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. 2000 Jun;120(3):526–531. doi: 10.1046/j.1365-2249.2000.01233.x

Suppression of ongoing experimental allergic encephalomyelitis (EAE) in Lewis rats: synergistic effects of myelin basic protein (MBP) peptide 68–86 and IL-4

L-Y Xu *, Y-M Huang *, J-S Yang *, P H Van Der Meide *, H Link *, B-G Xiao *
PMCID: PMC1905554  PMID: 10844533

Abstract

Mucosal myelin autoantigen administration effectively prevented EAE, but mostly failed to treat ongoing EAE. Patients with multiple sclerosis (MS), for which EAE is considered an animal model, did not benefit from oral treatment with bovine myelin. We anticipated that autoantigen, administered together with a cytokine that counteracts Th1 cell responses, might ameliorate Th1-driven autoimmune disease, and that nasal administration might considerably reduce the amounts of antigen + cytokine needed for treatment purposes. Lewis rats with EAE actively induced with myelin basic protein peptide (MBP 68–86) and Freund's complete adjuvant (FCA), received from day 7 post-immunization, i.e. after T cell priming had occurred, 120 μg MBP 68–86 + 100 ng IL-4 per rat per day for 5 consecutive days. These rats showed later onset, lower clinical scores, less body weight loss and shorter EAE duration compared with rats receiving MBP 68–86 or IL-4 only, or PBS. EAE amelioration was associated with decreased infiltration of ED1+ macrophages and CD4+ T cells within the central nervous system, and with decreased interferon-gamma (IFN-γ) and tumour necrosis factor-alpha (TNF-α) and enhanced IL-4, IL-10 and transforming growth factor-beta (TGF-β) responses by lymph node cells. Simultaneous administration of encephalitogenic peptide + IL-4 by the nasal route thus suppressed ongoing EAE and induced IL-4, IL-10 and TGF-β-related regulatory elements.

Keywords: experimental allergic encephalomyelitis, treatment, mucosal tolerance, IL-4, autoimmunity

Introduction

The nasal route of administration is effective for delivery of vaccines to induce mucosal immune responses [1]. Intranasally administered soluble antigens or peptides consisting of specific T cell epitopes can induce systemic tolerance and have been used as a strategy to develop antigen-specific therapy in several experimental autoimmune diseases including EAE, experimental autoimmune myasthenia gravis (EAMG), experimental autoimmune neuritis (EAN), experimental autoimmune uveoretinitis (EAU), collagen-induced arthritis (CIA) and murine insulin-dependent diabetes (for reviews, see [2,3]). Despite high efficacy in disease prevention, nasal antigen administration showed poor effects in suppressing ongoing disease [4–7].

In analogy with oral tolerance, the mechanisms of nasal tolerance are suggested to depend on the quality and quantity of the tolerogen used, and result in either anergy/deletion of antigen-specific T cells or induction of IL-4- and/or transforming growth factor-beta (TGF-β)-related regulatory mechanisms (for reviews, see [2,3]). Given the complexity of antigens involved in the pathogenesis of autoimmune diseases in humans, therapies based on anergy/deletion of antigen-specific T cells may not be feasible for clinical use. Instead, treatments with antigens or peptides that can induce regulatory elements which non-specifically suppress inflammation in target organs may be desirable.

Co-stimulation of CD4+ T cells with antigen and IL-4 plays a dominant role in the induction of IL-4-producing Th2 cells and TGF-β-producing Th3 cells in vitro and in vivo[8–12]. We therefore asked whether encephalitogenic peptide and IL-4 administered by the nasal route could be used to treat actively induced EAE. This approach might induce immune deviation by promoting the development of regulatory Th2/Th3 cells, thereby preferentially modulating autoantigen-specific T cell responses in vivo.

In this study, Lewis rats with EAE induced by immunizing with MBP 68–86 and Freund's complete adjuvant (FCA) were studied for clinical efficacy and immune alterations after MBP 68–86 + IL-4 administered nasally during days 7–11 post-immunization (p.i.). MBP 68–86 + IL-4-treated rats developed only mild EAE. Nasal administration of MBP 68–86 + IL-4 suppressed ongoing EAE, paralleled by suppressed Th1 cell responses and augmented Th2 and Th3 cell responses.

