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Modulation of effector cell functions in experimental autoimmune encephalomyelitis by leflunomide— mechanisms independent of pyrimidine depletion Thomas Korn,*,1 Tim Magnus,* Klaus Toyka,† and Stefan Jung* *Department of Neurology, Universita¨t des Saarlandes, Homburg, Germany; and †Department of Neurology, Julius-Maximilians-Universita¨t, Wu¨rzburg, Germany

Abstract: Leflunomide inhibits de novo pyrimidine synthesis and is a novel, immunosuppressive agent that has been successfully used to treat rheumatoid arthritis. Here, we investigated the efficacy of leflunomide and its mode of action in experimental autoimmune encephalomyelitis (EAE), which is a T helper cell type 1 cell-borne disease model to simulate inflammatory aspects of multiple sclerosis and was induced in Lewis rats by adoptive transfer of myelin basic protein (MBP)-specific T line cells. Given in vivo for 7 days after cell transfer, leflunomide suppressed clinical signs of disease even in uridine-substituted animals. MBP-specific T line cells that had been antigen-activated in vitro in the presence of A77 1726 (active metabolite of leflunomide) produced less interferon-␥, whereas interleukin (IL)-10 secretion had a tendency to be increased without changes in signal transducer and activator of transcription 6 trafficking. Furthermore, these T cells exhibited reduced chemotaxis and induced a significantly mitigated disease course upon transfer into naive rats. The effects of leflunomide on MBP-specific memory type T line cells in vitro may not be mediated by pyrimidine depletion, as they were not reversible by exogenous uridine. Moreover, A77 1726 led to increased expression of CD86 (B7-2) and secretion of IL-10 in cultured microglial cells in vitro, strengthening their down-modulatory impact on activated, autoantigen-specific T cells. In conclusion, our observations underline that the immunomodulatory potential of leflunomide in effector cells of EAE is clinically relevant and is not exclusively dependent on the depletion of cellular pyrimidine pools. J. Leukoc. Biol. 76: 950 –960; 2004. Key Words: clinical immunology sclerosis 䡠 cellular activation

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Th1 cells

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EAE

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INTRODUCTION Leflunomide [N-(4-trifluoromethyl-phenyl)5-methylisoxazol-4carboxamide] is a structurally and functionally new, immuno950

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modulatory drug [1]. Leflunomide is an inhibitor of the cyclooxygenase-2 and weakly decreases the expression of the inducible nitric oxide synthetase [2]. However, the main molecular target of A77 1726, which is the active, open-ring malononitrile metabolite of leflunomide, is the mitochondrial dihydroorotate dehydrogenase (DHODH), a key enzyme of the de novo pyrimidine synthesis [3, 4]. The enzyme is noncompetitively and reversibly inhibited by A77 1726 with a halfmaximal inhibitory concentration of 650 nM in humans and 15 nM in rats [5]. Consequently, the inhibition of immune cell proliferation appeared crucial to the effect of leflunomide in autoimmune diseases, as in case of activation, B and T cells may expand their pyrimidine pool by eightfold, depending on de novo pyrimidine biosynthesis [6]. During homeostatic proliferation, the so-called salvage pathway, which is independent of DHODH, seems sufficient for the cellular supply with pyrimidine bases [7]. To elucidate the semi-selective effects of leflunomide on B and T lymphocytes, a series of studies has been conducted to investigate modes of action beyond the inhibition of proliferation. Leflunomide was shown to affect nuclear factor (NF)-␬Bsignaling [8, 9] and is supposed to promote T helper cell type 2 (Th2) differentiation in vivo and in vitro [10]. These effects were assumed to also depend on the leflunomide-induced inhibition of the DHODH, as they were reversible by exogenous pryimidine supplementation. Other modes of action ascribed to tyrosine kinase inhibition by leflunomide [11–13] might be questioned regarding their relevance in vivo, as unphysiologically high drug concentrations were required for them to be induced in vitro [5]. Its presumed capacity to provoke Th2 skewing renders leflunomide an attractive agent to treat other Th1-mediated, autoimmune diseases apart from rheumatoid arthritis, where it has proven beneficial [14, 15]. Recently, a phase II study with teriflunomide, a closely related derivative of leflunomide, was implemented in multiple sclerosis (MS) patients. Leflunomide was tested in various neuroimmunological disease models such as actively induced experimental autoimmune myasthenia gravis [16], experimental autoimmune neu-

1 Correspondence: Dept. of Neurology, Universita¨t des Saarlandes, 66421 Homburg/Saar, Germany. E-mail: [email protected] Received May 27, 2004; revised July 27, 2004; accepted July 28, 2004; doi: 10.1189/jlb.0504308.

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ritis [17], and experimental autoimmune encephalomyelits (EAE) [18, 19], where, actively induced in Lewis rats by immunization with spinal cord homogenate in complete Freund’s adjuvant plus pertussis vaccine, preventive and therapeutic effects of leflunomide were demonstrated. Here, the model of adoptively transferred (AT)-EAE was used to identify underlying mechanisms of the autoimmune-suppressive effect of leflunomide. AT-EAE reflects effector pathways of autoaggressive inflammatory tissue damage in the central nervous system (CNS), which are relevant in the pathogenesis of MS lesions. We could show that the striking therapeutic potency of the compound in a setting of impending autoimmunity is based on the sustained reduction of interferon-␥ (⌱FN-␥) secretion and migratory capability of antigen-specific encephalitogenic T cells as well as on the strengthening of anti-inflammatory properties in microglial cells such as CNS-resident immunomodulators.

injection into the tail vein. Control rats received an equal number of T cell blasts stimulated with MBP following the conventional protocol.

