Â±6.4% (n = 9 Â±SD). effect when applied as a single stimulus, were sufficient to Peripheral blood-derived macrophages were isolated from buffy coats significantly enhance the effect of 7-interferon concerning of healthy donors by two-step density gradient centrifugation (Ficoll/ neopterin formation (P < 0.05, Student's t test). Paque, discontinuous Percoli, obtained from Pharmacia, Uppsala, Swe Dexamethasone significantly increased the effects of 7-inter den) and adherence to plastic as described (11). Cells were cultured Â¡n feron and LPS but did not alter the action of Â«-interferon and RPMI 1640 supplemented with 2 HIM L-glutamine, antibiotics (see 0-interferon (Figs. 1 and 2). above) and 10% human pooled serum from healthy donors and were Intracellular Pteridine Pattern. Concentrations of intracellu more than 99% esterase positive on Day 3 of culture. Buffy coats and lar pteridines in THP-1 and in monocyte-derived macrophages human serum were generously supplied by the blood transfusion unit of the University Hospital of Innsbruck. are given in Table 2, showing that on the basis of total cell Cell Extracts for Determination of Intracellular Pteridines. THP-1 protein both cell types produced similar amounts of neopterin, (seeding density 6 x lOVml) was cultured for 48 h with 250 units/ml monapterin, and biopterin upon 7-interferon treatment. The of 7-interferon or medium alone. After harvesting cells by centrifuga ratio of pteridines being formed was 51 to 2.2 to 1 for THP-1 tion, the pellet was washed twice in phosphate buffer (see above). and 62 to 2.5 to 1 for macrophages, as calculated from Table Viability of cells, as determined by trypan blue exclusion, was always 2. greater than 95%. Five times IO7 cells were resuspended in l ml of Characteristics of 7-Interferon-induced Tryptophan Metabo distilled water and frozen at -80Â°C for 30 min. After thawing rapidly, lism. Tryptophan metabolites detectable in supernatants upon cell debris was centrifuged at 10,000 x g. The supernatant was imme 7-interferon treatment were kynurenine, anthranilic acid, and diately oxidized with acid iodine as described (11). Extracts from 3-hydroxyanthranilic acid. Kynurenine was thereby formed to macrophages, treated with 250 units/ml of 7-interferon on Day 3 of 55.3 Â±0.9% of degraded tryptophan, anthranilic acid to 3.9 Â± culture for 48 h or medium alone, were obtained after washing cells 0.4%, and 3-hydroxyanthranilic acid to 1.3 Â±0.1% (n = 9 Â± with phosphate buffer (see above) when still adherent, by disrupting them by rigorous scraping in a small volume of distilled water as SD). described (11) and subsequent freezing and thawing. The sample was 7-Interferon-induced formation of neopterin and IDO activ further processed as described for THP-1. ity were enhanced with the increasing concentrations of L- Determination of Pteridines, Tryptophan, and Its Metabolites. Deter tryptophan added to the culture medium (Fig. 4). Activities mination of neopterin in supernatants and pteridines in cell extracts obtained with 25 Â¿IML-tryptophan, which is the standard con was carried out by solid-phase extraction over AASP-SCX cartridges centration of the used culture medium, were significantly lower (Analytichem, Harbor City, CA) and on-line elution HPLC as described than those obtained with 200 Â¿IML-tryptophan (P < 0.02 for (11, 18). Detection limits are 1 nM for neopterin and monapterin and neopterin formation, P < 0.001 for IDO; Student's t test). 2 nM for biopterin for a sample volume of 100 M'(corresponding to 0.1 Control cells had no detectable IDO activity. Only in the case and 0.2 pmol absolute). Values are related to total cell protein. For determination of intracellular biopterin, extract of at least IO7 cells, of 200 /UML-tryptophan could an activity of 9.6 Â±1.3 nmol per mg of protein be found. This is 3% of the 7-interferon-induced containing 800 to 1000 Mgof total cell protein in up to 1 ml of sample, activity in the presence of 200 Â¿JMi.-tryptophan. was applied for one determination. Tryptophan and its metabolites were determined in supernatants by In comparison, D-tryptophan was only weakly metabolized, HPLC with fluorescence and ultraviolet detection as previously re and neopterin formation was not increased significantly (Fig. ported (19). Ten p\ of crude supernatant were injected. Detection limits 4). 2864