Materials and methods

Reagents

Guinea pig MBP peptide covering the amino acid residues 68–86 (MBP 68–86) (YGSLPQKSQRSQDENPV) was synthesized in an automatic Tecan/Syro Synthesizer (Multisyntech, Bochum, Germany). Murine recombinant IL-4 (specific activity 5·98 × 107−1·98 × 108 U/mg; 61% homology with rat IL-4) was from Schering-Plough Research Institute (Kenilworth, NJ), and has been shown to act in rats [13,14]. Mouse anti-rat interferon-gamma (IFN-γ) MoAb (DB1) was purchased from Innogenetics (Ghent, Belgium). Anti-rat CD4 MoAbs were purified from culture supernatant of hybridoma (clone W3/25) [15]. Anti-rat macrophage MoAbs (ED1) were from SeroTec (Oxford, UK).

Animals

Lewis rats, 6–8 weeks old, were purchased from Zentralinstitut fur Versuchstierzucht (Hannover, Germany).

Induction of EAE

Each rat was immunized subcutaneously in the hind footpads with 200 μl inoculum containing 25 μg MBP 68–86, 2 mg Mycobacterium tuberculosis (strain H37RA; Difco, Detroit, MI), 100 μl saline and 100 μl Freund's incomplete adjuvant (FIA; Difco). Rats were weighed and evaluated daily in a blinded fashion by at least two investigators for the presence of clinical signs. Clinical scores of EAE were graded according to the following criteria: 0, asymptomatic; 1, flaccid tail; 2, loss of righting reflex with or without partial hind limb paralysis; 3, complete hind limb paralysis; 4, moribund; 5, dead.

Induction of nasal tolerance

Seven days p.i., four groups of rats received into nostrils 60 μl PBS pH 7·4 or PBS containing MBP 68–86 (120 μg/rat per day), or IL-4 (100 ng/rat per day) or MBP 68–86 (120 μg/rat per day) + IL-4 (100 ng/rat per day), respectively. The solution was administered daily for 5 consecutive days by micropipettes and the rats were gently anaesthetized with ether.

Immunohistochemistry

On day 14 p.i., the spinal cords from sacrificed animals were dissected, and segments of lumbar spinal cord were snap-frozen in liquid nitrogen. Cryostat sections were cut at 10 μm and fixed in acetone for 10 min. Endogenous peroxidase activity was inactivated with 0·3% H2O2 for 20 min. Non-specific binding sites were further blocked with 1% blocking reagent (Boehringer Mannheim, Mannheim, Germany). The sections were incubated overnight in primary anti-CD4 and ED1 antibodies at a dilution of 1:100. Reactivity was detected with ABC reactive system (Vector, Burlingame, CA). Specificity of the staining was tested by incubating sections without the primary antibodies. For each animal, three spinal cord sections were observed in a blinded fashion. Positive cells were counted by automatic video scanning using Leica Q500MC.

Preparation of mononuclear cells from draining lymph nodes

On day 14 p.i., popliteal and inguinal lymph nodes (PILN) were removed under aseptic conditions. Mononuclear cell (MNC) suspensions were obtained by grinding the organs through a nylon mesh in medium. Cells were then washed three times and resuspended in medium consisting of Dulbecco's modification of Eagle's medium (Gibco, Paisley, UK), supplemented with 1% minimum essential medium (Gibco), 2 mm glutamine (Flow Labs, Irvine, UK), 50 U penicillin and 50 μg/ml streptomycin (Gibco), and 10% (v/v) heat-inactivated fetal calf serum (FCS; Gibco). Cells were then adjusted to 2 × 106/ml.

Enumeration of MBP 68–86-reactive IFN-γ-secreting cells by ELISPOT

An enzyme-linked immunospot (ELISPOT) assay was used for detection of IFN-γ secretion at the single-cell level [16]. Nitrocellulose-bottomed microtitre plates (Millititre-HAM plates; Millipore Co., Bedford, UK) were coated with 100 μl aliquots of anti-rat IFN-γ MoAb (DB1) at 15 μg/ml. MNC suspensions (4 × 105 cells/200 μl) were added to individual wells, and incubated with or without MBP 68–86 (10 μg/ml). After 48 h of culture, the wells were extensively washed. The plates were incubated with 100 μl of polyclonal rabbit anti-rat IFN-γ antibody (Innogenetics) diluted 1:500 for 4 h at room temperature. After washing, the plates were incubated with biotinylated swine anti-rabbit IgG (1:500; Dakopatts, Copenhagen, Denmark) and then avidin-biotin peroxidase complex (1:200; Vector) followed by peroxidase staining. The red-brown immunospots which corresponded to the cells that had secreted IFN-γ were counted in a dissection microscope. Results are expressed as numbers of spots per 105 MNC after subtraction of values from ‘0 antigen’ background control cultures.

