Th2 balance and inhibited migration of inflammatory cells into the peripheral nerve tissue

Neuropharmacology 42 (2002) 731–739 www.elsevier.com/locate/neuropharm

Suppression of experimental autoimmune neuritis by ABR-215062 is associated with altered Th1/Th2 balance and inhibited migration of inflammatory cells into the peripheral nerve tissue L.-P. Zou a, N. Abbas a, I. Volkmann a, I. Nennesmo b, M. Levi c, B. Wahren c, B. Winblad a, G. Hedlund d, J. Zhu a,∗ a

Division of Geriatric Medicine, Department of Clinical Neuroscience, Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden b Division of Pathology, Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden c Microbiology and Tumorbiology Center, Karolinska Institute, Stockholm, Sweden d Active Biotech Research AB, Lund, Sweden Received 9 March 2001; accepted 21 December 2001

Abstract The therapeutic effects of ABR-215062, which is a new immunoregulator derived from Linomide, have been evaluated in experimental autoimmune neuritis (EAN), a CD4+ T cell-mediated animal model of Guillain-Barre´ syndrome in man. In previous studies, we reported that Linomide suppressed the clinical EAN and myelin antigen-reactive T and B cell responses. Here EAN induced in Lewis rats by inoculation with peripheral nerve myelin P0 protein peptide 180-199 and Freund’s complete adjuvant was strongly suppressed by ABR-215062 administered daily subcutaneously from the day of inoculation. ABR-215062 dose-dependently reduced the incidence of EAN, ameliorated clinical signs and inhibited P0 peptide 180-199-specific T cell responses as well as also the decreased inflammation and demyelination in the peripheral nerves. The suppression of clinical EAN was associated with inhibition of the inflammatory cytokines IFN-γ and TNF-α, as well as the enhancement of anti-inflammatory cytokine IL-4 in lymph node cells and periphery nerve tissues, respectively, in a dose-dependent manner. These effects indicate that ABR-215062 may mediate its effects by regulation of Th1/Th2 cytokine balance and suggest that ABR-215062 is potentially a new chemical entity for effective treatment of autoimmune diseases.  2002 Elsevier Science Ltd. All rights reserved. Keywords: Experimental autoimmnue neuritis; ABR-215062; Linomide; Guillain-Barre´ syndrome; Cytokines

1. Introduction Experimental autoimmune neuritis (EAN) is an inflammatory autoimmune demyelinating disease of the peripheral nervous system (PNS). EAN represents an animal model of the Guillain-Barre´ syndrome (GBS) which is a demyelinating disease in man. EAN can be induced in susceptible animals by immunization with heterogeneous peripheral nerve myelin or its components P2 or P0 proteins emulsified in Freund’s complete adjuvant (FCA) (Waksman and Adams, 1955; Kadlubowski and Hughes, 1979; Milner et al., 1987). Following

Corresponding author. Tel.: +46-8-58585494; fax: +46-858585470. E-mail address: [email protected] (J. Zhu). ∗

immunization, myelin-reactive T cells along with other inflammatory cells such as macrophages increase in the PNS. Linomide (roquinimex, LS-2616), which is a quinoline with pleiotropic immune modulating capacity, has shown therapeutic effects in a series of models for autoimmune disease (Tarkowski et al., 1986; Karussis et al., 1993; Karussis et al., 1994; Gross et al., 1994; Zhang et al., 1997; Diab et al., 1998; Pekarski et al., 1998; Zhu et al., 1998a) also including EAN (Bai et al., 1997a; Zhu et al., 1999). Linomide induces suppression of Th1 cytokines and enhancement of Th2 cytokine production, which may play an important role in the control of T cell-mediated autoimmunity (Zhu et al., 1999). Furthermore, Linomide which is orally active, has been evaluated in clinical trials for the indication of (MS) and has shown disease inhibitory effects (Andersen et al., 1996;

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Karussis et al., 1996; Noseworthy et al., 2000; Wolinsky et al., 2000; Tan et al., 2000). The clinical program was stopped though due to intolerable side effects (Noseworthy et al., 2000; Tan et al., 2000). A new chemical entity, ABR-215062, was discovered in a program aiming to define compounds effective in autoimmune diseases such as MS and devoid of side effects often recorded for immunomodulators (e.g. fever, muscle/joint pain, stiffness). ABR-215062 inhibits the development of disease in a mouse model for MS, experimental autoimmune encephalomyelitis, and it was furthermore shown that this new compound was significantly more potent, as compared to Linomide, in its disease inhibitory capacity. By direct comparison, based on dose and exposure, ABR-215062 was approximately 20 times more potent than Linomide (Brunmark et al., submitted for publication). ABR-215062 has also shown a highly favourable profile in a dog model used to elucidate pro-inflammatory signs (e.g. fever, muscle pain and joint pain) compatible to the side effects recorded in the clinical trials with Linomide (Noseworthy et al., 2000). We have in the present study examined the effects of ABR-215062 on the clinical course of EAN and the histopathological changes in the PNS.

