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The Journal of Immunology, 2002, 169: 3392-3399.
Copyright © 2002 by The American Association of Immunologists

Restriction of De Novo Pyrimidine Biosynthesis Inhibits Th1 Cell Activation and Promotes Th2 Cell Differentiation1

Petya Dimitrova*,{dagger}, Alla Skapenko*,{dagger}, Matthias L. Herrmann{ddagger}, Rudolf Schleyerbach{ddagger}, Joachim R. Kalden{dagger} and Hendrik Schulze-Koops2,*,{dagger}

* Nikolaus Fiebiger Center for Molecular Medicine, Clinical Research Group III, and {dagger} Department of Internal Medicine III and Institute for Clinical Immunology, University of Erlangen-Nuremberg, Erlangen, Germany; and {ddagger} DG Thrombotic Diseases/Degenerative Joint Diseases, Aventis Pharma, Frankfurt, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Leflunomide, an inhibitor of de novo pyrimidine biosynthesis, has recently been introduced as a treatment for rheumatoid arthritis in an attempt to ameliorate inflammation by inhibiting lymphocyte activation. Although the immunosuppressive ability of leflunomide has been well described in several experimental animal models, the precise effects of a limited pyrimidine supply on T cell differentiation and effector functions have not been elucidated. We investigated the impact of restricted pyrimidine biosynthesis on the activation and differentiation of CD4 T cells in vivo and in vitro. Decreased activation of memory CD4 T cells in the presence of leflunomide resulted in impaired generation and outgrowth of Th1 effectors without an alteration of Th2 cell activation. Moreover, priming of naive T cells in the presence of leflunomide promoted Th2 differentiation from uncommitted precursors in vitro and enhanced Th2 effector functions in vivo, as indicated by an increase in Ag-specific Th2 cells and in the Th2-dependent Ag-specific Ig responses (IgG1) in immunized mice. The effects of leflunomide on T cell proliferation and differentiation could be antagonized by exogenous UTP, suggesting that they were related to a profound inhibition of de novo pyrimidine biosynthesis. These results indicate that leflunomide might exert its anti-inflammatory activities in the treatment of autoimmune diseases by preventing the generation of proinflammatory Th1 effectors and promoting Th2 cell differentiation. Moreover, the results further suggest that differentiation of CD4 T cells can be regulated at the level of nucleotide biosynthesis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
CD4 T cells can be divided into at least two different subsets depending on the pattern of their cytokine production. Th1 cells preferentially produce IL-2 and IFN-{gamma}, activate macrophages, and stimulate production of the Ig subclasses IgG2a and IgG3 in mice and IgG1 and IgG3 in humans (1, 2). The signature cytokines of Th2 cells are IL-4, IL-5, and IL-13, which provide potent B cell help and induce isotype switching to IgE and IgG1 in mice or to IgE, IgG2, and IgG4 in humans (1, 2). Whereas Th1 cells mediate cellular immunity and are responsible for delayed-type hypersensitivity reactions, Th2 effector functions are related to allergic reactions and immune responses to helminths (3, 4, 5). CD4 T cells regulate immune responses not only in physiological but also in pathological conditions. Whereas Th2 cells play a role in the pathogenesis of atopy and asthma, Th1 effectors are involved in the pathogenesis of several autoimmune diseases, such as rheumatoid arthritis (RA)3 (6, 7, 8). Importantly, Th2 cells inhibit the generation and effector functions of Th1 cells (9), and consequently, a reduction in Th1 and an increase in Th2 cell activity are associated with resolution of inflammation in autoimmune diseases. Moreover, a switch in T cell differentiation from the Th1 to the Th2 direction has been successfully used in the treatment of autoimmune diseases in animals (10, 11).

Recent evidence suggests that differentiated Th1 or Th2 cells do not derive from different precommitted lineages, but may develop from a common uncommitted precursor cell (3, 12). Cytokines play a dominant role in regulating T cell differentiation (9), but other factors, such as the genetic background, the intensity of costimulatory signals, particularly costimulation via CD28 (13), and the intensity of TCR signaling, may also modify the generation of Th1 or Th2 effector subsets. Several in vivo and in vitro experiments emphasize the role of the intensity of T cell stimulation in the outcome of Th differentiation. For example, in an Ag-independent in vitro system, increasing TCR stimulation with a mAb to the {epsilon}-chain of the TCR complex in the presence of constant costimulation enhanced IFN-{gamma} production of memory CD4 T effectors and Th1 differentiation in a dose-dependent manner (14). Likewise, high doses of Ag (15), a high affinity of peptide/MHC class II/TCR interactions (16, 17), and increasing intensities of Ag presentation (18) favor Th1 differentiation. In contrast, limited TCR engagement increased Th2 differentiation in those experimental systems. The mechanisms, however, by which the intensity of TCR-mediated signals regulates T cell differentiation are currently unknown.

