Brain, Vol. 124, No. 9, 1791-1802,
September 2001
© 2001 Oxford University Press
Suppression of experimental autoimmune neuritis by leflunomide
1 Department of Neurology, Neuroimmunology Branch and MS Clinical Research Group, Julius-Maximilians-Universität Würzburg, Germany and 2 Department of Neurology, Karl-Franzens-Universität Graz, Austria
Correspondence to:
Dr Stefan Jung, Department of Neurology, Building 90, Universität des Saarlandes, D-66421 Homburg/Saar, Germany E-mail: nesjung{at}med-rz.uni-saarland.de
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Abstract |
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Leflunomide is a new immunosuppressive drug whose active metabolite, A77 1726, impairs cellular nucleotide metabolism by inhibiting the dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme of de novo pyrimidine synthesis. Furthermore, A77 1726 suppresses tyrosine kinases involved in signal transduction pathways. We investigated the immunosuppressive effects of leflunomide in experimental autoimmune neuritis (EAN) in rats, which is a model of immune-mediated neuropathies. In EAN that was actively induced by subcutaneous injection of peripheral nerve myelin, leflunomide completely prevented paraparesis if applied orally from the day of immunization. Leflunomide was much more effective than azathioprine, which did not mitigate EAN at all. Even when leflunomide was administered therapeutically after the appearance of the first neuropathical signs, it halted the progression and markedly reduced the severity and duration of EAN. Inflammatory infiltrates, demyelination and axonal degeneration in sciatic nerve sections of leflunomide-treated EAN rats were strongly reduced. Leflunomide-treated rats did not mount autoantibodies as specified by ELISA (enzyme-linked immunosorbent assay) with a mixture of peripheral myelin proteins, including P2 and myelin basic protein. In EAN that was adoptively transferred by injection of neuritogenic cells of a P2-specific T-helper line, application of leflunomide also clearly reduced signs of disease. Additional injection of uridine did not neutralize the effect of leflunomide. Similarly, transfer of neuritogenic P2-specific T cells, which were activated in the presence of A77 1726 plus uridine in vitro, still resulted in reduced severity of adoptive transfer EAN in vivo, although proliferation of these cells in vitro was identical to that of control cells. The T-cell receptor-mediated in vitro activatability of a P2-specific T-cell hybridoma was diminished by high concentrations of A77 1726, as evidenced by reduced Ca2+ flux into the cytosol. Together with the findings in adoptive transfer EAN, this indicates that the antiproliferative effect is probably not the only mechanism of immunosuppressive action by leflunomide. In summary, leflunomide suppresses EAN efficiently and may constitute a promising therapy for immune-mediated neuropathies.
immune-mediated neuropathy; pyrimidine metabolism; leflunomide; immunomodulatory therapy
AT-EAN = adoptive transfer EAN; BPM = bovine peripheral nerve myelin; CFA = complete Freund's adjuvant; DHODH = dihydroorotate dehydrogenase; EAN = experimental autoimmune neuritis; FCS = foetal calf serum; mAb = monoclonal antibody; PBS = phosphate-buffered saline; OD = optical density; PBST = PBS with Tween-20; p.i. = post-immunization; p.o. = per os; TCR = T-cell receptor
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionAcknowledgementsReferences |
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Leflunomide [N-(4-trifluoromethyl-phenyl)5-methylisoxazol-4-carboxamide] is an isoxazole derivative that is structurally and functionally unrelated to other known immunomodulatory drugs (Brazelton and Morris, 1996
). In the intestinal mucosa and in plasma, almost 100% of the compound is non-enzymatically converted into the active open ring malononitrile metabolite A77 1726. The main molecular target of A77 1726 is the dihydroorotate dehydrogenase (DHODH), a key enzyme of de novo pyrimidine synthesis (Davis et al., 1996; Liu et al., 2000). The enzyme is non-competitively and reversibly inhibited by A77 1726 with an IC50 of 650 nM in humans and 15 nM in rats (Herrmann et al., 2000). At the cellular level, there is evidence for a variety of additional effects independent of pyrimidine metabolism that might be of relevance in the treatment of autoimmune diseases: (i) prevention of isotype switching from IgM to IgG1 after stimulation of the interleukin-4 receptor in murine B cells (Siemasko et al., 1998); (ii) decreased Ca2+ mobilization and interleukin-2 production in human lymphoma cells upon triggering the T-cell receptor (TCR) complex (Xu et al., 1995); and (iii) diminished activation of inducible nitric oxide synthase in rat astrocytes (Miljkovic et al., 2001). These phenomena are compatible with the tyrosine kinase inhibitory properties of leflunomide (Mattar et al., 1993; Xu et al., 1995, 1996; Elder et al., 1997). However, regarding the IC50 values for enzyme inhibition, the potency of leflunomide to suppress tyrosine kinase activity is at least one order of magnitude lower than that for DHODH inhibition. Therefore, the functional importance of tyrosine kinase inhibition by leflunomide in vivo has been questioned in the context of its immunosuppressive action (Herrmann et al., 2000). Consequently, the main effect of leflunomide in immune-mediated diseases has been attributed to the inhibition of proliferation of B and T cells. When activation occurs, these cells may expand their pyrimidine pool up to eight-fold relying on de novo pyrimidine biosynthesis (Fairbanks et al., 1995). It is still not clear which of these modes of action are relevant to the immunosuppressive effect of leflunomide observed in various autoimmune mediated diseases.