2. Materials and methods 2.1. Antigens The neuritogenic P0 protein peptides, corresponding to the amino acid 180-199 of rat PNS myelin P0 protein (Adelmann and Linington, 1992) were synthesized by solid-phase stepwise elongation using a Tecansyro peptide synthesizer (Multisyntech, Bochum, Germany). Bovine peripheral myelin (BPM) was prepared according to the procedure of Norton and Poduslo (1973). 2.2. Induction of EAN and assessment of clinical signs A total of 50 male Lewis rats, 6–8 weeks old, purchased from Charles River Co. (Sulzfeld of Germany) and weighing 180–200 g were used in the present study. All animals were immunised by injection into both hind footpads of altogether 200 µl of inoculum containing 100 µg of P0 protein peptide 180–199 and 1.5 mg Mycobacterium (M) tuberculosis (strain H 37 RA; Difco, Detroit, MI) emulsified in 100 µl saline and 100 µl Freund’s incomplete adjuvant (FIA; Difco). The FIA+M. tuberculosis mixture was referred to as FCA. Rats were monitored blindly for clinical signs by two separated examiners. Body weights and clinical scores were assessed immediately before immunization (day 0) and thereafter every second day until day 40 p.i. Severity of paresis was graded as follows: 0=no illness; 1=flaccid tail; 2=moderate paraparesis; 3=severe paraparesis;

4=tetraparesis or death; intermediate scores of 0.5 increment were given to rats with intermediate signs. 2.3. In vivo treatment with ABR-215062 ABR-215062 (Fig. 1; Active Biotech Research AB, Lund, Sweden) was dissolved in PBS and administered by a daily subcutaneous injection from the day of immunization to day 35 p.i. Groups of 10 rats received ABR-215062 at 0.16, 1.6 or 16 mg/kg/day, respectively, while 10 rats received Linomide at 16 mg/kg/day. Ten rats which received PBS daily via subcutaneous injection served as a sham-treated control group. 2.4. Histopathological assessment Segments of sciatic nerves close to the lumbar spinal cord from sacrificed animals were dissected, fixed in 4% formaldehyde and embedded in paraffin. Multiple longitudinal sections (5–6 µm slices) of sciatic nerves were stained with haematoxylin-eosin and replicate section with luxol fast blue violet for evaluation of the extent of mononuclear cells (MNC) infiltration and of demyelination. Tissue areas were measured by image analysis and the numbers of inflammatory cells were counted at x20 magnification. The average results were expressed as cells per mm2 tissue section. To assess the severity and extent of demyelination, peripheral nerve sections were scored using a semiquantitative grading system: 0=normal; 1=demyelinated fibres less than 25%; 2=demyelinated fibres 25–50%; 3=demyelinated fibres 50–75%; 4=demyelinated fibres more than 75%. 2.5. Immunohistochemistry Segments of sciatic nerve were dissected and snapfrozen in liquid nitrogen. Cryostat sections (10 µm) after fixation in acetone at ⫺20°C, were exposed to the mouse monoclonal antibodies (mAb) ED1 (anti-rat, macrophages), W3/25 (anti-rat CD4, T helper cells) and Ox8 (anti-rat CD8, T cytotoxic/supressor cells) (Serotec, Oxford, UK), as well as anti-rat IFN-γ (DB1) and anti-rat

Fig. 1.

The chemical structure of ABR-215062.

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IL-4 antibody (the Central Laboratory Animal Institute (CLAI), Utrecht, The Netherlands), and anti-rat TNF-α (gift from Dr Bakhiet M, Division of Infectious Disease, Huddinge Hospital, Stockholm, Sweden) respectively. Sections were stained according to the avidin-biotin technique (Vectastain Elite Kit; Vector). Omission of the primary antibodies served as negative controls. Specificity of the staining was also controlled on sections of peripheral lymphoid organs. The tissue areas were measured by image analysis. The numbers of positive cells as well as infiltrates were counted at ×20 magnification in the entire section area. 2.6. Isolation of mononuclear cells from lymph nodes The popliteal and inguinal lymph nodes were removed under aseptic conditions. Single cell suspensions of MNC from individual rats were prepared separately. The cells were washed three times in culture medium before being suspended to 2×106 MNC/ml. The culture medium consisted of Iscove’s modification of Dulbecco’s medium (Flow Lab, Irvine, UK) supplemented with 1% (v/v) MEM (Flow), 50 IU/penicillin, 60 µg/ml streptomycin (Gibco, Paisley, UK), 2 mM glutamine (Flow) and 3% normal human AB+ serum. 2.7. Lymphocyte proliferation assay About 200 µl aliquots of MNC suspensions were cultured in triplicates in round-bottomed 96-well polystyrene microtitre plates (Nunc, Copenhagen, Denmark) at a cell density of 2×106 MNC/ml. For specific lymphocyte stimulation, 10 µl aliquots of P0 peptides 180-199 were added to cultures at a final concentration of 10 µg/ml. This concentration had high stimulatory effects as assessed in preliminary experiments. Triplicate wells without antigen served as background controls. After 60 h of incubation, cells were pulsed with [3H]-methylthymidine (1 µCi/well; Amersham, Little Chalfont, UK) and cultured for an additional 12 h. Cells were harvested onto glass fibre filters (Titertek, Skatron, Lierbyen, Norway). [3H]-methylthymidine incorporation was measured in a liquid β-scintillation counter and numbers expressed per 105 MNC. 2.8. Measurement of levels of IFN-g and TNF-a in supernatants IFN-γ and TNF-α productions were measured from 1 ml of cultures containing 2×106 MNC, which were stimulated with P0 peptide 180-199 at a final concentration of 10 µg/ml. Culture supernatants were collected after 24 h and stored at ⫺20°C until assayed. The levels of IFN-γ and TNF-α were determined using ELISA. Capture mAb and detecting polyclonal antibody reactive with rat IFN-γ and TNF-α were produced, and the speci-