Scoring of disease Rats were weighed daily and inspected for disease severity in an open-label setting by two independent observers. Clinical scores were given according to the following scale as described [23]: 0 ⫽ normal; 1 ⫽ reduced tone of the tail, hanging tail tip; 2 ⫽ limp tail, impaired righting; 3 ⫽ absent righting; 4 ⫽ gait ataxia, abnormal positioning; 5 ⫽ mild paraparesis of the hind limbs; 6 ⫽ moderate paraparesis; 7 ⫽ severe paraparesis or paraplegia of the hind limbs; 8 ⫽ tetraparesis; 9 ⫽ moribund; 10 ⫽death.

Isolation of microglial cells

Lewis rats, obtained from the Zentralinstitut fu¨r Versuchstierzucht (Hannover, Germany) or from our own breeding facility, were 6 – 8 weeks old and weighed 130 –240 g. All experiments were approved by the Bavarian and Saarland State Authorities.

Rat microglial cells were isolated from primary mixed brain glial cell cultures using a modification of methods described previously [24, 25]. In brief, microglial cultures were prepared from newborn Lewis rat brains (postnatal days 0 –2, Charles River, Sulzfeld, Germany), which were freed from their meninges and minced with scissors under a dissecting microscope (Wild, Heerbrugg, Switzerland). Mixed glial cell cultures were grown in microglia medium [basal medium Eagle’s supplemented with 10% FCS (Sigma, Deisenhofen, Germany) and 100 U/ml penicillin/100 ␮g/ml streptomycin] at 37°C for 10 –14 days. Then, microglial cells were isolated by shaking the culture flasks for 7 h and adherence to a FCS-coated culture flask (Primaria, Falcon, Franklin Lakes, NJ). After trypsinization, microglial cells were resuspended and reseeded in culture plates (Corning Inc., Corning, NY). To check the purity of the microglia cultures, we took a small fraction and performed immunocytochemistry with monoclonal antibodies (mAb) ED1 against CD68 (1:50, Serotec, Eching, Germany) and glial fibrillary acidic protein (GFAP; 1:100, Dako, Hamburg, Germany) as described [24]. Ninety-five percent or more of the cells were ED1-positive, and less than 5% were GFAP-positive.

Antigens and reagents

Proliferation assay

Myelin basic protein (MBP) was prepared from guinea pig brains according to the method of Eylar et al. [20]. Cell culture medium RPMI (Roswell Park Memorial Institute, Buffalo, NY) 1640 (Gibco, Eggenstein, Germany) was supplemented with 1% nonessential amino acids, 1% sodium pyruvate, 1% glutamine (all as 100⫻ solution from Gibco), and 100 U penicillin/100 ␮g streptomycin (Biochrom, Berlin, Germany) per ml. During restimulation, 2% fetal calf serum (FCS; Biochrom) and 10⫺5 M 2-mercaptoethanol (2-ME) were added. For the propagation of T cell blasts 10% (v/v), FCS and 10% (v/v) supernatant of concanavalin A (Con A)-activated syngeneic spleen cells (Con A sup) as an interleukin (IL)-2 source were added. For in vitro characterization of differentially activated MBP-specific T cells, human recombinant IL-2 (Proleukin, Chiron, Emeryvill, CA; and not Con A sup) was used at a concentration of 100 U/ml during the T cell expansion phase. Leflunomide, for in vivo application, was obtained from Aventis (Frankfurt/Main, Germany). As leflunomide is not converted to its active metabolite A77 1726 in vitro, we used the active metabolite A77 1726 by Aventis for all in vitro experiments.

In parallel to T line bulk activation for AT, proliferation of MBP-specific T line cells (MBP.2, MBP.4, and MBP.5) was assayed in flat-bottomed, 96-well microtiter plates (Nunc, Wiesbaden, Germany). T cell line expansion was measured by seeding 4 ⫻ 104 MBP-specific T cells and 2.5 ⫻ 105 irradiated, syngeneic splenocytes plus 20 ␮g/ml MBP in 100 ␮l medium per well. Furthermore, purified T cell blasts (50,000/well in 200 ␮l) that had been stimulated with MBP in the absence or presence of 20 ␮M A77 1726 plus 200 ␮M uridine were assessed for proliferation in IL-2-supplemented medium without A77 1726. Cells were labeled with 0.25 ␮Ci 3H-methyl-thymidine (NEN, Frankfurt/M, Germany) per well during the second day of culture and harvested 16 h later onto glass fiber filters by an Inotech 96-well harvester (PerkinElmer Wallac, Freiburg, Germany). Using melt-on scintillation sheets, 3 H-thymidine incorporation into the cells was measured by a Microbeta scintillation counter (1450 Microbeta Trilux, PerkinElmer Wallac). Data are given as mean counts per minute (cpm) of triplicate cultures (⫹SD).