Table 1 Neopterin formation and Â¡DOactivity of TUP-1 upon treatment with recombinant interfÃ©rons,asdetermined in supernatants Cells (6 x 10'/ml) were treated with various doses of interferons for 24,48. and 72 h. Tryptophan metabolites (kynurenine. anthranilic acid, and 3-hydroxyanthranilic acid) and neopterin were determined in supernatants by HPLC. IDO activity is expressed as the sum of tryptophan metabolites detected per mg of total cell protein after the indicated incubation period. Neopterin levels were also related to mg of cell protein. (nmol/mg)48hNDNDNDNDNDNDND10.9(pmol/mg)72hNDNDND3.4 Treatment (units/ml)NoneÂ«-Interferon502501000/Â¡-Interferon502501000-(-Interferon50250100024hNDÂ°N

DNDN Â±0.9*18.0Â± Â±0.917.5 Â±0.423.3 2.024.0 Â±1.327.7Â±0.925.0 Â±3.129.1 DNDN Â±0.3NDND5.1 Â±2.020.2 Â±2.840.0

Â±3.723.6 Â±2.840.1 Â±3.544.7 DNDND20.1 Â±2.726.5 Â±4.248.7 Â±1.656.3 Â±0.359.2 Â±5.829.6 Â±4.052.1 Â±1.767.2

Â±0.624.4 Â±0.531.8 Â±2.760.5 Â±0.8150.5 Â±2.9221.3 Â±0.732.7 Â±1.060.7 Â±3.196.5 Â±6.394.5 Â±9.2334.9 Â±13.7673.0 Â±2.7IDO Â±3.8Neopterin Â±3.224hND12.4 Â±3.648hND12.1 Â±14.872hND16.5 Â±32.0 ' ND. not detectable. *Mean Â±SD of 3 observations.

1000-500-,100-50-i[lOI2A3I4AX5-X1A2A3I4-1.5fi1O2A3Ar4A5Â«1S4 1000-

500 - u â€¢>. A1A2â€¢3I4&5Aâ€”1I243I4i5â€¢r..12i2A445JE1A4IFN-a Â« : < Z UE a;U 7 21 100 - < o E- I in CM O 50 -

IFN-a IFN-ÃŸ IFN-Y LPS 10 1000 U/ml 1000 U/ml 250 U/ml 10 ug/ml LPS1000 IFN-6 IFN-Y Fig. 1. Formation of neopterin upon treatment with interferons in THP-1 cells, i.e.. synergistic effect of TNF-Â«,LPS, and dexamethasone. Cells (6 x IO5/ a/ml 1000 U/ml 250 U/ml 10 ug/ml Fig. 2. IDO activity upon treatment with interferons with THP-1 cells, i.e., ml) were cultured in the presence of various stimuli for 72 h. Neopterin was synergistic effect of TNF-a, LPS, and dexamethasone. Cells (6 x 10!/ml) were determined in supernatants by HPLC. Values, calculated per mg of total cell protein, are expressed as the percentage of levels obtained with 250 units/ml of cultured in the presence of various stimuli for 72 h. Tryptophan and its metabolites 7-interferon (=100%) and are plotted on a logarithmic scale. Columns, mean of were determined in supernatants by HPLC. IDO activity (sum of detectable three observations; bars. SD. In supernatants of untreated cells and in cultures tryptophan metabolites per mg of cell protein) is expressed as the percentage of treated with dexamethasone no neopterin was detectable. In the case of 100 units/ levels obtained with 250 units/ml of -y-interferon (=100%) and plotted on a ml of TNF-tt, 37.5 Â±8.0 pmol/mg of neopterin were detectable, which is logarithmic scale. Columns, mean of three experiments; bars. SD. In supernatants equivalent to 6.23 Â±1.33%. Identification oÃcolumns: I, no costimulus; co- of untreated cells and in cultures treated with 100 units/ml of TNF-a or with stimulated with 100 units/ml of TNF-n (2), 10 ng/ml of LPS (3), 50 nM dexamethasone, no IDO activity was detectable. Identification oÃcolumns: 1, no dexamethasone (4), and 10 Mg/ml of LPS together with 50 nM dexamethasone costimulus; costimulated with 100 units/ml of TNF-a (2), 10 ng/ml of LPS (3), (5). In most cases the values obtained by combined application of stimuli are 50 nM dexamethasone (4), and 10 ^g/ml of LPS together with 50 nM dexameth significantly higher (Student's t test) than the sum of effects obtained by single asone (5). In most cases, values obtained by the combined application of stimuli are significantly higher (Student's / test) than the sum of effects obtained by single stimuli: O. P < 0.05; â€¢,P< 0.01 ; A, P < 0.001 ; A. P < 0.0001. For example, the effect achieved with Â«-interferonapplied in combination with LPS (Column 3) is stimuli: â€¢,P < 0.01; A. P < 0.001; A, P < 0.0001. For example, the effect significantly higher than the effect of Â«-interferon alone (Column I) plus that of achieved with a-interferon applied in combination with LPS (Column 3) is LPS alone (Column /). t' means unit. significantly higher than the effect of Â«-interferonalone (Column /) plus that of LPS alone (Column /). L' means unit.