Lymphocyte proliferation assays

Proliferative responses of MNC were examined by 3H-thymidine incorporation. Briefly, 200 μl of MNC suspensions (2 × 106/ml) were incubated in 96-well polystyrene microtitre plates (Nunc, Roskilde, Denmark) at 37°C in 5% CO2 with or without MBP 68–86 (10 μg/ml). After 60 h, cells were pulsed with 3H-thymidine (1 μCi/well; Amersham, Little Chalfont, UK) for 12 h. Cells were harvested and 3H-thymidine incorporation was measured in a liquid β-scintillation counter.

Preparation of lymph node cells for in situ hybridization

Aliquots (200 µ l) of lymph node MNC suspensions from individual rats were plated in round-bottomed microtitre plates (Nunc) at a density of 2 × 106 MNC/ml medium and stimulated with or without MBP 68–86 (10 μg/ml). A culture time of 24 h was selected for detection of cytokine mRNA. After washing in PBS, 105 MNC from each culture were dried onto restricted areas of ProbeOn slides (Fisher Scientific, Pittsburgh, PA) and stored in sealed boxes at −70°C.

In situ hybridization

In situ hybridization (ISH) was performed using 35S-labelled synthetic oligonucleotide probes (Scandinavian Gene Synthesis AB, Koping, Sweden). For each cytokine [17], a mixture of four different approximately 48 bp long oligonucleotide probes was used in order to increase the sensitivity of the method. The oligonucleotide sequences were obtained from GeneBank using the MacVector system. Control slides were hybridized with the same total amount of a sense probe with nucleotide sequence for exon 4 of rat IFN-γ. A constant ratio of the guanine/cytosine content of approx. 60% was employed. The oligonucleotide probes were checked for absence of palindromes and long sequences of homology within the species against available GenBank data. The labelling was performed with 35S-deoxyadenosine 5′-thiotri-phosphate (New England Nuclear, Cambridge, MA) with terminal deoxynucleotidyl transferase (Amersham). Cells were hybridized with 106 ct/min of labelled probe per 100 μl of hybridization mixture. After emulsion autoradiography, development and fixation, the coded slides were examined by dark field microscopy for positive cells containing > 15 grains per cell in a star-like distribution [17,18]. The intracellular distribution of the grains was always checked by light microscopy. In many positive cells, the grains were so heavily accumulated within and around the cells that it was not possible to count every single grain. In cells judged negative, the number of grains was mostly 0–2 per cell, and the grains were scattered randomly over the cells and not distributed in a star-like fashion. There was therefore no difficulty in differentiating between positive and negative cells. The control probe used in parallel with the cytokine probes on sections produced a uniformly weak background signal without revealing any positive cells. The number of cells used in ISH was not equal to the number that was ultimately detected on the slide. To compensate for cell losses, the total number of cells on the slides was regularly counted. With the help of a microscope grid used as a measuring unit, the radius (r) of the surface area (A) covered by cells was determined. The area A was calculated by the formula A = π × r2. Cells were usually counted in two grids at the periphery and one grid at the centre of the surface covered by cells. In cases of uneven distribution, cells in additional grids were counted. The mean value of the number of the cells per grid was determined and multiplied by A. Results are expressed as numbers of positive cells per 105 MNC after subtraction of values from ‘0 antigen’ background control cultures.

Statistical analysis

Differences between four groups were tested by one-factor analysis of variance (anova). The level of significance was set to α = 0·05. All tests were two-sided.

Results

Nasal administration of MBP 68–86 plus IL-4 suppresses ongoing EAE in Lewis rats

Nasal administration of MBP 68–86 (600 μg/rat) or of IL-4 (500 ng/rat) given alone before immunization has previously been shown to prevent the development of EAE in Lewis rats [6,19]. If the same dose of the MBP 68–86 or IL-4 was administered alone by the nasal route to Lewis rats daily on day 7–11 p.i., i.e. when encephalitogenic T cells were already primed and the animals had entered the preclinical phase of EAE, no clinical amelioration was observed compared with control rats receiving only PBS (Fig. 1a,b). However, when rats received MBP 68–86 (120 μg/rat per day) + IL-4 (100 ng/rat per day) on days 7–11 p.i., they developed EAE with later onset, lower mean clinical score (P < 0·05), less body weight loss, and shorter duration. Thus, co-administration of MBP 68–86 + IL-4 suppressed ongoing EAE. If the dose of IL-4 was instead reduced to 50 ng/day per rat, no clinical suppression was observed (data not shown).