MATERIALS AND METHODS Animals

Generation of encephalitogenic T cell blasts and treatment of AT-EAE Encephalitogenic CD4-positive T line cells specific for guinea pig MBP were established from popliteal lymph nodes of Lewis rats that had been immunized with MBP/complete Freund’s adjuvant antigen into the hind footpads [21]. For maintenance and adoptive intravenous (i.v.) transfer, T line cells (4⫻105/ml) were activated for 72 h in vitro with MBP (20 ␮g/ml) in the presence of irradiated (3000 rd), syngeneic, antigen-presenting spleen cells (APC; 2.5⫻106/ml; see also ref. [22]). To induce AT-EAE, naı¨ve Lewis rats were injected i.v. with 7 ⫻ 106 freshly activated and gradient-purified (Ficoll, Nycomed AS, Oslo, Norway), encephalitogenic, MBP-specific T line cells. Thereafter, rats were divided into control and treatment groups. Six rats (control animals) were sham-treated with 1.5% carboxymethyl-cellulose (vehicle for leflunomide), and 12 rats received leflunomide (20 mg/kg body weight/day per os). These animals were further subdivided into two groups of six rats each. One subgroup was administered 500 mg uridine intraperitoneally (i.p.) twice daily. In another set of experiments, AT-EAE was generated using MBP-specific T cell blasts that had been freshly activated with MBP and APC for 72 h in the presence of 20 ␮M A77 1726 plus 200 ␮M uridine before

Migration assay MBP-specific T line cells (MBP.5) were antigen-stimulated in the absence or presence of A77 1726 (20 ␮M) plus uridine (200 ␮M). Isolated T cell blasts were washed by centrifugation in serum-free medium [RPMI 1640, 0.25% bovine serum albumin (BSA), 100 U/ml IL-2] and placed in the upper chamber of a 24 well-transwell plate (Costar 6.5 mm transwell, polycarbonate membrane, pore size 5.0 ␮m). T cell blasts (5⫻105) were applied per insert in a volume of 100 ␮l. The bottom chamber contained rat recombinant macrophage inflammatory protein-1␣ (MIP-1␣; 100 ng/ml, PeproTech, Rocky Hill, NJ) or rat recombinant monocyte chemoattractant protein-1 (MCP-1; 10 ng/ml, PeproTech) in serum-free, IL-2- conditioned medium (600 ␮l). After 3 h (37°C, 5% CO2), cells that had migrated to the lower chamber were collected and counted by fluorescence-activated cell sorter. In parallel, CD4 (W3/25 mAb) and propidium iodide (PI) stains (Sigma) of the transmigrated cells were performed. All conditions were assessed in quadruplicates. Data are given as mean number of PI-negative cells (⫹SD) recorded in 90 s counting time.

Enzyme-linked immunosorbent assay (ELISA) For the detection of rat IFN-␥ and IL-10 in the supernatant of T cell cultures, OptEIA ELISA kits from PharMingen (Heidelberg, Germany) were used ac-

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cording to the manufacturer’s recommendations. Rat tumor necrosis factor ␣ (TNF-␣) was measured using a monoclonal anti-mouse/rat TNF-␣ [immunoglobulin G (IgG)] in 0.1 M carbonate buffer (pH 7.4) as capture antibody and a polyclonal rabbit anti-mouse/rat TNF-␣ in 1% BSA in phosphate-buffered saline (PBS) as detection antibody, which was biotinylated. Streptavidin-bound horseradish peroxidase and 3, 3⬘, 5, 5⬘ tetramethylbenzidine were used to visualize TNF-␣-dependent antibody binding.

Statistical evaluation Clinical scores were compared by the Wilcoxon rank sum test. For comparison of the means of migration data and ELISA data, the Student's t-test was used. Mean numbers of inflammatory cells per mm2 in the immunohistologically stained spinal cord sections were also compared by the Student’s t-test.

Immunoblot

RESULTS

MBP.4 cells (5⫻106) were preincubated for 1 h in the absence or presence of A77 1726 (50 ␮M) and stimulated with rat IL-4 (20 ng/ml, R&D, Wiesbaden, Germany) or phorbol 12-myristate 13-acetate (PMA; 10 ng/ml, Sigma) plus ionomycin (1 ␮M, Sigma) for 30 min in RPMI, 10% FCS, and 10 mM Hepes. The reaction was stopped by ice-cold PBS. Cells were centrifuged at 1000 g and lysed for 30 min on ice in cytoplasmic extract buffer [10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl2, 0.2% Nonidet P-40, 1 mM dithiothreitol (DTT), 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM leupeptin]. Lysates were centrifuged at 8000 g for 10 min at 4°C, and supernatants were collected as cytoplasmic extracts. Pelleted nuclei were reconstituted in nuclear extract buffer (20 mM Hepes, pH 7.9, 420 mM NaCl, 0.1 mM EDTA, 1.5 mM MgCl2, 25% glycerol, 1 mM DTT, 0.5 mM PMSF, 1 mM leupeptin) and lysed on ice for 15 min. After centrifugation (16,000 g, 4°C, 20 min), supernatants were removed as nuclear extracts. Protein contents were determined using the BioRad assay (Munich, Germany). Samples were normalized to equal protein concentrations, denatured in polyacrylamide gel electrophoresis-sample buffer (2% w/v) sodium dodecyl sulfate, 62.5 mM Tris/HCl, pH 6.8, 5% (v/v) 2-ME, and 10% glycerol, and resolved by electrophoresis on 4 –12% polyacrylamide gradient gels (NuPAGE威, Invitrogen, Carlsbad, CA). Proteins were transferred to nitrocellulose membranes, which were blocked in 3% skimmed milk/Tris-buffered saline/Tween 20 [TBST; 137 mM NaCl, 2.7 mM KCl, 50 mM Tris/HCl, pH 8.0, 0.05% (v/v) Tween 20] at room temperature for 1 h. Anti-signal transducer and activator of transcription (STAT)6 mAb (BD Transduction Laboratories, San Diego, CA) was diluted 1:500 in 3% skimmed milk/TBST. As secondary antibody, a biotinylated anti-mouse IgG (1:2000, Vector Laboratories, Burlingame, CA) was used. Specific signals were visualized using the streptavidin-biotin-peroxidase-complex reagent (Dako) and the enhanced chemiluminescence detection system (Amersham, Freiburg, Germany) with Fuji X-ray film RX NIF.