DISCUSSION isolated normal monocytes/macrophages. The effects presented A variety of human cells synthesize pteridines and degrade here for THP-1 could be observed with untreated cells growing tryptophan by IDO when treated with 7-interferon (11). It was in suspension culture. Further differentiation into adherent demonstrated previously that, concerning these metabolic ac cells, as it can be achieved by treatment with phorbol myristate tions of 7-interferon, peripheral blood-derived monocytes/mac- acetate (21), was not required. In other leukemic cell lines rophages have special characteristics, as compared with normal originating from the myelomonocytic lineage, such as U 937 or fibroblasts or a panel of tumor cell lines from different tissue HL-60, neither pteridine synthesis (12, 13) nor IDO activity originili, 14,20). (13, 22) can be induced, which could probably be explained by Here, we report for the first time that an established cell line, a diverse differentiation state of these cells. THP-1, which has distinct monocytic markers (15), shows the The most striking similarity between THP-1 cells and mon- same characteristics regarding these two pathways as do freshly ocyte-derived macrophages concerns the ratio of pteridines 2865

1000 - 700

500 - U fN E Ã• 200- a

>â€¢ i-i

100 - u > 5 25 50 100 200 5 25 50 100 200 M H TRYPTOPHAN added (UM) 50 Fig. 4. Influence of extracellular concentration of L-tryptophan (A) or D- tryptophan (A) on neopterin synthesis (left) and IDO activity (right) in THP-I cells treated with 250 units/ml of 7-interferon for 72 h. Cells (6 x 10'/ml) were cultured in tryptophan-free medium supplemented with various concentrations of L- or D-tryptophan. Due to the contribution of fetal calf serum, "tryptophan-free" medium contained 3 ti\t tryptophan as determined by HPLC. Neopterin, trypto phan. and its metabolites were determined in supernatants by HPLC. IDO activity is expressed as the sum of detectable metabolites per mg of cell protein. Points, mean of triplicate cultures; bars, SD. 10 -5 io-" ,-3 -2 10 10 10 0.1 10 pared with macrophages. THP-1 converts 55% of degraded LPS added (ug/ml) tryptophan into kynurenine and only 1.3% into 3-hydroxyan- Fig. 3. Neopterin formation ( ) and IDO activity ( ) of THP-1 upon thranilic acid, whereas macrophages form kynurenine to 28% treatment with various concentrations of LPS applied either as a single stimulus and 3-hydroxyanthranilic acid to 19% from degraded trypto (A) or in combination with 250 units/ml of-y-interferon (B). Cells (6 x ]0'/ml) were treated with stimuli for 72 h. Neopterin. tryptophan, and its metabolites phan (20). Detected amounts of 3-hydroxyanthranilic acid, were detected in supernatants by HPLC and were related to cell protein. Values which derives from monocytes rather than from lymphocytes, (n = 3) are expressed as the percentage of the effect obtained with 250 units/ml of-y-interferon and are plotted on a logarithmic scale. Points, mean: bars, SD. are thereby similar in freshly isolated mononuclear cells treated with 7-interferon to those formed by macrophages stimulated Table 2 Concentrations of intracellular pteridines formed in THP-1 and in after 3 days of adherence (19). Thus, the relatively low activity monocyre-derived macrophages of kynurenine 3-monooxygenase of THP-1 does not occur due Cells were cultured for 48 h with either medium alone or with 250 units/ml of 7-interferon, harvested by centrifugation (THP-1) or scraping cells from the to its monocyte character, but may be related to the transformed plates (macrophages), and extracted by distilled water and freezing/thawing. After state of the cell. Anthranilic acid is synthesized in THP-1 to immediate acid iodine oxidation, samples were