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ficity of antibodies were examined and did not show cross-reactivates with various other cytokines (Bakhiet et al., 1997). Ninety-six-well flat bottom polystyrene high binding plates (Costar, Cambridge, USA) were coated with 100 µl of anti-IFN-γ or anti-TNF-α mAbs diluted to 1 µg/ml in carbonate bicarbonate buffer (pH 9.6) and kept at 4°C overnight. After four washes with 0.05 PBS-Tween 20, the wells were blocked with 100 µl per well of 5% bovine serum albumin (BSA) for 90 min in room temperature (RT). After repeated washings with PBS-Tween 20, supernatants diluted 1:4 for IFN-γ and 1:2 for TNF-α in PBS were added in duplicate to each well. After overnight incubation at 4°C, plates were washed repeatedly in PBS-Tween 20. To detect any captured IFN-γ and TNF-α, the detecting anti-IFN-γ or antiTNF-α polyclonal antibodies were added at concentrations of 10 µg/ml for 1 h at 37°C. After washes, 100 µl of biotinylated rabbit anti-mouse IgG (Vector Lab) diluted 1: 2000 in PBS was added. After another 2 h of incubation in RT and 4 washes, 100 µl of avidin-biotin alkaline phosphates complex (ABC-AP; Vector Lab., Burlingame, USA) diluted 1: 200 in PBS was added for 45 min. Unbound ABC-AP was removed by five washes with PBS-Tween 20, and 100 µl/well of freshly prepared enzyme substrate solution was added. Absorbance was measured after 10 min incubation in the dark in the 405 Multiscan photometer (spectra Max 250; Molecular Devices, California). In order to quantify supernatant cytokines, standard curves were obtained simultaneously by incubating different known concentrations of IFN-γ and TNF-α (CLAI, Utrecht, The Netherlands) for 60 min at RT in wells pre-coated with anti-cytokine mAb. Development of the plate was performed as described above and the absorbance measured from the standard concentration of cytokines was used to plot cytokines standard curves using computer software. The absorbance obtained for the specimens were automatically converted to pg/ml by the computer from the standard curve. 2.9. Statistical methods Differences between pairs of groups were tested by Student’s t-test. Differences between three or more groups were tested by one-factor analysis of variance (ANOVA). The rejection limit was set to a ⫽ 0.05. 3. Results 3.1. Effects of ABR-215062 on EAN After immunization with P0 protein peptide 180-199, the clinical EAN started early. ABR-215062 delayed the onset of clinical EAN by 2–8 days and dramatically reduced the severity of the disease when compared with

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sham-treated control rats (1.6 and 16 mg/kg vs. control p ⬍ 0.01) (Fig. 2A). The effects on EAN achieved with ABR-215062 were clearly dose-dependent. Rats that received the ABR-215062 were relatively resistant to the development of EAN with only 20% incidence of disease in the group receiving the dose of 16 mg/kg/day, 30% incidence in the 1.6 mg/kg/day group and 60 % incidence in the 0.16 mg/kg/day group. All rats in the control group developed disease. No statistically confirmed difference regarding the effects on disease symptoms in EAN was shown between ABR-215062 and Linomide. Furthermore, there were no significant differences in changes of body weight during the course of EAN either in the groups treated with Linomide or ABR-215062 as compared to the sham-treated controls or in the group treated with Linomide and the groups treated with ABR-215062 (Fig. 2B).