In 1998, leflunomide was approved (as Arava) for the treatment of rheumatoid arthritis by the FDA (Food and Drug Administration, USA). It is the latest disease-modifying antirheumatic drug and the first to receive the indication of retarding the structural damage of rheumatoid arthritis (Schattenkirchner, 2000). In a phase III clinical study, leflunomide showed a rapid onset of action without major myelotoxic adverse events (Smolen et al., 1999). Leflunomide has been used successfully in animal models of neurological autoimmune diseases, including experimental myasthenia gravis (Vidic et al., 1995) and experimental autoimmune encephalitis (Bartlett et al., 1993). We studied the compound in experimental autoimmune neuritis (EAN), a model of inflammatory demyelinating immune-mediated neuropathies (Hughes, 1994; Hahn, 1996). We observed a striking therapeutic effect of leflunomide in EAN. From our investigations, we hypothesize that leflunomide provokes a substantial functional alteration of neuritogenic T cells besides inhibiting their proliferation.
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Material and methods |
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Animals
Female and male Lewis rats obtained from the Zentralinstitut für 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 State authorities.
Antigens and reagents
Bovine peripheral nerve myelin (BPM) was prepared from intra- and extradural spinal nerve roots according to Norton and Poduslo on sucrose gradients (Norton and Poduslo, 1973). After washing of the interphase, water-soluble myelin proteins (P2 and myelin basic protein) were extracted at pH 2.0 (acidic extract). P2 protein was isolated from acidic extract as described by Kitamura and colleagues (Kitamura et al., 1976). The neuritogenic peptide P2 53–78, representing the corresponding amino acids of bovine P2, was synthesized by Biotrend (Köln, Germany). Concanavalin A was obtained from Pharmacia (Freiburg, Germany).
Cell culture medium RPMI (Roswell Park Memorial Institute) 1640 (Gibco, Eggenstein, Germany) was supplemented with 1% non-essential amino acids, 1% sodium pyruvate, 1% glutamine (all as x100 solutions from Gibco), and 100 U penicillin/100 µg streptomycin (Biochrom, Berlin, Germany) per ml. During restimulation, 2% foetal calf serum (FCS) (Biochrom), 10 µg/ml indomethacin (Sigma, Deisenhofen, Germany) and 10–5 M 2-mercaptoethanol were added. During interleukin 2-dependent propagation of T-cell blasts, supplementation consisted of 10% FCS and 8–10% supernatant of Concanavalin A-activated syngeneic spleen cells.
Leflunomide for in vivo application was obtained from Aventis (Frankfurt am Main, Germany) (formerly Hoechst Marion Roussel). Since leflunomide is not converted to its active metabolite A77 1726 in vitro, we used the active metabolite A77 1726 by Aventis for in vitro experiments.
Monoclonal antibodies
The hybridoma R73 (Huenig et al., 1989), secreting mouse monoclonal antibody (mAb) R73 (IgG1) directed to the rat 
/ß TCR, was kindly provided by Thomas Hünig, Würzburg. Hybridoma culture supernatants containing 2% 
-globulin-depleted FCS (c.c. pro; Neustadt, W. Germany) were prepared conventionally or in the Techno-Mouse (Technomara, Fernwald, Germany). The mAb was isolated by protein A (Pharmacia) affinity chromatography, dialysed in phosphate-buffered saline (PBS), sterilized by filtration, and stored at –20°C.