In an attempt to down-regulate the activation and proliferation of CD4 T cells promoting chronic autoimmune inflammation, the limitation of newly synthesized pyrimidines has recently become a therapeutic principle in RA. While resting T cells use nucleotides from the salvage pathway for their steady state metabolic requirements, the pyrimidine pool expands 8-fold in activated T cells, and de novo biosynthesis of pyrimidines becomes essential for the expression of T cell effector functions (19, 20). Leflunomide is an izoxazole derivative that specifically inhibits the activity of dihydro-orotate dehydrogenase (DHODH) (21, 22), the rate-limiting enzyme of the de novo biosynthetic pathway of pyrimidines. It has been employed in several experimental animal models of allo- and xenotransplantations (23, 24, 25) and autoimmune diseases (26, 27, 28) with clinical benefit and has also been successfully used in the treatment of RA (29), resulting in a remission of inflammatory activity. Although the immunosuppressive ability of leflunomide has been well documented in these studies, the precise effects of a limitation of pyrimidine supply on T cell differentiation and effector functions have not been delineated.

We investigated the activation and differentiation of CD4 T cells in pyrimidine-limited conditions in vitro and in vivo. A restricted pyrimidine supply resulted in diminished T cell activation and decreased outgrowth and expansion of memory Th1 effectors. Notably, the inhibition of de novo pyrimidine biosynthesis by leflunomide promoted Th2 cell differentiation from uncommitted precursors in vitro, employing human cord blood naive cells, and in vivo, as assessed by the Ag-specific T cell response in immunized mice. The data suggest that the anti-inflammatory activity of leflunomide is not restricted to a simple inhibition of T cell activation, but is related to decreased Th1 cell generation and increased Th2 differentiation, shifting the balance toward immunomodulatory Th2 cells. Moreover, the data further suggest that T cell differentiation might be regulated at the level of de novo pyrimidine biosynthesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs and reagents

The following Abs and recombinant cytokines were used for purification of cells and for cell culture and staining: anti-CD3 (OKT3), anti-CD8 (OKT8), anti-HLA-DR (L243), anti-CD16, anti-CD19, anti-CD56, and anti-CD45RA (Dako Diagnostika, Hamburg, Germany); anti-CD45RO (111-1C5; a gift from Dr. R. Vilella, Barcelona, Spain); anti-human CD28 (28.2; BD PharMingen, San Diego, CA); neutralizing anti-IL-4 (25D2) and rIL-4 (Endogen, Woburn, MA); neutralizing anti-IL-12 and rIL-12 (R&D Systems, Wiesbaden, Germany); human rIL-2 (Life Technologies, Eggenstein, Germany); FITC-conjugated anti-CD3, PE-conjugated anti-CD4, PE-conjugated anti-CD45RA, FITC-labeled anti-CD45RO, FITC-labeled anti-CD25, FITC-conjugated anti-CD69, and FITC-conjugated anti-HLA-DR (Dako Diagnostika); and PE-labeled IL-4 (MP4-25D2), PE-labeled IL-2 (MQ1-17H12), and FITC-conjugated IFN-{gamma} (4S.B3; BD PharMingen). For determination of murine Igs, anti-mouse IgE (R35-72), mouse standard IgE (C48-2), biotinylated anti-IgE (R35-118), and biotinylated anti-mouse IgG1 (A85-1) and IgG2a (R19-15; BD PharMingen) were used. Murine cytokines were detected with high sensitivity Quantikine ELISA kits (R&D Systems), and mouse splenocytes were costimulated with anti-mouse CD28 (37.51; BD PharMingen). Immunoblotting was performed with anti-phosphotyrosine IgG (PY99; Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-STAT6, anti-STAT6, and anti-mouse or anti-rabbit HRP-linked IgG (New England Biolabs, Frankfurt, Germany).

Cell purification

Mononuclear cells were obtained from heparinized cord blood or from heparinized peripheral blood of healthy individuals after gradient centrifugation (40 min, 400 x g) over a Ficoll-Hypaque layer (Sigma-Aldrich, Deisenhofen, Germany). Cells were washed with PBS and incubated with SRBC (30). The rosette-positive cells were further purified by negative selection using saturating amounts of mAbs against CD8, CD16, CD19, CD56, HLA-DR, and CD45RA (for the purification of memory CD4 T cells) or CD45RO (for the purification of naive CD4 T cells), followed by panning on plastic petri dishes coated with goat anti-mouse Igs (Cappel, West Chester, PA) for 15 min at room temperature as previously described (14, 31). Recovered cells were washed with PBS and resuspended in RPMI 1640 (Life Technologies) containing 10% normal human serum (NHS). The purity and homogeneity of the recovered cells were assessed by flow cytometry. Typically, >=95% of the cells were positive for CD3 and CD4, >=90% of memory cells stained brightly for CD45RO, >=95% of the isolated naive cells were positive for CD45RA, and >=98% of the cells were viable after cell purification. Recovered cells were negative for the activation markers CD25, CD30, CD69, and HLA-DR (data not shown).

Culture conditions

All cell cultures were conducted in RPMI 1640 medium supplemented with penicillin G (100 IU/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), and 10% NHS at 37°C in a humidified atmosphere containing 5% CO2. Where indicated, the active metabolite of leflunomide, A77 1726, was added.