3.2. Histopathology Histopathologic data showing inflammation and demyelination within the PNS are presented in Fig. 3A and B. The results revealed that administration of ABR215062 or Linomide significantly reduced infiltration of macrophages and lymphocytes in the sciatic nerves compared to sham-treated control EAN rats when examined on day 16 p.i. Only low-grade inflammation was recorded within the PNS in the high dose of ABR215062 group (Fig. 3C). When compared with the low doses of ABR-215062 groups the grade of inflammation increased in a dose-depend manner. Similar results were seen when analysing demyelination. Administration of 16 mg/kg/day of ABR-215062 strongly reduced regional demyelination (Fig. 3D) in the sciatic nerves when compared with sham-treated controls rats (Fig. 3E and F). Additionally, markedly less infiltration of macrophages, CD4+ cells and CD8+ cells was found in sciatic nerve sections from the ABR-215062- or Linomidetreated rats compared with those from sham-treated control rats (Fig. 4). The rats treated with high dose of ABR215062 (16 mg/kg) had about one-fifth of the numbers of macrophages and one-sixth of the numbers of CD4+ cells identified in controls by immunohistochemistry assay on day 16 p.i. (Fig. 4). 3.3. Effects of ABR215062 on T cell responses P0 peptide 180-199-induced T cell proliferation was significantly suppressed in the rats receiving the ABR215062 when compared with sham-treated control EAN rats receiving PBS only (p ⬍ 0.01 for 1.6 and 16 mg/kg/day groups). In unstimulated MNC culture, there were also differences in lymphocyte proliferation between EAN rats receiving ABR-215062 and control EAN rats receiving PBS only (p ⬍ 0.01 for 1.6 and 16 mg/kg/day groups). There were no differences between the rats treated at the same dose ABR-210562 and Linomide (Fig. 5). 3.4. Cytokine expression in sciatic nerves

Fig. 2. Clinical scores (A) and body weight (B) of EAN rats (n ⫽ 50). EAN was induced in Lewis rats by immunization on day 0 with P0 peptide 180-199 plus FCA. Rats received ABR-215062 at doses of 0.16 (n ⫽ 10), 1.6 (n ⫽ 10), and 16 mg/kg/day (n ⫽ 10) and Linomide 16 mg/kg/day (n ⫽ 10) from day 0 to 35 post immunization by the subcutaneous route. Control rats (n ⫽ 10) received PBS only. Mean values are depicted.

The numbers of IFN-γ and TNF-α expressing cells were strongly decreased in the sciatic nerves of ABR215062 treated rats at 1.6 and 16 mg/kg/day doses compared to sham-treated control EAN rats (Fig. 6). The numbers of IL-4 expressing cells were increased in the sciatic nerves in 16 mg/kg/day of ABR-215062-treated rats when compared with sham-treated control EAN rats. With the size and design of the present study, no statistically confirmed difference regarding the number of cells expressing IFN-γ, TNF-α or IL-4 in the PNS was shown when comparing ABR-215062 to Linomide treated animals (Fig. 6). The data on the percentages of the numbers of cytokine producing cell from different groups in

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Fig. 4. Mean numbers of macrophages, CD4+ cells and CD8+ cells per mm2 sciatic nerve sections as measured by immunohistochemistry on day 16 p.i. from EAN rats receiving ABR-215062 at different doses (0.16, 1.6 and 16 mg/kg/day), Linomide at 16 mg/kg/day or PBS (n ⫽ 5, respectively). Mean values and SEM are depicted. p values refer to comparisons between EAN rats receiving ABR-215062 or Linomide and sham-treated control EAN rats receiving PBS only. ∗∗ p ⬍ 0.01; ∗∗∗p ⬍ 0.001.

Fig. 5. Proliferation of lymph node mononuclear cells (MNC) on day 16 p.i. from rats receiving ABR-215062 at different doses (0.16, 1.6 and 16 mg/kg/day), Linomide 16 mg/rat/day or PBS by the subcutaneous route (n ⫽ 5, respectively). MNC were cultured in the presence of P0 peptide 180-199 or without antigen. Mean values and SEM are indicated. p values refer to comparisons between EAN rats receiving different doses of ABR-215062 or 16 mg/kg/day of Linomide and sham-treated control EAN rats receiving PBS only. ∗∗p ⬍ 0.01.

Fig. 3. Inflammatory infiltrates composed of macrophages and lymphocytes and regional demyelination in sciatic nerve sections from EAN rats (Fig. 3A and B) (5 rats for each group). Only low-grade inflammation (Fig. 3C) and milder regional demyelination (Fig. 3D) within PNS were detected in rats treated with 16 mg/kg/day of ABR215062 as analysed on day 16 p.i. and as compared to sham-treated control (Fig. 3E and F, respectively). Mean values and SEM are depicted. p values refer to comparisons between EAN rats receiving different doses of ABR-215062 or 16 mg/kg/day of Linomide and the sham-treated control EAN rats receiving PBS only. ∗p ⬍ 0.05, ∗∗p ⬍ 0.01, ∗∗∗p ⬍ 0.001.