Active induction and therapy of EAN
For active induction of myelin EAN, each rat was immunized in the right hind footpad with 50 µl of an emulsion of equal volumes of PBS and Freund's incomplete adjuvant oil (Gibco) containing 3.3 mg of bovine peripheral nerve myelin and 25 µg Mycobacterium tuberculosis H37Ra (Difco, Detroit, Mich., USA). Preventive treatment was performed by oral administration of the indicated dose of leflunomide suspended in a solution of 1.5% carboxymethylcellulose, using a volume of 4 ml/kg body weight per day. Azathioprine (6-[1-methyl-4-nitroimidazol-5-yl]-thiopurine; Sigma) was dissolved in 0.9% NaCl solution and administered at a dose of 4 mg/kg body weight per day intraperitoneally. Administration was started on the day of immunization. Therapeutic treatment with leflunomide was initiated on the first day of clinically apparent EAN. Rats with similar clinical scores were assigned alternately to each of the two experimental groups. Animals in the therapy group (n = 8) received leflunomide at 20 mg/kg body weight per day. Nine sham-treated controls were fed corresponding volumes of the vehicle solution (1.5% carboxymethylcellulose).
Generation of neuritogenic T-cell blasts and treatment of adoptive transfer EAN (AT-EAN)
The P2-specific neuritogenic T-helper cell line P2.37 was established as described (Jung et al., 1996) from popliteal lymph nodes of a rat immunized in the hind footpads with P2 in complete Freund's adjuvant (CFA). For maintenance and adoptive intravenous transfer, the established cell line was activated as follows. Briefly, 4x105/ml line cells were incubated with P2 53–78 (2 µg/ml) and 2.5x106/ml irradiated (3000 rad) syngeneic spleen cells as antigen-presenting cells. Seventy-two hours later, activated T-cell blasts were separated from cell debris by centrifugation on Ficoll (Nycomed, Oslo, Norway) gradients at 4°C. Blasts from the interphase were washed twice and 8.5x106 cells/rat were injected into the tail vein of 18 animals (Jung et al., 1996). Thereafter rats were divided into control and treatment groups. Twelve rats received 20 mg leflunomide per kg body weight per day per os (p.o.). These animals were further subdivided into two groups of six rats each. One subgroup was treated with 500 mg uridine intraperitoneally twice daily. The other subgroup was sham-treated with 1.5% carboxymethyl- cellulose and served as the control.
In one experiment, AT-EAN was generated using P2-specific T-cell blasts that had been freshly activated with P2 and antigen-presenting cells for 72 h in the absence or presence of 20 µM A77 1726 plus 200 µM uridine before injection in the tail vein of six rats per group.
Generation of T-cell hybridomas
T-cell hybridomas were generated by fusion of activated T-cell blasts from T-cell lines with a variant of the mouse thymoma BW 5147 (B 1100.129.237) (Torres-Nagel et al., 1993) and screened for TCR expression by immuno- fluorocytometry.
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 by Hartung and colleagues (Hartung et al., 1988): 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.
Monoclonal antibodies Histology
Rats were anaesthetized deeply with pentobarbital (Narcoren; Iffa Merieux, Laupheim, Germany) and 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 0.1 M phosphate buffer, pH 7.4. Spinal cords with adjacent roots and right sciatic nerves were dissected and fixed for an additional 14 h. Material for the semiquantitative assessment of histopathological changes was embedded in Araldite (Serva, Heidelberg, Germany) after post-fixation in 2% osmium tetroxide for at least 4 h. Semithin sections from the cauda equina and sciatic nerves were stained with toluidine blue. Areas surrounding intraneural vessels were examined by a blinded investigator. All perivascular areas present in cross-sections were evaluated, and the degree of pathological alteration was graded semiquantitatively on the following scale: 0 = normal perivascular area; 1 = mild cellular infiltrate adjacent to the vessel; 2 = cellular infiltration plus demyelination in immediate proximity to the vessel; 3 = cellular infiltration and demyelination throughout the section. The mean histological score for individual animals was calculated by dividing the summed scores by the number of perivascular areas examined (Hartung et al., 1988).