extracted with AASP-SCX car tridges and directly eluted onto a reversed-phase HPLC column. Pteridines were 3.9% and in macrophages to 1.6%, indicating that kynureninase quantified by fluorescence detection (353/438 nm) and are expressed per mg of activities of both cell types are comparable. cell protein. Values are the mean Â±SD of 5 experiments for THP-1. In the case In a recent study (14) we showed that (a) LPS is a potent of macrophages the mean Â±SD obtained with cells from 3 different donors is given. inducer of pteridine synthesis and IDO activity in macrophages but only a weak one in normal fibroblasts, and (b) dexametha- Cell/treatmentTHP-1None (pmol/mg)4.7 (pmol/mg)NDÂ°5.3(pmol/mg)ND2.4 sone, which by itself did not induce these pathways, amplified the action of 7-interferon and of LPS in macrophages, whereas Â±4.0 in fibroblasts it enhanced only the action of 7-interferon but 7-Interferon (250 units/ml)Neopterin 123.2 Â±67.0MonapterinÂ±1.5Biopterin Â±0.5 decreased the effect of LPS. The results presented here clearly Macrophages indicate that THP-1 behaves like macrophages in this respect. None 2.8 Â±1.9 ND ND 7-Interferon (250 units/ml) Concerning the activating potential of the three interferon 131.4 Â±68.0 5.4 Â±1.0 2.1 Â±1.2 species, THP-1 is comparable to macrophages (14). IDO activ Â°ND, not detectable. ity of THP-1 has also been previously observed upon stimula tion with 7-interferon but, in contrast to our findings, not with formed, which is unusual as compared with a panel of other a-interferon (22). This difference may occur due to the subspe human cells (11). Further, addition of increasing L-tryptophan concentrations to the culture medium together with 7-inter cies of (v-interferon used. Further we show that, in THP-1, the effect of interferons is feron stimulation leads to a severalfold increase of IDO activity and of neopterin formation in THP-1 in a way comparable to synergized by cotreatment with TNF-Â«or with LPS, which can macrophages (11). In contrast, in fibroblasts IDO activity re also be seen with macrophages and fibroblasts (14, 23). It was demonstrated that glucocorticoids increase the number of 7- mains unchanged, and in a number of tumor cell lines, it is even significantly decreased by the addition of L-tryptophan interferon receptors (24) and that 7-interferon increases the (20). total number of TNF receptors (25). It is therefore conceivable IDO is the only enzyme activity of the kynurenine pathway, that enhanced formation of cytokine receptors could be involved which is regulated by 7-interferon, whereas activities further in some of the observed synergistic effects. down the pathway are constitutively present in the cells (20). The data presented here indicate that the monocytic cell line While most cells produce only kynurenine, macrophages form THP-1 can serve as an in vitro model for studying 7-interferon- kynurenine, anthranilic acid, and 3-hydroxyanthranilic acid (13, induced features of pteridine synthesis and tryptophan degra 20). As is shown here, THP-1 forms the same metabolites from dation characteristic for normal monocytes/macrophages, thus tryptophan. However, the relative activity of kynurenine 3- providing a useful tool for elucidation of the biological signifi monooxygenase (EC 1.14.13.9) is smaller in THP-1 as com- cance of these metabolic actions of 7-interferon. 2866

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 1990 American Association for Cancer Research. Neopterin Formation and Tryptophan Degradation by a Human Myelomonocytic Cell Line (THP-1) upon Cytokine Treatment

Gabriele Werner-Felmayer, Ernst Robert Werner, Dietmar Fuchs, et al.

Cancer Res 1990;50:2863-2867.

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