the total number of infiltrating cells are presented in the Fig. 7. The administration of high dose of ABR-215062 resulted in an enhancement of the relative ratios of IL4 producing cells, however, the same treatment did not reduce the ratios of IFN-γ and TNF-α producing cells in the total infiltrating cells of sciatic nerves. Important though the total counts of IFN-γ and TNF-α producing cells in sciatic nerves were significantly reduced (Fig. 7). The similar results were found with the Linomide treatment. 3.5. Levels of IFN-g and TNF-a in the supernatants Levels of IFN-γ and TNF-α in the MNC supernatants were significantly decreased in EAN rats treated with 1.6

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Fig. 6. Mean numbers of cytokine expressing cells in sciatic nerve sections as measured on day 16 p.i. from EAN rats receiving ABR215062 at different doses (0.16, 1.6 and 16 mg/kg/day), Linomide at 16 mg/kg/day or PBS (n ⫽ 5, respectively). Mean values and SEM are depicted. p values refer to comparisons between EAN rats receiving ABR-215062 or Linomide and sham-treated control EAN rats receiving PBS only ∗p ⬍ 0.05, ∗∗p ⬍ 0.01.

Fig. 7. The percentage of the numbers of cytokine producing cells from EAN rats receiving ABR-215062 at different doses (0.16, 1.6 and 16 mg/kg/day), Linomide at 16 mg/kg/day or PBS (n ⫽ 5, respectively) in the total number of infiltrating cells.

and 16 mg/kg/day of ABR-215062 when examined on day 16 p.i. as compared with sham-treated EAN-rats (p ⬍ 0.01 for both compositions). However there were no differences between the same dose of ABR-215062 and Linomide treated EAN rats (Figs. 8 and 9).

4. Discussion In the present study we show that ABR-215062 dosedependently suppresses EAN by delaying the interval between immunization and onset of clinical EAN and dramatically reducing the incidence and the severity of EAN symptoms. This suppression of clinical EAN is also associated with reduced inflammation and demyelination of the sciatic nerves, down-regulated P0 peptideinduced T cell responses and a changed Th1/Th2 balance toward Th2 cells. These data show that ABR-215062 treatment is beneficial in EAN.

Fig. 8. The levels of IFN-γ in supernatant obtained lymph node MNC on day 16 p.i. from rats receiving ABR-215062 at different doses (0.16, 1.6 and 16 mg/kg/day), Linomide 16 mg/rat/day or PBS by the subcutaneous route (n ⫽ 5, respectively). MNC were cultured in the presence of P0 peptide 180-199 for 24 h. Mean values and SEM are indicated. p values refer to comparisons between EAN rats receiving different doses of ABR-215062 or 16 mg/kg/day of Linomide and sham-treated control EAN rats receiving PBS only. ∗∗p ⬍ 0.01.

Fig. 9. The levels of TNF-α in supernatant obtained lymph node MNC on day 16 p.i. from rats receiving ABR-215062 at different doses (0.16, 1.6 and 16 mg/kg/day), Linomide 16 mg/kg/day or PBS by the subcutaneous route (n ⫽ 5, respectively). MNC were cultured in the presence of P0 peptide 180-199 for 24 h. Mean values and SEM are indicated. p values refer to comparisons between EAN rats receiving different doses of ABR-215062 or 16 mg/kg/day of Linomide and sham-treated control EAN rats receiving PBS only ∗∗p ⬍ 0.01.

The data of the present study revealed that ABR215062 strongly inhibited the homing and migration of inflammatory cells, especially inhibited T cells, into the sciatic nerves. Only a mild inflammation and demyelination in the sciatic nerves were detected in the EAN rats treated with ABR-215062. However, sham-treated EAN rats receiving PBS developed typical and severe histological changes. The findings suggest that after administration of ABR-215062, either the autoreactive T cells have reduced their capacity to enter the PNS or the T cells have lost their capacity to recruit additional inflammatory cells to migrate into the PNS. Infiltration of inflammatory cells into the PNS is a typical pathologic