Flow cytometry

Freshly isolated, in vitro activated, and in vitro differentiated CD4 T cells (1 x 105/sample) were washed with PBS containing 2% FCS, incubated with saturating amounts of directly labeled mAbs, and analyzed by flow cytometry (EPICS; Beckman Coulter, Fullerton, CA). To determine the phenotype of the cells, as defined by their ability to produce cytokines (32), cells were stimulated with ionomycin (1 mM; Calbiochem, San Diego, CA) and PMA (20 ng/ml; Sigma-Aldrich) for 5 h in the presence of 2 µM monensin (Sigma-Aldrich), harvested, washed, and fixed with 4% paraformaldehyde (Sigma-Aldrich) in PBS for 10 min at 37°C. Cells were permeabilized with 0.1% (w/v) saponin (Sigma-Aldrich) in 2% FSC/PBS, and the unspecific binding sites were blocked with 4% rat and mouse serum. Cells were incubated for 30 min at 4°C with saturating amounts of directly fluorochrome-labeled mAbs against IL-2, IL-4, and IFN-{gamma}. After washing with 0.1% saponin/2% FCS/PBS, cells were resuspended in 2% FCS/PBS and analyzed for cytoplasmic cytokines by flow cytometry (32).

Analysis of cell division

Purified naive and memory CD4 T cells (1 x 107/ml) in PBS were incubated with 10 µM CFSE (Molecular Probes, Göttingen, Germany) for 8 min at room temperature. Labeling was stopped by adding 1 vol NHS, and the cells were washed three times with RPMI 1640 supplemented as described above. CFSE-labeled CD4 T cells (1 x 106/ml) were stimulated with immobilized OKT3 (1 µg/ml in Tris-HCl buffer, pH 9.5) and soluble anti-CD28 (1 µg/ml) in the presence or the absence of different concentrations of leflunomide. At different time points, cells were harvested, washed, and analyzed by flow cytometry.

In vitro generation of T cell effectors

Freshly isolated resting memory CD4 T cells (CD4+CD45RO+) were resuspended at a concentration of 1 x 106/ml in 10% NHS/RPMI 1640 supplemented with rIL-2 (10 U/ml) and activated with immobilized OKT3 (1 µg/ml in Tris-HCl buffer, pH 9.5) and soluble anti-CD28 mAb (1 µg/ml) in the presence or the absence of 100 µM leflunomide. Activated memory CD4 T cells were daily harvested, washed, restimulated as indicated above, and analyzed for the production of cytoplasmic cytokines by flow cytometry.

In vitro T cell differentiation

T cell differentiation was assessed employing an in vitro, multiple-step differentiation system as previously described (14, 32). Briefly, freshly isolated, naive CD4 T cells (CD4+CD45RA+) (0.5 x 106/ml) were primed by plate-bound OKT3 (1 µg/ml) and anti-CD28 mAb (1 µg/ml) in the presence of rIL-2 (10 U/ml), in the presence or the absence of A77 1726, and where indicated in the presence of 50 µM UTP (Amersham Pharmacia Biotech, Piscataway, NJ). In some experiments freshly isolated naive CD4 T cells were primed in the presence of IL-12 (40 ng/ml) and anti-IL-4 (10 µg/ml; Th1-inducing conditions) or in the presence of IL-4 (31.5 ng/ml) and anti-IL-12 (100 ng/ml; Th2-inducing conditions). After 5 days of priming, the cells were harvested, washed, counted, and rested for 2.5 days at 37°C at a concentration of 1 x 106 cells/ml in medium supplemented with a nonmitogenic concentration of rIL-2 (10 U/ml) to maintain cell viability (14). Differentiated CD4 T cells were harvested, washed, and assessed for their ability to produce cytokines by intracellular flow cytometry.

In vivo T cell differentiation

Six-week-old female C57BL/6 mice were immunized s.c. at the base of the tail with 50 µg keyhole limpet hemocyanin (KLH; Calbiochem) in IFA (Sigma-Aldrich). Four weeks after the first immunization mice were boosted with 50 µg KLH in IFA, and sera and splenocytes were collected 1 wk later. Leflunomide was freshly prepared in a suspension of distilled water containing 1% carboxymethyl-cellulose (CMC; Sigma-Aldrich) with medium viscosity as a carrier and was given daily by gavage from 7 days before to 7 days after the primary and secondary immunizations to five mice at a dose of 5 mg/kg. Mice in the control group (n = 5) were fed 1% CMC only.

Measurement of Ig production

Total IgE and KLH-specific IgG, IgG1, IgG2a, and IgE were determined in the sera of mice by ELISA. Nunc Immuno-Maxisorp 96-well plates (Dynatech, Nürtingen, Germany) were coated with 2 µg/ml KLH or anti-mouse IgE capture mAb in coating buffer (0.1 M Na2HPO4, pH 8.9) overnight at 4°C. Plates were washed three times with PBS containing 0.05% Tween and were blocked for 30 min at room temperature with PBS supplemented with 5% NHS and 5% FCS. After washing, serum samples (50 µl/well) or a serial dilution of standard (in 4% FCS/PBS) were added and allowed to bind for 2 h at room temperature. The plates were washed three times with 0.05% Tween/PBS. Biotin-conjugated anti-mouse IgG, anti-mouse IgE, or isotype-specific anti-mouse IgG1 or IgG2a were added and incubated for 2 h at room temperature. After removal of unbound mAbs, streptavidin conjugated with peroxidase (Roche, Mannheim, Germany) was added and allowed to bind for 1 h at room temperature. ABTS (Roche) was used as a substrate, and the OD405 was determined with a SPECTRAmax 190 spectrophotometer (Molecular Devices, Sunnyvale, CA).