Material for immunohistological identification of T lymphocytes and macrophages was embedded in paraffin. Deparaffinized 5 µm sections were treated with 0.03% H2O2 to quench endogenous peroxidase and were preincubated with porcine serum to cover non-specific binding sites. Sections were stained indirectly with the avidin–biotin–peroxidase technique (Hsu et al., 1981). Slides were incubated overnight with the monoclonal antibodies B115–1 (1 : 100) (Sanbio, Beutelsbach, Germany) and ED1 (1 : 800) (Serotec, Eching, Germany) followed by incubation with affinity-purified biotinylated anti-mouse IgG (Vector Laboratories via Alexis, Grünberg, Germany) that had been preabsorbed with normal rat serum. The avidin–biotin–peroxidase complex reagent and diaminobenzidine were used as recommended by the manufacturer.
Proliferation assays
In parallel with T-cell line bulk activation for AT-EAN, proliferation of the P2-specific T cells was assayed in flat-bottomed 96-well microtitre plates (Nunc, Wiesbaden, Germany). T-cell line expansion was measured by seeding 2x104 P2-specific T cells and 1.25x105 irradiated syngeneic splenocytes per 100 µl of medium per well. BPM was used at a concentration of 50–100 µg/ml, P2 protein at 20 µg/ml, P2 53–78 at 2 µg/ml and Concanavalin A at 2.5 µg/ml in the absence or presence of the indicated concentrations of A77 1726, with or without 200 µM uridine.
Cells were labelled with 0.25 µCi [3H]methyl thymidine (Amersham, Braunschweig, Germany) per well during the second day of culture and harvested 16 h later onto glass fibre filters with a Wallac 96-well harvester (Pharmacia). [3H]Thymidine incorporation was measured by liquid scintillation counting (Wallac 1205 Betaplate; Pharmacia). Data are given as mean counts per minute of triplicate cultures (± standard deviation).
Immunofluorocytometric analysis of Ca2+ flux into P2-specific hybridoma cells
Indo-1 (Molecular Probes, Eugene, Oreg., USA) premix was prepared by dissolving Indo-1 dye at 2 mM in dimethylsulphoxide, adding Pluronic F-127 (Molecular Probes) to a concentration of 10% (w/v) and heat-inactivated FCS to a final concentration of 70% (v/v). The Indo-1 mix was allowed to stand at room temperature for 10 min for proper dissolution. Fifteen microlitres of Indo-1 mix was applied to 1 ml of cell suspension (5x106 cells). P2-specific T-hybridoma cells (P2.6.1) were loaded with Indo-1 in RPMI 1% FCS at 37°C for 45 min. The following incubation with the TCR mAb R73 (5 µg/ml) was performed in RPMI 1% FCS at 4°C for 15 min. Cells were washed once, resuspended (107/ml) and kept strictly on ice. During loading and the subsequent incubation with R73, A77 1726 or genistein was present at the concentrations indicated. For induction of Ca2+ flux, cells were diluted to 5x105/ml in prewarmed (37°C) medium and stimulated with cross-linking goat anti-mouse IgG (H+L) antibody (Dianova, Hamburg, Germany) at 2 µg/ml. Analysis was carried out in a FacsVantage cytometer (Becton Dickinson, Heidelberg, Germany) at an excitation wavelength of 
= 351–364 nm.
ELISA (enzyme-linked immunosorbent assay) for determination of peripheral nerve myelin protein specific antibodies
Each well of Maxisorb immunoplates (Nunc) was coated with 50 µl of acidic extract of BPM (20 µg/ml of acidic extract in borate buffer, pH 8.4/0.1% sodium dodecyl sulphate) overnight at 4°C. After several rinses with PBS containing 0.05% Tween-20 (PBST), blocking was performed with casein buffer containing 2.5% casein in 0.3 N NaOH pH 8.4 (200 µl/well) for 2 h at room temperature. Plates were rinsed with PBST. Serum samples were centrifuged to remove fibrin and particulate debris before dilution in PBS/0.5% bovine serum albumin (BSA)/0.05% Tween-20. Each serum sample (50 µl/well) was applied serially diluted in ascending 1 : 2 dilutions. Serum samples from BPM/CFA-immunized animals and PBS/CFA-immunized controls were incubated for 1 h at 37°C. Plates were washed five times with PBST. Fifty microlitres of peroxidase-conjugated goat anti-rat-IgG antibody (Dianova) was added at a dilution of 1 : 2000 in PBS/0.5% BSA/0.05% Tween-20 and the preparation was incubated for 1 h at 37°C. After five washes with PBST, plates were developed by adding 100 µl/well of ABTS substrate solution [0.05% of 2,2'-azino-bis(3-ethylbenz-thiazoline-6-sulfonic acid) (Sigma) and 0.008% H2O2 (30%) in citrate buffer, pH 4.6]. Optical density (OD) was read with an automated plate-reader at 405 nm. Titres were calculated by end-point dilution analysis as follows. The cut-off was defined as the mean of ODs from sera, diluted 1 : 4, from PBS/CFA-immunized control rats + 2 SD. The sample titre was defined as the highest titre of a serum sample whose corresponding OD was still above the cut-off OD.