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hallmark for EAN and GBS (Zhu et al., 1998b). The mechanism underlying the beneficial effect of ABR215062 in EAN may reside in an inhibitory effect on the migration of blood-derived MNC across the bloodnerve barrier or on the transduction of chemotactic signals for migration. The latter is a critical step in the migratory process of inflammatory cells across tight endothelial junctions (Stuve et al., 1996; Pan and Kastin, 2001). As central regulatory cells of the immune system, T cells mediate many of their functions via the secretion of cytokines. The effects of Th1 and Th2 subsets in EAN were defined by the secreted cytokine profile (Zhu et al., 1998b), and their relative role in GBS has also been examined (Dahle et al., 1997; Hadden et al., 2001; Jander and Stoll, 2001; Press et al., 2001). The inhibition of T cell proliferation following the administration of ABR-215062 has been associated with a down-regulation of Th1 cytokine IFN-γ and inflammatory cytokine TNF-α in supernatants of the lymph node cells, as well as a marked increase in the production of Th2 cytokine IL-4 in the sciatic nerves. The drug may act by affecting T cell differentiation and shifting the cytokine profile from a Th1-like to a Th2-like pattern, which was shown as the high ratios of IL-4 producing cells in the sciatic nerves. This phenomenon has previously also been shown for Linomide (Diab et al., 1998; Zhu et al., 1999). In additional studies we have shown that certain types of treatment resulting in inhibition of clinical manifestations of EAN was associated with down-regulated Th1 cytokine and up-regulated Th2 cytokine production (Zhu et al., 1998b,c; Zou et al., 1998, 2000). Kunzendorf et al. (1998) suggest that generating a balance between expression of Th1 and the Th2 type of cytokines may be an optimal goal for inhibiting severe autoimmune disease. However, the observations also indicate that the role of cytokines in immune regulation and autoimmune disease is more complex than a simple Th1-Th2 dichotomy would suggest (Zhu et al., 1998b). This is a complex process that can be influenced by the microenvironment. Therefore new treatments to counteract this complex cytokine imbalance need to be considered. The balance between Th1 and Th2 cytokines may determine the outcome of an autoimmune disease (Zhu et al., 1998b). According to their cytokine production CD4+ Th cells are divided in to the two populations Th1 and Th2 with contrasting and cross-regulating cytokine profiles. CD4+ Th1 cells are involved in the pathogenesis of EAN by releasing inflammatory cytokines, such as IFN-γ, which can activate macrophages that directly attack the myelin sheath through phagocytosis and release of injurious factors and other proinflammatory cytokines, such as TNF-α (Zhu et al., 1998b). There is convincing evidence that the level of IFN-γ producing cells in blood, lymph nodes and PNS tissue roughly parallels clinical EAN and IFN-γ receptor-deficient mice are

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resistant to induction of EAN (Zhu et al., 1994, 1996; Zhu et al., 2001), consistent with an inflammatory role of Th1-promoting cytokines in the pathogenesis of EAN (Zhu et al., 1998b). Shifting the Th1/Th2 balance toward Th2 cells by the in vivo administration of IL-4 (Deretzi et al., 1999) and IL-10 (Bai et al., 1997b) markedly suppressed EAN. Spontaneous recovery from EAN in rats correlates with an expansion of Th2-like cells and Th2 cytokines as well as TGF-β (Zhu et al., 1998b). Kiefer et al. (1996) also indicated that the spontaneous recovery observed in Lewis rat EAN might be mediated by the endogenous elaboration of TGF-β1 within the peripheral nerve, and that macrophages might control their own cytotoxicity by expressing TGF-β1. Our present results suggest that EAN suppression by ABR-215062 may be due to insufficiency of antigen specific T cell to differentiate into Th1 effector cells in the periphery altering the in vivo Th1/Th2 balance in favour of a Th2 selection. In conclusion, the development of EAN is successfully suppressed in a dose-dependent manner by treatment with the new immunoregulator ABR-215062. ABR-215062 counteracts EAN by suppressing the T cell proliferation, Th1 proinflammatory cytokines IFN-γ and TNF-α production in lymph node cells as well as increasing IL-4 production in PNS tissues. ABR-215062 inhibits migration of inflammatory cells into the PNS tissue and reduces demyelination of nerves. Furthermore, ABR-215062 has shown a highly favourable profile in a dog model used to elucidate drug-induced symptoms compatible to the side effects recorded in the clinical trials with Linomide. ABR-215062 is therefore a potential candidate for effective treatment of autoimmune diseases.

Acknowledgements This study was supported by grants from Active Biotech Research AB, Lund, Sweden, the Swedish Medical Research Council (project numbers: K199971X-013133-01A, K1999-99P-012720-02B, K200071X-13133-02B and K2000-99P-12720-03A) and funds ˚ ke Wibergs foundation and Kapten Artur from A Erikssons foundation.

References Adelmann, M., Linington, C., 1992. Molecular mimicry and the autoimmune response to the peripheral nerve myelin P0 glycoprotein. Neurochemical Research 17, 887–891. Andersen, O., Lycke, J., Tollesson, P.O., Svenningsson, A., Runmarker, B., Linde, A.S., Astrom, M., Gjorstrup, P., Ekholm, S., 1996. Linomide reduces the rate of active lesions in relapsingremitting multiple sclerosis. Neurology 47, 895–900. Bakhiet, M., Diab, A., Mustafa, M., Zhu, J., Lindqvist, L., Link, H., 1997. Potential role of autoantibodies in the regulation of cytokine