Detection of cytokine secretion from splenocytes by ELISA

Splenocytes (1 x 106/ml) from mice that were immunized with KLH and treated or untreated with leflunomide were stimulated with KLH (10 µg/ml) and anti-CD28 (5 µg/ml) or PMA (1 mM) and ionomycin (20 ng/ml) for 72 h. IL-4 and IFN-{gamma} were assessed in the culture supernatants by high sensitive Quantikine ELISA kits (R&D Systems) according to the manufacturer’s instructions. The detection limit of the ELISA kits was <2 pg/ml murine IL-4 or IFN-{gamma}, respectively.

Detection of tyrosine phosphorylation of intracellular substrates in CD4 T cells

Freshly isolated naive CD4 T cells (2 x 106/ml) were primed with anti-CD3 (1 µg/ml) and anti-CD28 (1 µg/ml) mAbs in the presence of rIL-2 (10 U/ml) and in the presence or the absence of 25 µM A77 1726 for 48 h. Polarizing CD4 T cells were harvested, counted, and resuspended in ice-cold RPMI 1640. Recovered cells (1 x 106/ml) were restimulated with anti-CD3 (10 µg/ml) and anti-CD28 (10 µg/ml) mAbs and, where indicated, with rIL-4 (31.2 ng/ml) for 10 min at 4°C. After cross-linking with goat anti-mouse IgG (10 µg/ml; Cappel) for 2 min at 37°C, cells were washed with ice-cold PBS, lysed (62.5 mM Tris-HCl (pH 6.8), 2% SDS, 10% glycerol, 0.01% bromophenol blue, and 50 mM DTT) and boiled for 5 min. Cell lysates were separated in 10% polyacrylamide gels and transferred onto nitrocellulose membranes. The immunoblots were probed with specific Abs to phosphotyrosine (PY99), phospho-STAT6, or STAT6, and specific binding was detected with ECL (Santa Cruz Biotechnology).

Statistical analysis

The distributions of frequencies of activated or differentiated CD4 T cells producing cytokines in the presence or the absence of leflunomide were compared by paired two-tailed Student’s t test. Serum levels of total IgG, IgG1, IgG2a, and IgE were compared between the groups of mice using the Mann-Whitney test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Leflunomide inhibits proliferation of naive and memory CD4 T cells

To evaluate the impact of leflunomide on T cell proliferation, freshly isolated naive and memory CD4 T cells were labeled with CFSE and stimulated with anti-CD3 and anti-CD28 mAbs in the presence of increasing concentrations of the active metabolite of leflunomide, A77 1726. The drug inhibited the proliferation of naive and memory CD4 T cells in a dose-dependent manner. Concentrations >50 µM (naive T cells) and >200 µM leflunomide (memory T cells) were associated with increasing toxicity, as assessed by cell viability assays (data not shown). A maximum inhibitory effect of leflunomide on T cell proliferation without imposing toxicity was achieved with 25 µM in naive T cells and with 100 µM in the memory T cell subset (Fig. 1Go). Inhibition of T cell proliferation was evident from day 3 after T cell stimulation, as determined in kinetic experiments (data not shown). The presence of the pyrimidine nucleotide, UTP, during stimulation antagonized the inhibitory effect of leflunomide on T cell proliferation, indicating that the anti-proliferative activity of leflunomide on T cells was related to the inhibition of DHODH activity (data not shown).



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  		FIGURE 1. Leflunomide inhibits the proliferation and activation of naive and memory CD4 T cells. Purified naive and memory CD4 T cells (1 x 107/ml) were labeled with 10 µM CFSE, stimulated with mAbs to CD3 and CD28 mAbs in the absence or the presence of 25 µM (naive CD4 T cells) or 100 µM leflunomide (memory CD4 T cells) for 5 days, and analyzed by flow cytometry.

 
Leflunomide prevents the generation of Th1 effector cells

Upon activation, CD4 T cells regulate immune responses by generating T cell effectors that exert their function largely via the production of cytokines. To delineate whether the impairment of T cell activation by leflunomide was associated with an alteration of the generation of CD4 T cell effectors, resting memory CD4 T cells were stimulated with mAbs to CD3 and CD28 in the presence or the absence of 100 µM leflunomide. CD4 T cells were harvested daily and analyzed for their effector functions. To address the impact of leflunomide on particular T cell subsets, the ability of individual T cells to produce cytokines was assessed by flow cytometry. The numbers of recovered CD4 T cells in leflunomide-treated populations were reduced compared with the control values (data not shown), confirming data from the proliferation experiments (Fig. 1Go). Fig. 2GoA shows typical profiles of cytokine production of freshly isolated memory CD4 T cells and of T cell effectors that were activated in the presence or the absence of leflunomide. Stimulation of memory CD4 T cells induced an increase in single IFN-{gamma} frequencies from 3 to 42% on day 4. In contrast, the frequencies of CD4 effectors producing IL-2 gradually declined from 46 to 6% on day 4. Interestingly, the presence of 100 µM leflunomide significantly reduced the frequencies of specialized, well-differentiated, single IFN-{gamma} producers (Fig. 2GoA). Conversely, the frequencies of IL-2-producing cells were markedly increased in the presence of the drug. In accordance, the inhibitory effect of leflunomide on the generation of well-differentiated Th1 effectors was significant from day 2 after T cell activation, as determined in 11 individual experiments (Fig. 2GoB). By contrast, leflunomide did not influence the activation and/or outgrowth of IL-4-producing Th2 effector cells (Fig. 2GoB).