Leflunomide suppresses the effector phase of EAE in vivo To investigate the in vivo therapeutic potential of leflunomide under the conditions of an impending autoimmune encephalomyelitic attack, we transferred MBP-activated encephalitogenic T cells into naive animals. After cell transfer, rats were sham-treated (vehicle suspension) or received leflunomide (20 mg/kg/day) or leflunomide plus uridine for a period of 7 days. Uridine was given in excess (500 mg i.p. twice/day) and at this dose, certainly replenished the cellular pyrimidine pools in the liver and the secondary lymphoid tissue [27]. Therefore, leflunomide effects that were independent of DHODH inhibition should be unmasked. In previous trials, leflunomide at a dose of 20 mg/kg/day had proven to suppress virtually any clinical signs in the model of experimental autoimmune neuritis in Lewis rats [17]. As expected, the sham-treated rats were struck by a bout of severe paraparesis starting on day 3 after AT, from which they recovered by day 16 (Fig. 1). In contrast, the leflunomide group did not experience any clinical signs of EAE as long as leflunomide was being given. The suppression of clinical disease by leflunomide was not reversed by uridine supplementation. When leflunomide was discontinued, the

Flow cytometry After a washing step in PBS with 1% BSA and 0.1% sodium azide, the cells (2⫻105 per sample) were incubated at 4°C for 30 min with the mAb OX-6 [anti-major histocompatibility complex (MHC) class II (RT1B), IgG1, hybridoma supernatant, undiluted], 3H5 (anti-CD80, IgG1, 1:100), 24F (anti-CD86, IgG1, 1:100), or isotype control. A fluorescein isothioycanate-conjugated rat anti-mouse Ig F(ab⬘)2 fragment (Dianova, Hamburg, Germany) was used for detection of cell-bound, primary antibodies in a FACScan machine (Becton Dickinson, Heidelberg, Germany).

Histology Rats were anesthetized i.p. with Xylazine (Rompun威, Bayer, Leverkusen, Germany) 0.2%/ketamine (Ketanest威, Parke-Davis, Berlin, Germany) 5 ␮g/␮l in a dose of 2 ␮l/g body weight and were perfused through the left cardiac ventricle with Ringer solution (Fresenius, Bad Homburg, Germany) containing 20,000 U/l heparin, followed by 4% paraformaldehyde (Merck, Darmstadt, Germany) in a 0.1-M phosphate buffer, pH 7.4. Spinal cords were dissected and fixed for an additional 14 h. For immunohistological identifcation of T lymphocytes, spinal cord tissue was embedded in paraffin. Deparaffinized, 5 ␮m sections were treated with 0.03% H2O2 to quench endogenous peroxidase and were preincubated with porcine serum to cover unspecific binding sites. Sections were indirectly stained by the avidin-biotin-peroxidase technique [26]. Slides were incubated overnight with the mAb B115-1 (1:100, Sanbio, Beutelsbach, Germany) followed by incubation with affinity-purified, biotinylated, anti-mouse IgG (Vector Laboratories via Alexis, Gru¨nberg, Germany), which had been preabsorbed with normal rat serum. The avidin-biotin-peroxidase complex reagent and diaminobenzidine were used as recommended by the manufacturer.

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Fig. 1. In vivo treatment of AT-EAE with leflunomide. Lewis rats (six per group) were injected with 7 ⫻ 106 MBP-activated T cell blasts and treated in vivo as indicated (see bar on the time axis), receiving vehicle suspension (sham), leflunomide (lef; 20 mg/kg/day), or leflunomide plus uridine (2⫻500 mg/day i.p.), respectively. The clinical signs were scored as described in Materials and Methods and are given as mean clinical sores ⫹ SD.

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Fig. 2. Antigen stimulation of MBP-specific T cells in the absence and presence of A77 1726 plus uridine. (A) MBP.5 cells were MBP-stimulated (20 ␮g/ml) with increasing A77 1726 concentrations without or with uridine (200 ␮M). (B) Three different T line cells (MBP.5, MBP.2, and MBP.4) were antigen-stimulated in the absence (5-1, 2-1, 4-1) or in the presence (5-2, 2-2, 4-2) of A77 1726 (20 ␮M) plus uridine (200 ␮M) and expanded in conventional IL-2 medium devoid of A77 1726. (C) A conventional reactivation cycle (without A77 1726) was performed in MBP.5, MBP.2, and MBP.4 cells (TC) titrating the number of APC. In the previous cycle, 5-2, 2-2, and 4-2 cells had been antigen-activated in the presence of A77 1726/uridine. Proliferation was measured by 3H-thymidine incorporation and plotted as mean cpm ⫹ SD of triplicate cultures.