738

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responses during bacterial infections. Infection and Immunity 65, 3300–3303. Bai, X.F., Shi, F.D., Zhu, J., Xiao, B.G., Hedlund, G., Link, H., 1997a. Linomide-induced suppression of experimental autoimmune neuritis is associated with down-regulated macrophage functions. Journal of Neuroimmunology 76, 177–184. Bai, X.F., Zhu, J., Zhang, G.X., Kaponides, G., Ho¨ jeberg, B., van der Meide, P.H., Link, H., 1997b. IL-10 suppresses experimental autoimmune neuritis and down-regulates Th1-type immune responses. Clinical Immunology and Immunopathology 83, 117–126. ˚ stro¨ m, M., Hedlund, G. The Brunmark, C., Ohlsson, L., Brodin, T., A new orally active immunoregulator ABR-215062 effectively inhibits development and relapses of experimental autoimmune encephalomyelitis (submitted for publication). Dahle, C., Ekerfelt, C., Vrethem, M., Samuelsson, M., Ernerudh, J., 1997. T helper type 2 like cytokine responses to peptides from P0 and P2 myelin proteins during the recovery phase of Guillain-Barre syndrome. Journal of the Neurological Sciences 153, 54–60. Deretzi, G., Zou, L.P., Pelidou, H.S., Nennesmo, I., Levi, M., Wahren, B., Mix, E., Zhu, J., 1999. Nasal administration of recombinant rat IL-4 ameliorates ongoing experimental autoimmune neuritis and inhibits demyelination. Journal of Autoimmunity 12, 81–89. Diab, A., Levi, M., Wahren, B., Deng, G.M., Bjork, J., Hedlund, G., Zhu, J., 1998. Linomide suppresses acute experimental autoimmune encephalomyelitis in Lewis rats by counter-acting the imbalance of pro-inflammatory versus anti-inflammatory cytokines. Journal of Neuroimmunology 85, 146–154. Gross, D.J., Sidi, H., Weiss, L., Kalland, T., Rosenmann, E., Slavin, S., 1994. Prevention of diabetes in non-obese diabetic mice by Linomide, a novel immunomodulating drug. Diabetologia 37, 1195– 1201. Hadden, R.D., Karch, H., Hartung, H.P., Zielasek, J., Weissbrich, B., Schubert, J., Weishaupt, A., Cornblath, D.R., Swan, A.V., Hughes, R.A., Toyka, K.V., Plasma Exchange/Sandoglobulin Guillain-Barre Syndrome Trial Group., 2001. Preceding infections, immune factors, and outcome in Guillain-Barre syndrome. Neurology 56, 758–765. Jander, S., Stoll, G., 2001. Interleukin-18 is induced in acute inflammatory demyelinating polyneuropathy. Journal of Neuroimmunology 114, 253–258. Kadlubowski, M., Hughes, R.A., 1979. Identification of the neuritogen for experimental allergic neuritis. Nature 277, 140–141. Karussis, D.M., Lehmann, D., Slavin, S., Vourka-Karussis, U., Mizrachi-Koll, R., Ovadia, H., Kalland, T., Abramsky, O., 1993. Treatment of chronic-relapsing experimental auto-immune encephalomyelitis with the synthetic immunomodulator linomide (quinoline3-carboxamide). Proceedings of the National Academy of Sciences USA 90, 6400–6404. Karussis, D.M., Lehmann, D., Brenner, T., Wirguin, I., Mizrachi-Koll, R., Sicsic, K., Abramsky, O., 1994. Immuno-modulation of experimental autoimmune myasthenia gravis with linomide. Journal of Neuroimmunology 55, 187–193. Karussis, D.M., Meiner, Z., Lehmann, D., Gomori, J.M., Schwarz, A., Linde, A., Abramsky, O., 1996. Treatment of secondary progressive multiple sclerosis with the immunomodulator linomide: a double-blind, placebo-controlled pilot study with monthly magnetic resonance imaging evaluation. Neurology 47, 341–346. Kiefer, R., Funa, K., Schweitzer, T., Jung, S., Bourde, O., Toyka, K.V., Hartung, H.P., 1996. Transforming growth factor-beta 1 in experimental autoimmune neuritis. Cellular localization and time course. American Journal of Pathology 148, 211–223. Kunzendorf, U., Tran, T.H., Bulfone-Paus, S., 1998. The Th1-Th2 paradigm in 1998: law of nature or rule with exceptions. Nephrology, Dialysis, Transplantation 13, 2445–2448. Milner, P., Lovelidge, C.A., Taylor, W.A., Hughes, R.A., 1987. P0 myelin protein produces experimental allergic neuritis in Lewis rats. Journal of the Neurological Sciences 79, 275–285.