Statistical evaluation
Clinical scores were compared with the Wilcoxon rank sum test. Student's t-test was used to compare mean weights and histological findings. The t-test could be applied to histological scores because the large number of samples (perivascular areas) permitted the assumption of a normal distribution according to the central limit theorem (Hartung et al., 1988).
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Results |
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Prevention of EAN by leflunomide
The potency of leflunomide to suppress the generation of a pathogenic PNS-specific autoimmune response was tested in myelin-induced EAN in Lewis rats by administration of the compound starting on the day of immunization with BPM. The animals were distributed into one sham-treated control and three treatment groups receiving 1.5, 10 and 20 mg leflunomide/kg per day p.o., respectively. The control animals all developed severe disease with paraplegia or tetraparesis (score 7 or 8). Reluctant and incomplete clinical recovery was indicative of substantial inflammation and axonal damage in this group. Leflunomide delayed the onset and strikingly suppressed EAN in a dose-dependent manner (Fig. 1
). Even in the low-dose group (1.5 mg/kg per day), the animals that were affected showed only minor neuropathic signs not exceeding Score 4 (ataxia and mild paraparesis. Leflunomide given at 20 mg/kg per day completely protected the rats from clinically apparent EAN.
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After this dose-finding trial, the severity and course of actively induced EAN were monitored with leflunomide being administered preventively at a dose of 12.5 mg/kg per day starting on the day of immunization. Compared with the sham-treated control group, which rapidly developed paraplegia from day 10 post-immunization (p.i.) (Fig. 2
), the leflunomide-treated rats again did not show any clinical sign of EAN. On day 16 p.i., only one animal showed weakness of the tail tip, which progressed to atactic gait (EAN score 4) by day 21. A further group was treated with azathioprine at 4 mg/kg per day intraperitoneally, the first treatment being given on the day of immunization. In contrast to leflunomide, azathioprine did not mitigate disease progress, and the treated rats were paraplegic by day 15 p.i.
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When it became obvious, by day 20 p.i., that leflunomide completely halted EAN, the rats in the leflunomide treatment group were taken off this drug to address the question whether leflunomide-treated animals would develop a delayed form of EAN. After discontinuation of leflunomide, the rats worsened rapidly to become paraplegic within 5 days, but the duration of complete hind-limb paresis was significantly shorter than in sham-treated animals (Fig. 2
).
Therapy of overt EAN with leflunomide
To assess its therapeutic potency, leflunomide was administered orally at a dose of 20 mg/kg per day after the first clinical signs of EAN had become apparent, namely a hanging tail-tip or incomplete paresis of the tail (EAN score 1 or 1.5 on day 10 in Fig. 3). Therapy with leflunomide was able to suppress the progression of disease significantly within 48 h and allowed the animals to recover clinically within 10 days, whereas control animals still suffered from paraparesis or paraplegia after 10 days. When rats were in complete remission, they were taken off leflunomide (day 20 in Fig. 3). Without therapy, they experienced a relapse 6 days later and showed EAN with the same degree of severity as the control group but with faster and more complete recovery, giving rise to the assumption that axonal damage was decreased by the preceding treatment. Leflunomide therefore prevented EAN for as long as it was being given to the animals and tended to shorten the time course of the disease after the cessation of treatment, but obviously did not abate the propensity of the rats to become ill with EAN.
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Myelin antibody formation
T-cell-dependent production of autoantibodies against basic PNS proteins, as measured by ELISA on day 21 p.i., was high in sham- and azathioprine-treated animals. In contrast, autoantibody titres were reduced to the cut-off limit in rats treated preventively with leflunomide (Table 1
). Even in the case of one animal in the leflunomide treatment group that suffered from atactic gait (score 4), virtually no autoantibodies were detected, indicating that leflunomide might profoundly affect T-cell effector functions.