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treated rats developed, with a delay of 4 days after stopping therapy, some weakness of the tail but did not become paraparetic. The recovery was complete after 6 days. Thus, in vivo, leflunomide was able to suppress clinical signs of autoimmunity completely. The underlying mechanism did not seem to depend on pyrimidine depletion.

Antiproliferative effects of A77 1726 on memory T cells are reversible by uridine Encephalitogenic, MBP-reactive T cells initiate and regulate the inflammation of the CNS in Lewis rat EAE. We were interested in the modulatory potential of A77 1726 on these cells apart from its well-known, antiproliferative effects. The presence of the pyrimidine-base uridine during in vitro stimulation antagonized the antiproliferative effect of A77 1726 on MBP-specific T cells up to a concentration of 30 ␮M (Fig. 2A). This indicated that in terms of T cell proliferation, DHODH suppression by A77 1726 could be fully compensated for by uridine. To reveal immunomodulatory effects of A77 1726, T line cells were antigen-activated for 3 days in the absence or presence of 20 ␮M A77 1726 plus 200 ␮M uridine. T cell blasts were isolated and compared for IL-2-dependent proliferation and MBP-triggered reactivatability in vitro. As expected, IL-2-dependent proliferation rates did not differ as to whether the T cells had been antigen-activated in the absence or presence of A77 1726/uridine [Fig. 2B, data displayed for three different encephalitogenic T cell lines (MBP.5, MBP.2, MBP.4)]. Also, there was no relevant difference in APC dependency during the following antigen-activation cycle of MBP.5 cells that had previously been antigen-activated in the absence or presence of A77 1726/uridine (Fig. 2C). MBP.2 and MBP.4 cells even proliferated slightly better on antigen-triggered reactivation when they had seen A77 1726/uridine during the preceding cycle (Fig. 2C). Thus, leflunomide did not exhibit a delayed or prolonged inhibition of T cell proliferation in our in vitro system. By these experiments, bias coming from impaired expandability of A77 1726-exposed T cells could be excluded for the analysis of further effector functions of encephalitogenic T line cells.

A77 1726 modulates the cytokine pattern of MBP-specific T cells Cytokine secretion as the most crucial effector mechanism of CD4-positive T cells was investigated in three different MBPspecific T cell lines. Once antigen-stimulated in the presence of A77 1726/uridine, all MBP-specific T cell lines proliferated equally (see Fig. 2B) but secreted significantly less IFN-␥ during expansion in the IL-2-supplemented medium (Fig. 3). There was a tendency for IL-10 to be increased. TNF-␣ appeared unchanged. This suggested that in a line cell population, which exhibits memory cell properties and is heavily Th1-biased, the selective decrease of the hallmark Th1 cytokine IFN-␥ by leflunomide might not be dependent on DHODH inhibition. Furthermore, it must be considered a sustained effect of the compound, as leflunomide had no longer been present for at least 12 h when the supernatants for cytokine assessment were collected. 954

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A77 1726 does not enhance STAT6 signaling in antigen-specific T line cells It is well established that activated STAT6 has a key role in IL-4-dependent, Th2-lineage commitment. After Th1 (and more so, Th2) commitment has been established, there is no reversibility on the single-cell level [28]. However, within a T lymphocyte population, IL-2-producing precursors might still be driven in either direction. As we observed a decline in IFN-␥ production and a tendency toward higer levels of IL-10 secretion in A77 1726-exposed T cells, we wanted to know whether there was further evidence for the up-regulation of Th2-associated pathways within the T line population. In vitro, IL-10 production seems dependent on IL-4-mediated STAT6 activation [29]. Therefore, we focused on the STAT6 expression level and activation state. Cytosolic and nuclear fractions were analyzed for STAT6 by Western blot. Conventionally cultured MBP.4 cells (4-1) and MBP.4 cells that had been chronically exposed to A77 1726/uridine (4-2) were stimulated with PMA/ ionomycin or with 20 ng/ml IL-4 in the absence or presence of A77 1726. Both populations responded to IL-4 by increasing the nuclear fraction of STAT6 (Fig. 4). However, no obvious difference in the nuclear or cytosolic amount of STAT6 was detectable when comparing identical stimulation conditions between normal and chronically A77 1726-exposed cells. Therefore, it might be a suppression of Th1-related signaling pathways and not an enhancement of STAT6 that is responsible for the A77 1726-induced alteration in the cytokine pattern of antigen-primed T cells.

Chemokine-induced migration is impaired in activated T cells after A77 1726 pretreatment We next asked whether MBP-specific T cell activation in the presence of A77 1726 plus uridine had an impact on the chemotactic mobility of T line cells. T cell blasts stimulated under either condition, conventionally (5-1) or in the presence of A77 1726/uridine (5-2), were isolated and cultured in IL2-conditioned medium without A77 1726/uridine. We did not find an altered expression of adhesion molecules including ␣4-integrin or intercellular adhesion molecule on A77 1726pretreated T cells (data not shown). However, when testing the migratory capability using a transwell system, chemotaxis toward MIP-1␣ (P⬍0.005) and MCP-1 (P⬍0.02) was clearly reduced in A77 1726 T cells (5-2) as compared with conventionally stimulated, control cells (5-1, Fig. 5), suggesting that fewer encephalitogenic T cells might invade the CNS when antigen activation in the peripheral lymphoid tissue has taken place in the presence of leflunomide.