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  		FIGURE 2. Inhibition of the generation of proinflammatory Th1 effectors by leflunomide. Freshly isolated memory CD4 T cells were stimulated with mAbs to CD3 and CD28 in the presence or the absence of 100 µM leflunomide. Memory T cells were assessed daily for cytokine production by intracellular flow cytometry. A, Cytoplasmic cytokines in effector cell populations generated in the presence (upper panels) or the absence (lower panels) of leflunomide from one experiment. B, Demonstrated are the mean ± SD of the frequencies of CD4 T cell effectors primed in the presence ({circ}) or absence ({blacktriangleup}) of leflunomide producing IFN-{gamma} or IL-4 after stimulation with PMA and ionomycin from 11 individual experiments. *, Significant differences from control cultures (p < 0.01).

 
Leflunomide favors Th2 cell differentiation from naive precursor CD4 T cells

The previous data suggested that the generation of Th1 effectors from precommitted memory T cells was particularly sensitive to a restriction of de novo pyrimidine synthesis. To evaluate the impact of pyrimidine restriction on T cell differentiation of uncommitted precursors, human naive CD4 cells were primed with anti-CD3 and anti-CD28 mAbs in the presence of leflunomide. Differentiation of T cells was analyzed by assessing the production of cytokines indicative of polarized Th1 or Th2 subsets in individual cells from the primed cell populations. Freshly isolated naive CD4 T cells did not produce IL-4 and IFN-{gamma}, but in the population of the cells that were primed with anti-CD3 and anti-CD28 mAbs, 3.5% of the cells became IL-4 producers (Fig. 3GoA). The presence of 25 µM leflunomide during priming resulted in a significant increase in Th2 frequencies from 3.5 to 6.1% (Fig. 3GoA). By contrast, the frequencies of IFN-{gamma}-producing cells were unchanged, indicating that the commitment of naive CD4 T cells to the Th1 direction was not affected by leflunomide (Fig. 3Go). Thus, the increase in Th2 cell frequencies was the result of enhanced Th2 differentiation, but not of preferential inhibition of Th1 polarization.



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  		FIGURE 3. Leflunomide promotes Th2 differentiation from uncommitted precursor CD4 T cells. Freshly isolated naive CD4 T cells were primed with mAbs to CD3 and CD28 in the presence or the absence of leflunomide. After 2.5 days of rest the cells were stimulated with PMA and ionomycin, and cytoplasmic cytokines were assessed by flow cytometry. A, Frequencies of IL-4- and IFN-{gamma}-producing cells in T cells primed in the presence or the absence of leflunomide from one experiment. B, Demonstrated are mean ± SD of the frequencies of freshly isolated and primed naive T cells producing IL-4 or IFN-{gamma} from nine individual experiments. *, Significant differences from control cultures (p < 0.05).

 
As cytokines play a dominant role in directing T cell differentiation (9), the effect of leflunomide on T cell differentiation was assessed in naive T cells that were primed in the presence of rIL-12 and anti-IL-4 (to favor Th1 polarization) or in the presence of rIL-4 and anti-IL-12 (to drive Th2 differentiation). Augmentation of Th2 differentiation from naive precursors by leflunomide was observed independently of the cytokine milieu present during priming (Fig. 4Go). In contrast, leflunomide had no effect on Th1 cell differentiation regardless of the cytokine conditions provided. Whereas the effect of leflunomide on Th2 cell differentiation appeared to be inferior to the cytokine-mediated direction of T cell polarization, there was a significant increase in Th2 frequencies in each priming condition (Th1-inducing, neutral, or Th2-inducing) when leflunomide was present during priming.



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  		FIGURE 4. Leflunomide favors Th2 differentiation independently of the priming conditions. Naive CD4 T cells were primed in neutral, Th1-inducing (IL-12, anti-IL-4), or Th2-inducing (IL-4, anti-IL-12) conditions in the presence ({square}) or the absence ({blacksquare}) of 25 µM leflunomide. The cytokine secretion profile of individual cells was assessed after restimulation with PMA and ionomycin for 5 h as described. Demonstrated are the mean ± SD of the frequencies of freshly isolated and primed naive T cells producing IL-4 or IFN-{gamma} from three individual experiments.

 
UTP antagonizes the effect of leflunomide on Th2 cell differentiation

As UTP antagonizes the suppressive activity of leflunomide on T cell proliferation (28, 33) (data not shown), we evaluated whether the effect of the drug on Th2 cell differentiation was also related to the inhibition of de novo pyrimidine biosynthesis. Naive precursors were primed with anti-CD3 and anti-CD28 in the presence or the absence of 25 µM leflunomide and in the presence or the absence of 50 µM UTP. UTP itself did not alter Th2 cell differentiation compared with that of the control cultures that were primed in the absence of leflunomide and UTP (Fig. 5Go). However, whereas Th2 cell differentiation was increased by the presence of leflunomide, UTP completely antagonized this effect and blocked the increase in Th2 frequencies (Fig. 5Go). The data indicate that the effect of leflunomide on Th2 cell differentiation was related to the inhibition of de novo pyrimidine synthesis.



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  		FIGURE 5. UTP inhibits the effect of leflunomide on Th2 differentiation. Freshly isolated naive CD4 T cells were primed as described in Fig. 3Go. Where indicated 50 µM UTP was added to the priming cultures. Cytoplasmic cytokine production of differentiated CD4 T cells was assessed by flow cytometry. Demonstrated are the mean ± SD of the frequencies of freshly isolated and primed naive T cells producing IL-4 or IFN-{gamma} after PMA and ionomycin stimulation from four individual experiments.