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Histopathology
Sections of the right sciatic nerve removed 16 days p.i. from three or four representative animals within each group in the last two experiments (Figs 2 and 3
) were examined histologically (Fig. 4). Compared with the controls (Fig. 4A), the preventive application of leflunomide markedly decreased the incidence of perivascular demyelination (grade 2), and both prophylaxis and therapy with leflunomide protected the nerves from more widespread demyelination and axonal degeneration (grade 3) (Fig. 4B). A higher percentage of perivascular fields examined in the leflunomide-treated rats showed no or only mild histopathological changes (grade 0 or 1) (Fig. 4C). Mean histological scores, which were calculated as outlined in Material and methods, were 0.5 ± 0.15 in the preventively treated group, 0.97 ± 0.09 in the therapeutically treated group and 2.1 ± 0.45 in the control animals (Fig. 4D). The difference compared with the controls was significant for both treatment groups (P < 0.01 and P < 0.02, respectively).
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Effects of leflunomide on adoptive transfer EAN
On one hand, the striking efficacy of leflunomide in preventing EAN by administration from the day of immunization argued in favour of the compound being able to suppress the generation of an immune response completely. On the other hand, the strong and rapid action of leflunomide on ongoing myelin-induced EAN was indicative of a direct effect on the effector functions of neuritogenic T cells. To dissect these two mechanisms and to determine the importance of pyrimidine depletion by leflunomide, administration of uridine during leflunomide treatment was investigated in the experimental setting of AT-EAN, which reflects the effector phase of disease. AT-EAN was produced by adoptive transfer of the neuritogenic T-helper (CD4+) cell line P2.37. The sham-treated control group became severely paraparetic by day 6 after cell transfer, whereas leflunomide alone (20 mg/kg per day p.o.) suppressed AT-EAN significantly (Fig. 5
) and protected the rats from losing weight (not shown). Intraperitoneal administration of uridine (500 mg twice daily) in addition to the leflunomide treatment reversed the suppressive effect of leflunomide only to a minor extent, implying a pronounced action of leflunomide on effector functions of neuritogenic T cells other than proliferation.
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Immunohistochemically, we observed massive infiltration of T cells and macrophages in sciatic nerves of sham-treated AT-EAN rats when they were killed for histology on day 8 after transfer of neuritogenic T-cell blasts. The number of T cells and activated macrophages was reduced impressively within the endoneurium when the animals had been treated with leflunomide (data not shown).
The in vivo findings in AT-EAN suggested anti- inflammatory effects of leflunomide in addition to its antiproliferative properties. Since full restoration of lymphocyte proliferation in vivo by injection of uridine could not be guaranteed, we performed an AT-EAN experiment in which we controlled for proliferation of the transferred neuritogenic T cells. In vitro and in the absence of exogenous uridine, we found that A77 1726 concentrations as low as 0.3 µM significantly inhibited the proliferation of the neuritogenic P2-specific (Fig. 6A) T-line cells P2.37. This inhibition could be reversed completely by addition of 200 µM uridine (Fig. 6B). The rescue effects of uridine on proliferation abated when A77 1726 concentrations >30 µM were applied (Fig. 6B). We induced AT-EAN by transfer of identical numbers of P2.37 T-line blasts that had been P2-activated in the absence or presence of 20 µM A77 1726 plus 200 µM uridine and that were not impaired in terms of proliferation in vitro (Fig. 6B). Nevertheless, the resulting AT-EAN was clearly milder in severity (maximal disease score 4) and delayed in onset of clinical signs by 1–2 days (Fig. 6C).
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Interestingly, the course of the AT-EAN produced by T cells that had been activated in vitro in the presence of leflunomide and uridine (Fig. 6C
) was the same as that of AT-EAN animals that were treated with leflunomide and uridine in vivo (Fig. 5). These two AT-EAN experiments can be compared since the severity and course of disease in control rats were similar in both of them.