Norton, W.I., Poduslo, S.E., 1973. Myelination in rat nerve: Method of myelin isolation. Journal of Neurochemistry 21, 749–757. Noseworthy, J.H., Wolinsky, J.S., Lublin, F.D., Whitaker, J.N., Linde, A., Gjorstrup, P., Sullivan, H.C., 2000. Linomide in relapsing and secondary progressive MS: part I: trial design and clinical results, North American Linomide Investigators. Neurology 54, 1726– 1733. Pan, W., Kastin, A.J., 2001. Changing the chemokine gradient: CINC1 crosses the blood-brain barrier. Journal of Neuroimmunology 115, 64–70. Pekarski, O., Bjork, J., Hedlund, G., Andersson, G., 1998. The inhibitory effect in experimental autoimmune encephalomyelitis by the immunomodulatory drug Linomide (PNU-212616) is not mediated via release of endogenous glucocorticoids. Autoimmunity 28, 235–241. Press, R., Deretzi, G., Zou, L.P., Zhu, J., Fredman, P., Lycke, J., Link, H., 2001. IL-10 and IFN-gamma in Guillain-Barre syndrome. Network Members of the Swedish Epidemiological Study Group. Journal of Neuroimmunology 112, 129–138. Stuve, O., Dooley, N.P., Uhm, J.H., Antel, J.P., Francis, G.S., Williams, G., Yong, W.V., 1996. Interferon β-1b decreases the migration of T lymphocytes in vitro: effects on matrix metalloproteinase-9. Annals of Neurology 40, 853–863. Tan, I.L., Lycklama, A., Nijeholt, G.J., Polman, C.H., Ader, H.J., Barkhof, F., 2000. Linomide in the treatment of multiple sclerosis: MRI results from prematurely terminated phase-III trials. Multiple Sclerosis 6, 99–104. ˚ , Lindholm, L., Sta˚ lhandTarkowski, A., Gunnarsson, K., Nilsson, L.A ske, T., 1986. Successful treatment of autoimmunity in MRL/1 mice with LS-2616, a new immuno-modulator. Arthritis and Rheumatism 29, 1405–1409. Waksman, N.H., Adams, R.D., 1955. Allergic neuritis: an experimental disease of rabbits induced by the injection of peripheral nervous tissue and adjuvant. Journal of Experimental Medicine 102, 213– 225. Wolinsky, J.S., Narayana, P.A., Noseworthy, J.H., Lublin, F.D., Whitaker, J.N., Linde, A., Gjorstrup, P., Sullivan, H.C., 2000. Linomide in relapsing and secondary progressive MS: part II: MRI results. MRI Analysis Center of the University of Texas-Houston, Health Science Center, and the North American Linomide Investigators. Neurology 54, 1734–1741. Zhang, G.X., Yu, L.Y., Shi, F.D., Xiao, B.G., Bjork, J., Hedlund, G., Link, H., 1997. Linomide suppresses both Th1 and Th2 cytokine in experimental autoimmune myasthenia gravis. Journal of Neuroimmunology 73, 175–182. Zhu, J., Mix, E., Olsson, T., Link, H., 1994. Cellular mRNA expression of interferon-gamma, IL-4 and transforming growth factor-beta (TGF-beta) by rat mononuclear cells stimulated with peripheral nerve myelin antigens in experimental allergic neuritis. Clinical and Experimental Immunology 98, 306–312. Zhu, J., Mix, E., Issazadeh, S., Link, H., 1996. Dynamics of mRNA expression of interferon-γ, interleukin 4 and transforming growth factor ß in sciatic nerve and lymphoid organs in experimental allergic neuritis. European Journal of Neurology 3, 232–240. Zhu, J., Diab, A., Mustafa, M., Levi, M., Wahren, B., Bjork, J., Hedlund, G., 1998a. Linomide suppresses chronic-relapsing experimental autoimmune encephalomyelitis in DA rats. Journal of the Neurological Sciences 160, 113–120. Zhu, J., Mix, E., Link, H., 1998b. Cytokine production and the pathogenesis of experimental autoimmune neuritis and Guillain-Barre syndrome. Journal of Neuroimmunology 84, 40–52. Zhu, J., Deng, G.M., Levi, M., Wahren, B., Diab, A., van der Meide, P.H., Link, H., 1998c. Prevention of experimental autoimmune neuritis by nasal administration of P2 protein peptides 57-81. Journal of Neuropathology and Experimental Neurology 57, 291–301. Zhu, J., Bai, X.F., Hedlund, G., Bjork, J., Bakhiet, M., van der Meide, P.H., Link, H., 1999. Linomide suppresses experimental auto-

L.-P. Zou et al. / Neuropharmacology 42 (2002) 731–739

immune neuritis in Lewis rats by inhibiting myelin antigen-reactive T and B cell responses. Clinical and Experimental Immunology 115, 56–63. Zhu, Y., Ljunggren, H.G., Mix, E., Li, H.L., van der Meide, P., Elhassan, A.M., Winblad, B., Zhu, J., 2001. Suppression of autoimmune neuritis in IFN-gamma receptor-deficient mice. Experimental Neurology 169, 472–478. Zou, L.P., Zhu, J., Deng, G.M., Levi, M., Wahren, B., Diab, A., Hiller,

739

J., Link, H., 1998. Treatment with P2 protein peptide 57-81 by nasal route is effective in Lewis rats experimental autoimmune neuritis. Journal of Neuroimmunology 85, 137–145. Zou, L.P., Deretzi, G., Pelidou, S.H., Levi, M., Wahren, B., Quiding, C., Zhu, J., 2000. Rolipram suppresses experimental autoimmune neuritis and prevents relapses in Lewis rats. Neuropharmacology 39, 324–333.