 
Leflunomide increases Th2 differentiation in vivo

Our in vitro observations imply that leflunomide favored the differentiation of naive precursors to the Th2 direction. To elucidate whether the inhibition of de novo pyrimidine synthesis resulted in altered Th cell differentiation and T cell effector functions in vivo, C57BL/6 female mice were treated with leflunomide and subsequently immunized with KLH. Splenocytes were isolated and stimulated in vitro with KLH, and the cytokine production of Ag-specific T cells was determined by flow cytometry at an individual cell level and by ELISA in the supernatants of stimulated cells. The frequencies of Ag-specific T cells were only marginally above the detection limit of the FACS analysis (data not shown). However, determination of IL-4 and IFN-{gamma} in the supernatants of stimulated splenocytes revealed that the cells from leflunomide-treated animals produced significantly increased amounts of IL-4 and decreased amounts of IFN-{gamma} in response to challenge with KLH compared with controls (Table IGo). A similar pattern was observed when splenocytes were activated with PMA and ionomycin (Table IGo). Thus, the data show that leflunomide treatment in vivo shifted the balance of Th1/Th2 cells toward the later.


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  		Table I. Leflunomide induces a shift in the balance of Th1/Th2 cells in vivoa

 
To delineate the effect of a leflunomide-induced increase in Th2 cell differentiation on the outcome of an immune response in vivo, the KLH-specific Ig response was measured in the sera of the mice as an indicator of T cell effector functions. In this regard Th2 cells in mice stimulate the production of IgG1 Abs, whereas the production of IgG2a is a function of Th1-mediated B cell help (2). Leflunomide significantly decreased the level of total KLH-specific IgG (Fig. 6GoA), emphasizing its profound immunosuppressive activity. Interestingly, despite reduced total KLH-specific IgG, the level of Th2-dependent KLH-specific IgG1 in the mice treated with leflunomide was significantly increased (Fig. 6GoB). In marked contrast, the level of Th1-dependent IgG2a Abs in the sera of treated mice was decreased in a manner similar to that of total IgG (Fig. 6GoC). Consequently, the ratio of Th2-dependent KLH-specific IgG1 to Th1-dependent IgG2a increased >3-fold in treated animals, suggesting that leflunomide induced a shift from the Th1 to the Th2 direction in vivo (Fig. 6GoD). Notably, KLH-specific IgE was not detectable in the serum of any animal, and the level of total IgE was not significantly altered by leflunomide (Fig. 6GoE).



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  		FIGURE 6. Leflunomide increases Th2 effector functions in vivo. Six-week-old C57BL/6 mice were fed 1% CMC ({square}; n = 5) or 5 mg/kg leflunomide dissolved in 1% CMC/day ({blacksquare}; n = 5) and immunized with KLH as described in Materials and Methods. Sera were collected 1 wk after the secondary immunization, and the levels of total IgE and KLH-specific total IgG, IgG1, and IgG2a were determined by ELISA. A, Leflunomide inhibited KLH-specific IgG production. The mean KLH-specific IgG levels of the control mice were defined as 100 relative units. B, Leflunomide significantly increases the level of Th2-dependent KLH-specific IgG1. The mean KLH-specific IgG1 levels of the control mice were defined as 100 relative units. C, The level of Th1-dependent, KLH-specific IgG2a Abs decreases in leflunomide-treated animals. The mean KLH-specific IgG2a levels of the control mice were defined as 100 relative units. D, Leflunomide shifts the balance of Th2-dependent KLH-specific IgG1/Th1-dependent KLH-specific IgG2a toward IgG1. E, Leflunomide did not significantly alter the total production of IgE. *, Significant differences from control cultures (p < 0.05).

 
Leflunomide does not alter tyrosine phosphorylation of intracellular substrates in polarizing CD4 T cells

To elucidate whether the effect of leflunomide on Th2 differentiation was related to an alteration of tyrosine phosphorylation of intracellular substrates, naive CD4 T cells were primed in the presence or the absence of 25 µM leflunomide and restimulated with mAbs to CD3 and CD28, and whole cell lysates were analyzed by Western blot for tyrosine phosphorylation of intracellular substrates. No apparent difference was detected between cells that were primed in the presence of leflunomide and cells that were primed in the absence of leflunomide (data not shown).

As STAT6 is required for Th2 differentiation (34), and constitutively active STAT6 induces both Th2 differentiation and enhanced cell expansion (35), we examined whether leflunomide treatment resulted in altered expression or activation of STAT6. Whole cell lysates were prepared from naive CD4 T cells that were primed in the presence or the absence of leflunomide and restimulated with mAbs to CD3 and CD28 and with rIL-4. No differences in the expression of STAT6 and its phosphorylation in response to stimulation were found between leflunomide-treated and control cells (Fig. 7Go).