A77 1726-treated, antigen-specific T cells are less encephalitogenic To test the in vivo relevance of the impaired T cell effector functions, MBP-specific T cells that had been antigen-activated in vitro in the absence or presence of A77 1726 (20 ␮M)/uridine (200 ␮M) were transferred into naı¨ve rats that did not receive any subsequent in vivo treatment. As shown in Figure 6A, rats that were injected with MBP-specific T cells http://www.jleukbio.org

Fig. 3. Cytokine secretion of differentially activated T cell blasts. MBP-specific T line cells were antigen-activated in the absence (5-1, 2-1, 4-1) or presence of A77 1726/uridine (5-2, 2-2, 4-2), gradient-purified and expanded in conventional IL-2 medium. After the indicated time intervals, supernatants were analyzed for cytokine concentration by ELISA. Significant differences in cytokine secretion are marked by asterisks for the individual T cell lines (*, P⬍0.02, and **, P⬍0.002).

that had been routinely activated (control group) exhibited a typical monophasic disease course, culminating in severe tetraparesis on day 7 post-AT. In contrast, the recipients of T cells that had been MBP-activated in the presence of A77 1726/uridine showed a significantly mitigated disease course, confirming a reduced encephalitogenicity of A77 1726-exposed, MBP-specific T cells. The immunohistological analysis of spinal cord sections from control animals revealed scattered T cell infiltrates in the

spinal cord parenchyma (Fig. 6B, a), whereas T cell blasts that had previously been activated in the presence of A77 1726/ uridine appeared essentially confined to the meningeal space (Fig. 6B, b). The failure of A77 1726/uridine-pretreated T cell blasts to massively invade the CNS parenchyma led to significantly reduced intraparenchymal T cell numbers (Fig. 6C). This was strong evidence for the in vivo relevance of the impaired chemotactic mobility of A77 1726/uridine-exposed T cell blasts that had been measured in vitro previously (Fig. 5). Korn et al. Leflunomide in EAE

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DISCUSSION

Fig. 4. STAT6 signaling in MBP.4 cells after chronic A77 1726 exposure. MBP-specific T line cells were antigen-activated and expanded in the absence (4-1) or presence of A77 1726/uridine (4-2). During 30 min, both cell populations were stimulated with IL-4 (20 ng/ml) or PMA (10 ng/ml) plus ionomycin (Iono; 1 ␮M) in the absence or presence of 50 ␮M leflunomide as indicated. Cytosolic and nuclear extracts were prepared, and equal amounts of protein (10 ␮g per lane for cytosolic extracts and 1 ␮g per lane for nuclear extracts) were analyzed by Western blot. The specific STAT6 signal of 100 kD is shown.

A77 1726 alters the properties of activated microglial cells Obviously, it was essentially different whether conventionally MBP-activated T cells were transferred into animals that were treated with leflunomide in vivo or whether T cells that had been MBP-activated in the presence of A77 1726 in vitro previously were transferred into rats that were subsequently left untreated. In the first setting, signs of disease were suppressed (Fig. 1); in the second, they were only mitigated (Fig. 6). To find explanations for this discrepancy, we investigated A77 1726-induced effects on microglial cells. Microglia is an important immune effector population that resides in the CNS and is also supposed to have the capacity to reactivate and modulate pathogenic T cells in situ [30]. In vitro, microglial cells obviously changed their phenotype in the presence of A77 1726; i.e., they flattened, formed processes, and adhered more tightly to the culture dish when activated by lipopolysaccharide (LPS; Fig. 7A). After 3 days of LPS stimulation, microglia exhibited an up-regulated surface expression of MHC class II and of the costimulatory molecules CD80 and CD86. This was also observed in microglial cells that had been cultured in the presence of LPS plus A77 1726. However, A77 1726 appeared to preferentially enhance the up-regulation of CD86 as opposed to CD80 or MHC class II (Fig. 7B). To substantiate the hypothesis of A77 1726-induced modulation of microglial cells, we measured TNF-␣ and IL-10 secretion into the supernatant. It is interesting that A77 1726 led to a significantly increased IL-10 production in LPS-stimulated microglia, whereas TNF-␣ was unchanged when compared with activated microglia that had been maintained in the absence of A77 1726 (Fig. 7C). Moreover, A77 1726-exposed microglial cells were significantly more inhibitory than naı¨ve microglia when present as third-party cells in a spleen–APC-driven, antigen-specific restimulation assay of encephalitogenic Th1 cells (Fig. 8). In contrast to the conventional restimulation protocol (see Fig. 2A), the A77 1726-induced decrease in proliferation of the T responder cells was not reversible by exogenous uridine when microglial cells were cocultured (Fig. 8). This indicates that A77 1726 might strengthen the capability of microglial cells to down-regulate the activation of pathogenic Th1 cells. 956