Fig. 1.

Fig. 1

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Nasal administration of MBP 68–86 plus IL-4 suppresses ongoing EAE in Lewis rats. Between days 7 and 11 post-immunization (p.i.), four groups of rats (n = 4/group) received PBS (▪), MBP 68–86 (120 μg/rat per day; ▾), IL-4 (100 ng/rat per day; ▴) or MBP 68–86 (120 μg/rat per day) + IL-4 (100 ng/rat per day; •) by the nasal route. Results are representative of two independent experiments.

MBP 68–86 + IL-4 reduced inflammatory infiltration within the CNS

EAE is characterized by large numbers of infiltrating cells within the central nervous system (CNS). These cells are thought to contribute to disease pathogenesis [20]. In the present study, levels of infiltrating ED1+ macrophages and CD4+ T cells examined on day 14 p.i. were clearly reduced in sections of lumbar spinal cords from the MBP 68–86 + IL-4-treated rats compared with PBS-, or MBP 68–86- or IL-4-treated control EAE rats (Fig. 2). The reduced levels of immune cells in spinal cord sections from MBP 68–86 + IL-4-treated rats are thus consistent with the reduced clinical signs in the animals.

Fig. 2.

Fig. 2

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Inflammatory infiltration within the central nervous system (CNS). On day 14 post-immunization, lumbar spinal cords from different groups were dissected, and infiltrating ED1+ macrophages and CD4+ T cells were detected by immunohistochemical staining. *P < 0·05; **P < 0·01.

Treatment with MBP 68–86 + IL-4 reduced antigen-specific T cell responses

To gain further insight into the protection conferred by the nasal administration of MBP 68–86 + IL-4, MBP 68–86-induced IFN-γ secretion by MNC obtained on day 14 p.i. was evaluated employing ELISPOT analysis. PBS-, MBP 68–86- and IL-4-treated control EAE rats had high levels of MBP 68–86-reactive IFN-γ-secreting cells among LN MNC (Fig. 3a). By contrast, such cells were strikingly lower in MBP 68–86 + IL-4-treated rats, reflecting suppression of IFN-γ-secreting Th1 cell responses.

Fig. 3.

Fig. 3

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Antigen-specific T cell responses by lymph node (LN) mononuclear cells (MNC). On day 14 post-immunization, LN MNC (2 × 106/ml) were prepared from different groups and their antigen-specific responses were evaluated by measuring (a) IFN-γ-secreting MNC; (b) 3H-thymidine incorporation upon stimulation with MBP 68–86 (10 μg/ml). *P < 0·05; **P < 0·01.

LN MNC obtained on day 14 p.i. from the different groups of rats were further incubated with MBP 68–86, followed by 3H incorporation measurement 72 h later. Compared with PBS-treated EAE rats, MBP 68–86 as well as IL-4 treatment suppressed antigen-specific LN MNC cell proliferation (Fig. 3b). Interestingly, LN MNC from MBP 68–86 + IL-4-treated rats showed high proliferative responses, which revealed levels similar to those from PBS-treated EAE rats. Therefore, co-inhalation of MBP 68–86 + IL-4 did not suppress, but rather promoted the expansion of certain subsets of MNC.

Antigen-driven cytokine mRNA expression in LN MNC

To define further the manner in which combined administration of MBP 68–86 + IL-4 suppressed ongoing EAE, LN MNC obtained on day 14 p.i. from the different groups of rats were first cultured in vitro and then harvested and evaluated by ISH for frequencies of cells expressing cytokine mRNA. As shown in Fig. 4, treatment with MBP 68–86 or IL-4 or MBP 68–86 + IL-4 reduced the levels of MBP 68–86-reactive tumour necrosis factor-alpha (TNF-α) mRNA-expressing LN MNC compared with vehicle treatment. However, only the parallel nasal administration of MBP 68–86 + IL-4 decreased the numbers of MBP 68–86-reactive IFN-γ mRNA-expressing cells and, at the same time, increased the numbers of MBP 68–86-reactive IL-4, IL-10 and TGF-β mRNA-expressing LN MNC. Thus, MBP 68–86 + IL-4 administered together induced a dramatic shift of a Th1 response to Th2 and Th3 cell expansion. Consistent with their increased proliferative capacity, T cells from rats receiving MBP 68–86 + IL-4 exhibited significantly higher levels of MBP 68–86-reactive IL-4, IL-10 and TGF-β mRNA-expressing cells.

Fig. 4.

