Modulation of neopterin formation and tryptophan degradation by Th1- and Th2-derived cytokines in human monocytic cells

INTRODUCTION

Upon stimulation with interferon-gamma (IFN-γ) human monocytes/macrophages produce and release large amounts of neopterin [1], 6-d-erythro-1′,2′,3′-trihydroxypropyl-pterin, which is synthesized from guanosine triphosphate (GTP) by GTP-cyclohydrolase I (EC 3.5.4.16) [2]. Further, human monocytes/macrophages degrade the essential amino acid tryptophan [3,4], which is initiated by the induction of the enzyme indoleamine 2,3-dioxygenase (IDO) (EC 1.13.11.17) by IFN-γ. The enzyme cleaves tryptophan to form N-formyl-kynurenine and, as a subsequent product in the degradation pathway, l-kynurenine and other products like anthranilic acid and quinolinic acid accumulate. Other cytokines than IFN-γ have been found to be less important as a trigger for neopterin formation and tryptophan degradation in vitro [5]. In humans, an increased formation of neopterin and enhanced degradation of tryptophan have been found in viral infections, malignant disorders and autoimmune diseases [6–11], and significant correlations between the enhanced endogenous production of IFN-γ and of neopterin and the increased degradation of tryptophan have been found [6,12–17].

Recent data indicate that both the increased formation of neopterin and the accelerated degradation of tryptophan may interact with the immunological network. Neopterin derivatives have been found to interfere with reactive oxygen, nitrogen and chlorine species [18], thereby on the one hand possibly modulating the cytotoxic capacity of the activated monocyte/macrophage, and on the other hand influencing redox-sensitive intracellular signal transduction pathways [19], including the expression of NF-κB or tumour necrosis factor-alpha [20]. Degradation of tryptophan was found to be associated with the development of neuropsychiatric deviations in patients treated with cytokines or during states of chronic immune activation [6,7,15]. More recently degradation of tryptophan was described to be crucial for establishing immune tolerance during pregnancy [21].

It is well accepted that there exists a cross-regulation between the Th1-type and Th2-type immune response, by which Th1-type cytokines like IFN-γ and IL-2 and Th2-type cytokines like IL-4, IL-5, IL-10 and IL-13 negatively regulate each other [22]. IL-12 is the major regulatory stimulus for Th1-type immunity, triggering development of Th1-type cells from precursor cells and IFN-γ production by Th1- and natural killer (NK) cells [23,24]. IL-4 and IL-10 have been demonstrated to be the most effective anti-inflammatory cytokines, and many of these actions are due to counteracting IFN-γ- and Th1-mediated pathways [22].

This study was performed to determine the effects of Th1/Th2 induction on cytokine-inducible neopterin formation and degradation of tryptophan.

MATERIALS AND METHODS

Peripheral blood mononuclear cells (PBMC) were freshly prepared from whole blood of healthy volunteers by separation on Ficoll–Paque (Pharmacia, Uppsala, Sweden) as described previously [5]. PBMC were resuspended with RPMI 1640 (Biochrom, Schoeller, Vienna. Austria) supplemented with 10% (v/v) heat-inactivated fetal calf serum (FCS; Biochrom), 2 mml-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin (Sigma, Munich, Germany), and then seeded in 24-well plates (Falcon, Becton Dickinson, Lincoln Park, NJ) at a final density of 10⁶ cells/ml. For separation of macrophages, cells were incubated for 24 h, and after this non-adherent cells were removed.

Human myelomonocytic cells (THP-1; American Type Culture Collection, Bethesda, MD) were seeded in 24-well plates at a final density of 5 × 10⁵ cells/ml supplemented RPMI.

Human recombinant IFN-γ (250 U/l, corresponding to approx. 10 ng/ml; gibco Life Technologies, Eggenstein, Germany), IL-4 (10 ng/ml; R&D, Vienna, Austria), IL-10 (10 ng/ml; R&D), and IL-12 (5 U/ml corresponding to approx. 10 ng/ml; Genetic Institute, Cambridge, MA) were added as single agents or in various combinations to cells for 24 h, before stimulation with other cytokines was performed for another 48 h. After 72 h of incubation at 37°C in humidified air with 5% (v/v) CO₂, supernatants of cultures were harvested, centrifuged for 10 min at 400 g and 4°C, and frozen at −20°C.

Neopterin in supernatants was quantified by ELISA (ELItest Neopterin Screening; BRAHMS Diagnostica, Berlin, Germany) with a detection limit of 2 nmol/l. Optical density (OD) was read using an Anthos 2001 microplate reader (Laevosan, Linz, Austria).

For monitoring tryptophan degradation, supernatants were analysed by high performance liquid chromatography (HPLC) on a Varian 9010 (Varian, Palo Alto, CA) as described previously [25]. In addition to the determination of kynurenine and tryptophan concentrations, the ratio of kynurenine, the first product of IDO, to tryptophan, the substrate of IDO, was calculated, which allows a better estimate for IDO activity [15].

Extraction of RNA and Northern blots was carried out exactly as described [26]. For hybridization with huIFN-γ cDNA (in pBluescript M13+/KS) the 1.2-kb insert (PstI/PstI) was used which was kindly provided by Dr J. Böhm (Hannover, Germany). Probing with the rat cDNA of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (in PBR322; 1.4-kb PstI/PstI insert) was taken as a control.

For statistical comparisons between groups paired t-test was calculated.