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  		FIGURE 7. Leflunomide does not alter STAT6 expression or phosphorylation in polarizing CD4 T cells. Naive CD4 T cells were primed with anti-CD3 and anti-CD28 in the presence or the absence of leflunomide for 48 h and restimulated with mAbs to CD3 and CD28 and with rIL-4. Whole cell lysates were separated by SDS-PAGE and were analyzed by Western blotting with Abs to phospho-STAT6 or STAT6.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Inflammatory Th1 cells play a central role in the immunopathogenesis of several autoimmune diseases in animals and humans (6, 7, 8, 32). A modulation of the predominantly activated Th cell subset from proinflammatory Th1 to immunomodulatory Th2 effectors is associated with the resolution of autoimmune inflammation and has been employed successfully in the treatment of autoimmune diseases in animals (10, 11). Here, we demonstrate that leflunomide inhibits activation of Th1 effectors and promotes Th2 cell differentiation both in vitro and in vivo. Thus, leflunomide might exert its anti-inflammatory activity in patients with RA by shifting the Th1/Th2 balance toward Th2 cells. As UTP completely antagonized the effect of leflunomide on T cell proliferation and differentiation, the data further suggest that the activity of leflunomide on T cell function was related to its potent inhibition of de novo pyrimidine synthesis. Therefore, the data provide evidence for the conclusion that a limited pyrimidine supply might favor Th2 cell development and hence that Th cell differentiation might be modulated at the level of nucleotide synthesis, without directly interfering with specific pathways, such as those elicited by cytokines, those mediated by engagement of costimulatory molecules, or those involving particular transcription factors.

Besides the inhibitory effect of leflunomide on DHODH activity that results in deprivation of pyrimidines in activated CD4 T cells, recent studies have indicated that leflunomide might also inhibit the phosphorylation of various protein kinases by different mechanisms. For example, leflunomide inhibited NF-{kappa}B-associated kinases with an IC50 of 10 µM (36). This effect on kinase activity could be antagonized by UTP (37), suggesting that it was secondary to the inhibition of DHODH activity. In contrast, inhibition of the Scr kinases p59fyn and p56lck (38, 39), which are involved in TCR transduction pathways, and Janus kinase (JAK)1 and JAK3 (40, 41), which are essential for signaling via IL-2 and IL-4 receptors, required concentrations of the drug that were 10- to 500-fold higher than those inhibiting DHODH and, subsequently, T cell proliferation (39). As the effect on p59fyn, p56lck, JAK1, and JAK3 could not be antagonized by UTP (38, 39, 41), it was reasoned that leflunomide directly affected the phosphorylation of those protein tyrosine kinases. In the current study UTP completely blocked the increase in Th2 frequencies of CD4 T cells primed in the presence of 25 µM leflunomide. Thus, a direct inhibition of tyrosine kinase activity by leflunomide as shown for p59fyn and p56lck is unlikely to contribute to enhanced Th2 cell differentiation. Moreover, the expression and activation of STAT6 were not affected by leflunomide (Fig. 7Go). Rather, inhibition of pyrimidine biosynthesis is sufficient to explain the facilitation of Th2 activity of leflunomide.

A simple explanation for the increase in Th2 cell differentiation from uncommitted naive CD4 T cells that were primed in the presence of leflunomide would be that the generation of Th1 cells was more sensitive to the action of leflunomide, and therefore, the increase in Th2 cell frequencies was secondary to a decrease in Th1 cell numbers. In fact, in memory effector CD4 T cells, 100 µM leflunomide significantly inhibited T cell activation and proliferation and also diminished the generation of Th1 effectors (Fig. 2GoB). However, in contrast to memory CD4 T cells, leflunomide did not alter the frequencies of IFN-{gamma}-producing CD4 T cells in primed naive T cell populations (Fig. 3Go). Thus, the increase in Th2 cell frequencies in naive CD4 T cell populations that were primed in the presence of leflunomide was not secondary to decreased Th1 cell proliferation, but was the result of increased Th2 differentiation. Th1 cell differentiation was not affected by the drug regardless of priming in Th1-promoting, neutral, or Th2-favoring cytokine conditions (Fig. 4Go). In contrast, Th2 cell frequencies were increased ~2-fold when leflunomide was present during priming independently of the cytokine environment (Fig. 4Go). The data, therefore, confirm the hypothesis that Th2 cell differentiation can be regulated at the level of pyrimidine synthesis independently of the surrounding cytokine milieu.

In support of this hypothesis, leflunomide significantly increased Ag-specific Th2 cell differentiation in vivo (Table IGo). Consequently, the level of Th2-dependent KLH-specific IgG1 in mice challenged with KLH was increased in leflunomide-treated animals (Fig. 6GoB). In marked contrast, the levels of Th1-dependent, KLH-specific IgG2a and KLH-specific total IgG were significantly reduced by leflunomide, emphasizing its profound immunosuppressive effect on CD4 T cell-dependent IgG production (42). Importantly, the ratio of Th2-dependent KLH-specific IgG1 to Th1-dependent KLH-specific IgG2a increased >3-fold in leflunomide-treated animals. The data, therefore, indicate that leflunomide increased Th2 cell differentiation and enhanced Th2 effector functions in vitro and in vivo and consequently induced a switch in the Th1/Th2 balance toward immunomodulatory Th2 cells. It should be noted that a similar increase in the Th2-dependent KLH-specific IgG1/Th1-dependent KLH-specific IgG2a ratio was observed in mice treated with a higher dose of leflunomide (35 mg/kg/day; data not shown).