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As leflunomide has been successfully used in rheumatoid arthritis, there is a high interest in expanding its application toward other Th1-mediated, organ-specific, autoimmune diseases such as MS. Here, we provide evidence that leflunomide is effective in AT-EAE. This is a setting where antigen priming and cytokine commitment of T cells have already taken place, a situation likely to be present at the time when treatment decisions have to be taken in the human scenario. In vivo treatment of AT-EAE with leflunomide prevented clinical signs of disease as long as the drug was being administered. Discontinuation of leflunomide led to an attenuated flare-up of encephalitic signs within 4 days. Therefore, it is unlikely for leflunomide to produce apoptosis of antigen-specific T cells in vivo. Accordingly, neither naı¨ve thymocytes nor T line cells showed increased apoptosis rates during exposure to A77 1726 in vitro as confirmed by Annexin staining (data not shown). This is in line with the fact that A77 1726 inhibits

Fig. 5. Chemotaxis of MBP-specific T cells is reduced after MBP stimulation in the presence of A77 1726/uridine. MBP.5 cells were antigen-activated in the absence (5-1) or presence (5-2) of 20 ␮M A77 1726 plus 200 ␮M uridine, gradient-purified, controlled for equal proliferation, and tested for migratory capability in a transwell system in the absence of A77 1726. Previous treatment during antigen stimulation prompted T cell blasts to migrate significantly less toward MIP-1␣ and MCP-1. Chemokinesis in the presence of MIP-1␣, MCP-1, or plain medium in the upper (UC) and lower chamber (LC) was also significantly reduced in A77 1726-pretreated (5-2) T cells (*, P⬍0.02; **, P⬍0.005; ***, P⬍0.0005). Transmigration assays were performed in quadruplicates.

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Fig. 6. A77 1726/uridine-pretreated, MBP-specific T cells (T-MBP) are less encephalitogenic. (A) After MBP stimulation in the absence (control) or presence of A77 1726/uridine, naive Lewis rats (six per group) were injected with either cell type without subsequent in vivo treatment. The mean clinical score ⫹ SD of each group during the disease course is displayed. Significant differences between groups are marked by asterisks (*, P⬍0.005). (B) In a parallel experiment, control animals (n⫽6) and recipients of A77 1726/uridinepretreated, MBP-specific T cells (n⫽5) were killed for histological analysis on day 7 after cell transfer. Lumbar spinal cord sections were stained for T cells. (B) Representative sections of a severely paraparetic control rat (a) and an A77 1726/uridine cell recipient (b) are shown. Some of the invading T cells, which were found within the spinal cord parenchyma, are highlighted by arrows (bars, 20 ␮m). (C) Strictly intraparenchymally located T cells were counted. To this end, three lumbar spinal cord sections per animal were analyzed by at least 11 low-power magnification fields covering the entire spinal cord cross-section. For comparison of the T cell numbers per mm2 between the groups, the t-test was used (*, P⬍0.0004).

TNF-␣-induced caspase activation and thus prevents apoptotic cell death in vitro [8]. Rather, two other substantial effects of A77 1726 on MBP-specific T cells were identified. First, a decreased IFN-␥ production and second, a reduced migratory capability by T cell blasts that had been antigen-activated in the presence of A77 1726. We noticed a consistent and sustained reduction of IFN-␥ secretion when T line cells were antigen-activated in the presence of A77 1726, even if the culture medium was devoid of the drug during subsequent T cell expansion. The reduced secretion of this key Th1 effector cytokine was not a result of the lack of IL-2, as during antigen stimulation (without addition of exogenous IL-2) and during T cell expansion (with addition of exogenous IL-2), T cells proliferated as well in the presence of A77 1726 plus uridine as in their absence, indicating sufficient IL-2 concentrations and responsiveness, respectively. The IFN-␥ gene contains a NF-␬B binding site in the promotor region as well as intronic enhancers that bind the NF-␬B protein c-Rel [31, 32], and A77 1726 is known to suppress signaling through NF-␬B in Jurkat cells [9]. This

mechanism could account for the reduced secretion of IFN-␥ after T cell activation. The MBP-specific T line cells used in these experiments were CD4-positive, Th1-type cells. All the more surprising was the finding that IL-10 tended to be increased after stimulation in leflunomide-containing medium. In a recent report, A77 1726 was shown to shift the balance of uncommitted T cell precursors toward Th2 [10], although the STAT6 signaling pathway did not seem to be up-regulated. A77 1726-modulated preactivation of T cells led to a clearly decreased chemotactic response. This was confirmed in vivo, as T cell blasts that had been antigen-activated previously in the presence of A77 1726/uridine certainly were viable and proliferated well but were hardly capable to invade the CNS parenchyma upon transfer into naive rats. It is, at this point, unclear which is the underlying molecular mechanism of the decreased chemotactic T cell mobility. We did not recognize a down-regulation of the MCP-1 receptor (CC chemokine receptor 2) on activated T cells in vitro (data not shown). In previous reports, A77 1726 has been found to inhibit homotypic T lymphocyte aggregation by blocking signaling through CD43 Korn et al. Leflunomide in EAE

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Fig. 7. A77 1726 modulates the properties of microglial cells in vitro. (A) LPS-stimulated (100 ng/ml) microglial cells were cultured in the absence (a) or presence (b) of A77 1726. Microphotographs of typical cultures are shown (bars, 25 ␮m). Note the intensified ramification and adhesion of A77 1726-exposed microglia (b). (B) Surface markers of conventionally cultured (shaded curves) and A77 1726-exposed microglia (overlays) after 3 days of LPS stimulation. (C) Supernatants of microglial cells that had been LPS-activated for 3 days without or with 30 ␮M A77 1726 were analyzed for TNF-␣ and IL-10. One representative of two experiments performed in triplicates is shown (*, P⬍0.02).