Ca2+ flux studies
Since the AT-EAN experiments indicated that the mechanism of leflunomide was not merely antiproliferative, we tried to narrow down the stages of T-cell function affected by leflunomide. The T-cell hybridoma P2.6.1 generated from the neuritogenic P2-specific T-cell line P2.6 (Jung et al., 1991) expresses a P2-recognizing /ß TCR and was used as an in vitro system to test cell activation upon triggering the TCR by analysing the rise of intracellular Ca2+. After stimulation with the anti-TCR mAb R73, P2.6.1 cells showed a considerable Ca2+ influx, as measured by Indo-1 complexation. If incubated with A77 1726 in the range of 100–300 µM for 2 h before analysis, Ca2+ flux was markedly reduced. A77 1726 concentrations in the range of 30 µM, which inhibited cell proliferation in previous experiments, did not entail any detectable reduction of Ca2+ uptake (Fig. 7). At 300 µM, however, A77 1726 suppressed Ca2+ uptake to an extent comparable with that mediated by genistein at a concentration of 200 µM. Genistein is an inhibitor of src tyrosine kinases and protein kinase C (O'Dell et al., 1991). P2.6.1 T-cell hybridoma cells tolerated A77 1726 concentrations as high as 300 µM for 2 h without damage, as evidenced by the observation that the characteristics of the cells in the forward and sidewards scatters were not altered and that the cells still pumped Ca2+ out of the cytosol efficiently after stimulation (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterial and methodsResultsDiscussionAcknowledgementsReferences |
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In all our experiments on actively induced and adoptively transferred EAN, leflunomide markedly suppressed signs of disease and autoimmune inflammatory demyelination in the PNS. Even after the onset of clinical signs, leflunomide proved effective in preventing further progression of disease and shortened disease duration, an effect that was associated with a reduction in histopathological signs of nerve damage.
A dose- and time-dependent effect of leflunomide was noticed in the inhibition of actively induced EAN. A clear reduction in disease severity and delay of onset was observed at an oral dose of 1.5 mg/kg per day, the lowest dose used. When treatment was discontinued, the rats developed the first signs of EAN or relapsed 5–6 days later, irrespective of whether they had received leflunomide from the day of immunization or from the time when clinical signs of EAN had first appeared. Since none of the animals was treated for >20 days p.i., it cannot be completely excluded that the EAN bout that flared up between days 25 and 30 would also have appeared under treatment. However, there are two points that strongly argue against this notion. (i) Between days 16 and 21, some animals in the group treated preventively had already experienced a delayed and fully reversible episode of the usually monophasic Lewis-EAN when they were still under leflunomide treatment (Fig. 2). (ii) A clear temporal relationship between the discontinuation of leflunomide and the onset of clinical signs, with identical latency and dynamics of disease development, was also observable in the therapeutically treated animals in which the induction phase of EAN had taken place without the influence of leflunomide.
Compared with the time lag of 10–12 days until onset of EAN in naive myelin-immunized rats, the reduced latency until relapse or the first signs of disease after discontinuation of leflunomide suggests that neuritogenic cells also developed under preventive treatment with leflunomide but could not generate active disease as long as the drug was being administered. Together with the rapid clinical effect of interventionally applied leflunomide and our finding in AT-EAN, this points to suppression of the efferent limb of the immune response in EAN.
Autoantibody titres of the IgG isotype against basic PNS myelin proteins were virtually abrogated in the sera of leflunomide-treated rats. This indicates that T-cell-dependent production of autoantibodies was heavily impaired. Here, a direct suppression of B cell function seems to play a role since leflunomide has been shown to inhibit B-cell differentiation and subsequent isotype switching from IgM to IgG1 (Siemasko et al., 1998). This is of great relevance since pathogenic B-cell responses can intensify several human autoimmune-mediated diseases, including acute and chronic immunoneuropathies (Hartung et al., 1996; Toyka and Hartung, 1996). The pathogenesis of Lewis-EAN as a model of acute immune-mediated neuropathies is mainly T-cell-based. Thus, the reduction of autoantibodies does not contribute to the clinical efficacy of leflunomide in Lewis-EAN. Nevertheless, the marked reduction in autoantibodies against peripheral basic myelin proteins by leflunomide could be the rationale for the application of leflunomide in the therapy of chronic immune-mediated neuropathies in which antibodies against peripheral nerve tissue are pathogenetically more relevant. This might also be an advantage of leflunomide over azathioprine, which did not show a similar pronounced effect on autoantibody production.
The effect of most immunosuppressive agents relies on their ability to block more or less selectively the proliferation of immune cells. The first-generation immunosuppressant azathioprine, which has been used widely in immune- mediated neuromuscular diseases, acts by inhibition of purine nucleotide metabolism. Inhibition of T-cell proliferation by azathioprine can be measured immediately in vitro and is independent of TCR signalling (Dayton et al., 1992). Regarding the preponderance of the antiproliferative action of azathioprine against T cells, the events of the early effector phase of EAN, such as migration and invasion of pathogenic cells into the target organ, may not be halted. The lack of efficacy of azathioprine in preventing EAN in our experimental setting is in line with the clinical experience of a latency until onset of effects of ~3 months and highlights the need for faster-acting compounds.