RESULTS

In PBMC, THP-1 and macrophages neopterin formation was inducible after stimulation of cells with IFN-γ dosages between 50 and 100 U/l and exceeded baseline levels by approximately three- to 10-fold, corresponding to mean neopterin concentrations of 12.3 ± 1.7 nmol/l after stimulation with IFN-γ (Table 1). All regulatory effects of interleukins were similar in THP-1 and human macrophages (Table 1), while in PBMC effects were sometimes different (see below). In PBMC (Fig. 1a) but not in THP-1 cells (Fig. 1b), IL-12 was able to superinduce neopterin formation in response to IFN-γ by about 50% (P < 0.05). In contrast, IL-4 and IL-10 reduced the effects of IFN-γ on monocytic cells, and both cytokines were additively active in PBMC and THP-1 cells. However, the effects were greater in PBMC when preincubation with interleukins was performed. In THP-1 cells and primary macrophages, addition of cytokines to IFN-γ at the same time was more effective than preincubation. In PBMC, preincubation with IL-12 was able to reverse somewhat the inhibitory effects of IL-4 and IL-10 on neopterin formation, which was not the case in THP-1 cells or macrophages.

IDO activity was induced in all three cell types upon treatment with IFN-γ. While mean tryptophan concentrations in control cells ranged from 22.9 to 43.7 μmol/l (mean 37.4 ± 8.2 μmol/l), kynurenine was hardly detectable (range 0.5–2.1 μmol/l; mean 0.9 ± 0.6 μmol/l) thus leading to a rather low kynurenine per tryptophan ratio (0.038 mmol/mol; Table 2). Treatment of cells with IFN-γ resulted in a significant decrease of tryptophan (mean 22.4 ± 7.3 μm) and an increase of kynurenine levels (18.3 ± 6.2 μm) associated with an increase in the kynurenine per tryptophan ratios compared with controls (P < 0.05; Table 2). The effects of IL-12, IL-4 and IL-10 as well as the regulatory differences between the various cell types were similar, as described above for neopterin (Fig. 2).

To see whether differences in neopterin and kynurenine formation in response to Th1/Th2-derived cytokines could be referred to different expression of IFN-γ we performed a Northern blot for this cytokine mRNA. As can be seen in Fig. 3, IFN-γ mRNA was not detectable in untreated cells (control). Treatment of cells with IFN-γ and to a greater extent stimulation with IL-12 induced IFN-γ mRNA expression (Fig. 3, lanes 2 and 3), which—according to the Th1/Th2 paradigm—should be due to a positive effect of both cytokines on IFN-γ production by Th1 cells. Neither cytokine at the indicated concentrations had any synergistic effect on IFN-γ mRNA expression. Concomitant treatment with IL-4 was able to reduce IFN-γ mRNA expression in response to the two other stimuli (IL-12 and IFN-γ) (Fig. 3, compare lanes 3 and 4 with lane 5).

DISCUSSION

In agreement with known effects of IL-12 on T-cells [23,24], our results provide evidence that IL-12 may up-regulate neopterin formation and tryptophan degradation via induction of additional production of Th1 cytokines by either NK cells or Th1 cells, as is suggested by our Northern blot results showing increased IFN-γ expression under such conditions. A direct effect of IL-12 on monocytes/macrophages seems unlikely, as this up-regulation could not be seen in THP-1 cells or primary macrophages. Similarly, according to the Th1/Th2 paradigm [22], IL-4 and IL-10 inhibit neopterin production and tryptophan degradation in PBMC, probably via direct inhibition of Th1-type cytokine production [27] and additionally also via direct deactivation of IFN-γ effects towards monocytes/macrophages (see also Fig. 3). The latter observation has also been made in THP-1 cells, providing further evidence for the direct down-regulatory potential of anti-inflammatory cytokines on the Th1-type immune response involving macrophages. Moreover, IL-4 and IL-10 at the same concentrations were additive in down-regulation of macrophage effector functions. The reduced inhibitory effect of preincubation with IL-4 and IL-10 on the investigated IFN-γ-mediated pathways of macrophages and THP-1, compared with addition of the interleukins together with IFN-γ, could be influenced by loss of bioactivity of interleukins with increasing incubation time.

Determinations of neopterin and kynurenine in patients' sera have been widely used as clinically reliable markers for monitoring of Th1-mediated immune effector functions, involving the induction of IFN-γ [6–17]. Based on our in vitro results, it appears more likely that these clinical parameters rather reflect the net effect of Th1- and Th2-mediated immune effects. This may also be a reason why the determination of neopterin [8,9,11] and also of kynurenine [6,7,16] in various diseases has turned out to be a better predictive parameter for the progression of cancer, HIV infection or the clinical course of autoimmune disease than determination of single cytokines [13,14]. It seems that especially the sum effect of cytokines, generated within the Th1/Th2 interplay, on the population of macrophages is of importance for the development and the course of these diseases.

The balance of forward- and backward-regulatory cytokines within the cell-mediated immune response is crucial for the production of microbicidal and cytocidal metabolites like oxygen-free radicals or nitric oxide by activated macrophages [28]. In addition, these reactive compounds have the potential to interfere with intracellular pathways, further modulating cytokine production pathways and triggering cell death [19]. Interestingly, neopterin production may not only serve as an indicator of activation of monocytes/macrophages, but can itself also contribute to modulate the degree of oxidative stress elicited during the immune response [18]. Also, tryptophan degradation appears to have a role within the immunological network, being related to the development of immunotolerance [21]. Consequently, the modulating impact of forward- and backward-regulatory cytokines on the activation status of monocytes/macrophages may have an additional impact on immune system-related pathways in addition to their direct impact on the regulation of cytokine networks.