[33]. In vivo, CD43 is involved in homing of T cells to lymph nodes via high endothelial venules [34]. As in AT-EAE too, further activation of transferred encephalitogenic T cells takes place in lymph nodes and spleen [35], impaired migration of A77 1726-exposed T cells to these secondary lymphoid organs might eventually have an additional impact on the number of pathogenic T cells ready to invade the CNS. The therapeutic effects of leflunomide in vivo were not reversed by simultaneous administration of uridine. As the uridine dose was certainly high enough to replenish cellular pyrimidine pools even under conditions of increased pyrimidine needs [27], the antiproliferative effect of leflunomide on lymphoid cells might not be essential in vivo. Altered cytokine pattern and impaired migration of MBP-specific line cells in 958

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vitro were not dependent on depletion of pyrimidine nucleotides either. Conversely, the A77 1726-induced Th2 skewing of naı¨ve T cells in vitro was reported to be reversible by exogenous pyrimidine nucleotides [10]. Thus, there appears to be a split between A77 1726-induced effects that can be rescued by exogenous pyrimidines brought into the salvage pathway for the generation of pyrimidine nucleotides and others that are not reversible by exogenous pyrimidines. It is in line with this idea that uridine reversed in vivo effects of leflunomide on B cells and Th cell activity but not the simultaneously induced suppression of cytotoxic effector T cells in a mouse model for autoimmune diabetes [36]. In homogeneous cell populations such as T line cells, A77 1726 might act on the single-cell level, inhibiting signaling pathways for certain effector cytohttp://www.jleukbio.org

In conclusion, we proved that the spectrum of actions of leflunomide in treating CNS-specific autoimmunity includes the modulation of effector T cells, directly and indirectly via alteration of the T cell stimulatory milieu by CNS resident microglia. The fundamental impact of leflunomide on effector T cells could be shown in vitro and in vivo. Neither in vitro nor in vivo was the suppression of antigen-specific Th cell effector functions dependent on DHODH inhibition. This leads to the hypothesis that in contrast to the situation during T cell priming [10], the cellular pyrimidine load might no longer be a target to alter immune functions of firmly established effector cells. Our results are promising in terms of the application of leflunomide in chronic, Th1-mediated, autoimmune diseases such as MS, where safe drugs that combine immunosuppression with immunomodulation are still urgently needed.

ACKNOWLEDGMENTS

Fig. 8. A77 1726 enhances Th1 cell-suppressive effects of microglia (MG). MBP-specific responder T cells (MBP.5, 40,000/w) were activated with MBP and irradiated splenic APC (250,000/w) in the absence or presence of microglia (10,000/w). Where indicated, the culture medium was supplemented with A77 1726 (30 ␮M) or A77 1726 plus uridine (urid; 200 ␮M). Proliferation was measured by 3H-thymidine incorporation and plotted as mean cpm ⫹ SD of triplicate cultures (*, P⬍0.01, compared with culture in the presence of untreated microglia).

This work was supported by university funds of the University of the Saarland and by grants of the Gemeinnu¨tzige HertieStiftung (1.319.110/02/08). We are indebted to Dr. J. Pu¨nter from Aventis for providing leflunomide and A77 1726. We thank S. Seubert, A. Jerges, and A. Sabo for skillfull technical assistance. We also thank our chairman Prof. Georg Becker for his continuous support. Recently, he died in an accident. We are deeply grieving this.

REFERENCES kines and leaving cell-cycle progression of the same cell unchanged when only sufficient pyrimidine nucleotides are available from exogenous sources. Further investigations are necessary as to whether given a DHODH block by A77 1726, there are differences in the access to salvaged pyrimidines of the various pyrimidine-dependent, cellular synthesis pathways. The attenuation of EAE was not as strong when A77 1726exposed, MBP-activated T cells were transferred as in in vivo therapy after transfer of untreated, antigen-stimulated T cells. It is quite obvious that under in vivo therapy, a lot of effector cell populations such as bystander T cells or macrophages might be modulated by leflunomide, which does not apply for AT-EAE induced by A77 1726-pretreated T cells. Here, we looked more closely at microglia, the local immune cells of the CNS. In an inflammatory surrounding, microglial cells promote effector cell functions but are also down-modulatory [37, 38]. It is interesting that leflunomide prompted cultured microglia cells to enhance IL-10 secretion and increase expression of the costimulatory molecule CD86 (B7-2), which has been associated with the induction of less harmful or even protective T cell responses [39, 40]. In vitro, A77 1726-exposed microglia inhibited antigen/APC-driven proliferation of encephalitogenic T cells to a greater extent than untreated microglia. Therefore, it is conceivable that in vivo treatment with leflunomide might create a microglial environment in the CNS that is less favorable for activation of encephalitogenic Th1 cells, thus turning down destructive autoimmunity.

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