Second-generation immunosuppressive agents, including cyclosporin A and rapamycin, interfere with signal transduction and cell cycle regulation in immune cells (for review, see Allison, 2000). Cyclosporin A has been used in autoimmune-mediated neuropathies (Waterston et al., 1992; Ballare et al., 1995; Ryan et al., 2000). However, severe nephrotoxic adverse effects limit the doses that can be used (de Mattos et al., 2000). Furthermore, cyclosporin A prevents the elimination of autoreactive T cells by apoptosis. Accordingly, chronification of EAN has been induced by the administration of suboptimal doses of cyclosporin A (McCombe et al., 1992; Wilson et al., 1994). This dilemma of adverse effects of both high-dose and low-dose cyclosporin A treatment is of great clinical relevance. In the development of new immunosuppressants, interest has focused on compounds that do not impair naturally occurring tolerization against self (Allison, 2000). In addition, compounds are being sought that will either act more specifically on immune cells, such as mycophenolate mofetil, which blocks purine nucleotide synthesis only in activated lymphocytes, or combine inhibition of proliferation with the suppression of effector functions of pathogenic cells, as has been claimed for leflunomide.
Several modes of action have been proposed for leflunomide (Xu et al., 1995; Silva and Morris, 1997; Rueckemann et al., 1998; Manna and Aggarwal, 1999). The most consistent reports refer to the depletion of pyrimidine nucleotides in activated lymphocytes by inhibition of DHODH. We have shown that leflunomide is still therapeutically active when uridine is supplied exogenously: (i) AT-EAN in leflunomide-treated rats remained clearly reduced despite high-dose administration of uridine; (ii) disease also took a mild course after adoptive transfer of neuritogenic P2-specific T cells that had been activated under A77 1726 in the presence of a uridine concentration sufficient to restore proliferation properties fully in vitro. The neuritogenicity of these A77 1726-treated T cells was therefore reduced by the agent independently of the available cellular pyrimidine nucleotide pool. Various cellular consequences of leflunomide-induced pyrimidine depletion, e.g. deficiencies in membrane biosynthesis, glycosylation and therefore altered cell adhesion and activation properties (Déage et al., 1998), may be sustained and even preclude the full restoration of cellular functions by exogenous uridine (Rueckemann et al., 1998). Functionally, this could account for (i) the increased propensity of neuritogenic cells to apoptotic elimination, (ii) the reduction in migration of pathogenic cells into the nerve and (iii) the modulation of cytokine secretion. Finally, when incubated for 2 h with high concentrations of A77 1726, P2-specific T-hybridoma cells exhibited altered activatability in vitro, as evidenced by reduced Ca2+ influx into the cytosol on TCR stimulation, substantiating the hypothesis that a mechanism other than DHODH inhibition might contribute to the immunosuppression by leflunomide. After triggering of the TCR, Ca2+ mobilization requires the activation of src-family tyrosine kinases as proximal signalling events (Chan et al., 1995; Finco et al., 1998). Since the src tyrosine kinase p56lck has been reported to be directly inhibited by A77 1726 (Xu et al., 1996), the decreased Ca2+ mobilization after TCR triggering through mAb R73 binding in the presence of A77 1726 seems a plausible finding. Although it has been shown that shortage of pyrimidine nucleotides as a result of DHODH inhibition also depletes the purine nucleotide pool, a relevant decline in intracellular ATP concentration would only occur after 24 h (Rueckemann et al., 1998). The observed short-term effect in the TCR-triggered cellular Ca2+ response in our experiments is indicative of a more direct action of leflunomide, which might be mediated by tyrosine kinases.
In summary, leflunomide effectively suppressed EAN and proved to be an immunomodulatory agent at several stages of the immune reaction to peripheral nerve tissue, showing strong and rapid action at the cellular and humoral levels. Like mycophenolate mofetil, which has recently been used with benefit in chronic inflammatory demyelinating polyradiculoneuropathy (Chaudhry et al., 2001), leflunomide can be considered a promising alternative for the therapy of human autoimmune-mediated neuropathies.
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We wish to thank Drs Jürgen Pünter and Matthias Herrmann from Aventis for critical discussion and the supply of leflunomide, Dr Lars Nitschke and Sonja Rotzoll from the Institute of Virology of the University of Würzburg for their expertise in fluorocytometric Ca2+ measurement and Gabriele Köllner for expert technical assistance.
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Received March 22, 2001. Revised May 16, 2001. Accepted May 21, 2001.
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