In memory T cells leflunomide predominantly diminished the activation and outgrowth of Th1 effectors when present in an anti-proliferative concentration (100 µM), indicating that the generation of Th1 effectors from precommitted T cells might be particularly sensitive to a restriction of de novo pyrimidine synthesis. Analysis of the cytokine secretion pattern of individual activated effector cells revealed that leflunomide significantly reduced the frequencies of specialized IFN-{gamma}-producing Th1 cells. In marked contrast the frequencies of IL-2/IFN-{gamma}-coexpressing early Th1 cells remained elevated, and the frequencies of IL-4-producing Th2 cells were not altered by leflunomide (Fig. 2Go). As undifferentiated memory Th1 cells express IL-2, but no other cytokines, early Th1 cells coexpress IL-2 and IFN-{gamma}. Well-differentiated, late Th1 cells produce exclusively IFN-{gamma}, but not IL-2 (14, 31). Thus, the data imply that leflunomide inhibited the generation and/or outgrowth of specialized, IFN-{gamma}-producing, late memory Th1 cells.

In several recent studies vigorous T cell activation was shown to be required for IFN-{gamma} production and the expression of Th1 effector functions. For example, high doses of Ag (15), increasing concentrations of anti-CD3 mAb during priming of CD4 T cells (14), and high affinity TCR interactions (43, 44) preferentially induced IFN-{gamma} production, whereas, in contrast, low affinity of peptide/MHC class II/TCR interactions caused by changes in amino acid sequences either of the TCR (17) or the peptide (43, 44) led to a partial activation of T cells that was sufficient for IL-4 production, but did not alter the frequencies of IFN-{gamma}-producing cells. Together, the data support the idea that a decrease in CD4 T cell activation induced by limited de novo pyrimidine synthesis affected Th1 cells and their effector functions in a more pronounced manner than Th2 cells, which do not require a powerful T cell stimulation to develop. In concordance with this hypothesis, low doses of the purine inhibitor methotrexate favored IL-4 production of TCR-primed naive T cells (CD4+CD45RA+) in vitro (45), suggesting that limitation of both purine and pyrimidine biosynthesis might interfere not only with T cell proliferation, but also with Th cell differentiation. Together, the data suggest that leflunomide affects subsequent immune responses by reducing the number of CD4 T cells and by inhibiting the generation of specialized Th1 effectors.

An alternative explanation for the apparent different sensitivity of Th1 and Th2 effectors to leflunomide might derive from the recent observation that leflunomide is eliminated from the cells via multidrug-resistant pumps (46). As it has been suggested that Th1 and Th2 cells might express different multidrug-resistant pumps (47), a variation in the capacity to eliminate leflunomide from the cytosol of Th1 and Th2 cells might result in higher intracellular concentrations of the drug in Th1 cells that, consequently, would lead to enhanced anti-stimulatory activity.

The regulation of T cell differentiation is critical for the outcome of specific immune responses in both protective and destructive immunity, such as autoimmune diseases. Thus, the identification of mechanisms involved in the control of T cell differentiation might provide new targets for the development of novel effective treatments of autoimmune diseases. The current observation that leflunomide facilitates the induction of Th2 cell differentiation provides convincing evidence for the hypothesis that the restriction of pyrimidine biosynthesis might provide a mechanism to control inappropriate T cell activation in Th1-driven diseases.

In summary, we have demonstrated that restriction of de novo pyrimidine synthesis by leflunomide inhibits the generation of proinflammatory memory Th1 effectors and promotes the differentiation of immunomodulatory Th2 cells from uncommitted precursors in vivo and in vitro. As a modulation of the Th1/Th2 balance is associated with clinical benefit in autoimmune diseases, the therapeutic effect of leflunomide in RA might be related to these profound effects on the development of Th subsets. Inhibition of T cell activation and modulation of T cell differentiation by leflunomide were antagonized by the presence of UTP, suggesting that those effects were mediated via the inhibition of DHODH. Consequently, the data indicate that T cell differentiation might be regulated by de novo pyrimidine synthesis in activated CD4 T cells.


   Acknowledgments
 
We thank Dr. P. E. Lipsky for stimulating discussions and critical reading of the manuscript. We are grateful to Dr. R. Vilella (Barcelona, Spain) for his generous gift of the anti-CD45RA mAb, to Dr. P. Heise (Furth, Germany) for providing fresh cord blood, and to Dr. R. Voll (Erlangen, Germany) for his help with the animal experiments.


   Footnotes
 
1 This work was supported by the Interdisciplinary Center for Clinical Research at the University of Erlangen-Nuremberg (Project B27) and the Deutsche Forschungsgemeinschaft (Grants DFG Schu 786/2-2, 2-3, and 2-4). Back

2 Address correspondence and reprint requests to Dr. Hendrik Schulze-Koops, Nikolaus Fiebiger Center for Molecular Medicine, Clinical Research Group III, University of Erlangen-Nuremberg, Department of Internal Medicine III and Institute for Clinical Immunology, Glueckstrasse 6, 91054 Erlangen, Germany. E-mail address: schulze-koops{at}med3.imed.uni-erlangen.de Back

3 Abbreviations used in this paper: RA, rheumatoid arthritis; CMC, carboxymethyl-cellulose; DHODH, dihydro-orotate dehydrogenase; KLH, keyhole limpet hemocyanin; NHS, normal human serum; JAK, Janus kinase. Back

Received for publication February 8, 2002. Accepted for publication July 3, 2002.


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