Fig. 4

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Antigen-driven cytokine mRNA expression in lymph node (LN) mononuclear cells (MNC). On day 14 post-immunization, LN MNC (2 × 106/ml) were prepared from different groups. Cells were first cultured in vitro in the presence of MBP 68–86 (10 μg/ml), and were then evaluated by in situ hybridization for frequencies of cells expressing cytokine mRNA. *P < 0·05; **P < 0·01; ***P < 0·001.

Discussion

Antigen-specific therapy can induce regulatory elements, such as Th2 and/or Th3 cells. This kind of induction has been suggested to require two signals both in vitro and in vivo: antigenic signal as well as costimulatory signals, e.g. IL-4 [8]. Priming CD4+ T cells in vivo with staphylococcal enterotoxin A (SEA) or IL-4 alone did not result in Th2 cells capable of producing IL-4. However, CD4+ T cells derived from animals primed with both SEA and IL-4 produced large quantities of IL-4 when re-stimulated in vitro with SEA [21]. The natural development of Th2 cells was completely inhibited by anti-IL-4 antibody [22]. Priming conditions favouring high IL-4 production greatly enhanced TGF-β production from naive CD4+ T cells in secondary cultures [11]. Oral administration of IL-4 enhanced tolerance in EAE by inducing TGF-β-secreting Th3 cells [12]. These data indicate that IL-4 plays a dominant role in the differentiation of T cells towards Th2- and/or Th3-like phenotypes.

In this study, 7 days after EAE induction, nasal administration of MBP 68–86 or IL-4 at the doses used for effective EAE prevention showed neither ameliorating nor aggravating effects on the clinical course of EAE. In contrast, nasal administration of MBP 68–86 + IL-4 suppressed the severity of clinical EAE and reduced levels of infiltrating ED1+ macrophages and CD4+ T cells within the CNS. The suppression of clinical EAE in these rats was accompanied by strongly reduced levels of MBP 68–86-reactive IFN-γ-secreting Th1-like cells, which most probably reflects down-regulated Th1 immunity in these rats. On the other hand, the high proliferative responses and the augmented levels of MBP 68–86-reactive IL-4, IL-10 and TGF-β mRNA-expressing cells in MBP 68–86 + IL-4-treated rats indicate the induction and expansion of Th2 and Th3 cells. The combined administration of MBP 68–86 + IL-4 thus induced a shift of Th1 cell responses to Th2 and Th3 cell development, resulting in amelioration of myelin-destroying immune responses and of clinical EAE. When using ELISA to determine antigen-specific IgG isotype antibodies in sera obtained on day 14 p.i., no significant differences were found between the different groups (data not shown).

Previous reports have indicated that ongoing autoimmune diseases are difficult to suppress by nasal autoantigen delivery [4–7]. Reasons for this and the general complexity of the treatment of autoimmune diseases include: (i) antigen-specific T cells have been activated and the proportion of autoantigen-specific T cells within the T cell repertoire becomes highly elevated; (ii) activated antigen-specific T cells induce or secrete proinflammatory cytokines such as IFN-γ, which do not favour, but even inhibit the development of Th2 and Th3 cells [9,10]; (iii) during disease progression, intra- and/or intermolecular epitope spreading may occur [23]; (iv) the effector arms of the immune response, such as inflammatory macrophages, have come into play. Thus, increased amounts of tolerogens are required to suppress ongoing disease. However, concerning the same structure of tolerogens and disease-inducing antigens, the administration of large amounts of autoantigen may increase the risk of immunity induction. Administration of large amounts of proteins could also increase the risk of inflammatory reactions in the nasal mucosa and possibly in the lungs as well and, thereby, preclude their usage for human therapeutic trials. The success of IL-4 in synergistically enhancing low-dose mucosal tolerance shown by Inobe et al. [12] and our study indicates a new therapeutic strategy for autoimmune diseases.

Mucosal tolerance induction is affected by many factors, including the dosage of tolerogen, the genetic background of animals, the immunogens used for disease induction and the time window of tolerance induction. The efficacy of combined administration of antigen plus cytokine(s) in other autoimmune disease models needs further exploration. Since the suppression of clinical EAE that we obtained in this study was not complete, nasal tolerogen mixtures need further modifications by involving, for instance in EAE treatment, reagents promoting apoptosis of encephalitogenic T cells, non-specific effector immunocytes and/or neurotrophic factors promoting myelin regeneration.

Acknowledgments

This work was supported by grants from the Swedish MS Society (NHR), the Swedish Medical Research Council and Karolinska Institute Research Funds.

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