Nasal tolerance in experimental autoimmune myasthenia gravis (EAMG): induction of protective tolerance in primed animals

Nasal tolerance in experimental autoimmune myasthenia gravis (EAMG): inductionof protective tolerance in primed animals

F.-D. SHI, X.-F. BAI, H.-L. LI, Y.-M. HUANG, P. H. VAN DER MEIDE* & H. LINK Division of Neurology,Karolinska Institute, Huddinge University Hospital, Huddinge, Stockholm, Sweden, and*Biomedical Primate Research Centre,

Rijswijk, The Netherlands

(Accepted for publication 8 November 1997)

SUMMARY

Nasal administration ofmg doses of acetylcholine receptor (AChR) is effective in preventing thedevelopment of B cell-mediated EAMG in the Lewis rat, a model for human MG. In order to investigatewhether nasal administration of AChR modulates ongoing EAMG, Lewis rats were treated nasally withAChR 2 weeks after immunization with AChR and Freund’s complete adjuvant. Ten-fold higheramounts of AChR given nasally (600mg/rat) were required to ameliorate the manifestations of EAMGcompared with the amounts necessary for prevention of EAMG. In lymph node cells from rats receiving600mg/rat of AChR, AChR-induced proliferation and interferon-gamma (IFN-g) secretion werereduced compared with control EAMG rats receiving PBS only. The anti-AChR antibodies in ratstreated nasally with 600mg/rat of AChR had lower affinity, reduced proportion of IgG2b and reducedcapacity to induce AChR degradation. Numbers of AChR-reactive IFN-g and tumour necrosis factor-alpha (TNF-a) mRNA-expressing lymph node cells from rats treated nasally with 600mg/rat of AChRwere suppressed, while IL-4, IL-10 and transforming growth factor-beta (TGF-b) mRNA-expressingcells were not affected. Collectively, these data indicate that nasal administration of AChR in ongoingEAMG induced selective suppression of Th1 functions, i.e. IFN-g and IgG2b production, but noinfluence on Th2 cell functions. The impaired Th1 functions may result in the production of lessmyasthenic anti-AChR antibodies and contribute to the amelioration of EAMG severity in rats treatedwith AChR 600mg/rat by the nasal route.

Keywords mucosal tolerance experimental autoimmune myasthenia gravis Th1 cells Th2cells cytokine

INTRODUCTION

Mucosal antigen exposure without adjuvant results in immuno-logical unresponsiveness upon subsequent parenteral challenge,i.e. mucosal tolerance. Oral tolerance induction involves multiplemechanisms, including the induction of apoptosis and anergy ofantigen-specific T cells following administration of high antigendoses, while induction of regulatory Th2 and Th3 cells followslow-dose antigen feeding (reviewed in [1]). Exploitation of the oralroute for prevention and treatment of autoimmune disease has dealtwith experimental models of cell-mediated conditions, such asexperimental autoimmune encephalomyelitis (EAE), uveitis(EAU), or arthritis. Myasthenia gravis (MG) and its animalmodel EAMG are caused by autoantibodies against nicotinicacetylcholine receptors (AChR) at the neuromuscular junction

[2]. The production of anti-AChR antibodies is mediated bycytokines produced by T helper (Th) [3,4]. Immunotherapiesaimed at targeting disease-inducing AChR-specific Th cell popula-tions should be effective in EAMG. We and others have used oraltolerance induction as a strategy to prevent EAMG, and found thatoral administration of AChR inhibits clinical EAMG and sup-presses both humoral and cellular immune responses [5–7]. AChRadministration by the nasal route, although only 1/1000 of the doseof AChR needed, is still as effective as oral tolerance induction [8].

To determine whether nasal administration of AChR can beused to treat ongoing EAMG, i.e. a situation more likely to beencountered in human MG, we administered AChR nasally toLewis rats 2 weeks after immunization with AChRþ Freund’scomplete adjuvant (FCA). The effects of nasal administration ofAChR in ongoing EAMG were evaluated following several impor-tant aspects: (i) clinical course of EAMG; (ii) AChR-induced T cellresponses; (iii) anti-AChR antibody specificity; (iv) cytokineprofiles.

Clin Exp Immunol 1998;111:506–512

506 q 1998 Blackwell Science

Correspondence: Dr Fu-Dong Shi, Division of Neurology, KarolinskaInstitute, Huddinge University Hospital, S-141 86 Huddinge, Stockholm,Sweden.

MATERIALS AND METHODS

Antigen preparationsAChR was purified from the electroplax tissue ofTorpedo cali-fornica (Pacific Biomarine, Venice, CA) by affinity chromatogra-phy ona-cobrotoxin-agarose resin (Sigma, St Louis, MO) [9]. Theproduct was pure as judged by SDS–PAGE. The control antigenmyelin basic protein (MBP) was purified from guinea pig spinalcord [10]. Purity was confirmed by SDS–PAGE.

ImmunizationFemale Lewis rats, 8 weeks of age, were purchased from CharlesRiver Co. (Sulzfeld, Germany). Each rat was immunized subcuta-neously in both hind footpads and base of tail with 50mg of AChRemulsified in FCA in a total volume of 200ml. The clinical severityof EAMG was blindly graded [11] as follows: 0, no weakness; 1þ ,mildly decreased activity, weak grip or cry, with fatigue; 2þ ,markedly decreased activity and body weight, hunched posture atrest, head down and forelimb digits flexed, tremulous ambulation;and 3þ , severe generalized weakness, no cry or grip. Rats werekilled at day 49 post-immunization (p.i.).

Nasal tolerance inductionThe schedule previously described for rats nasally tolerized withAChR before immunization [8] was modified. Two weeks p.i., ratsreceived into each nostril 30ml PBS pH 7·4 containing AChR atconcentrations of 100mg/ml, 500mg/ml or 1000mg/ml using amicropipette. Control rats received PBS only. At each administra-tion, rats were gently anaesthetized with ether. The administrationswere performed daily for 10 days. In all, each rat received AChR atamounts of 60, 300 or 600mg.

Radioimmunoassay for muscle AChR contentTriplicate 2 pM aliquots of125I-a-bungarotoxin (a-BT; AmershamCorp., Arlington Heights, IL)-labelled Triton X-100 solubilized ratmuscle extract were mixed with standard pooled rat anti-AChRantiserum. After incubation, rabbit anti-rat immunoglobulin(Dakopatts, Copenhagen, Denmark) was added. The precipitateswere counted in a Packardg-counter. The percentage loss ofmuscle AChR in test rat carcass was calculated as described [12].

Enumeration of antigen-reactive interferon-gamma-secreting cellsThe rats were killed on day 49 p.i. Popliteal and inguinal lymphnode (PILN) cells were prepared and adjusted to a cell concentra-tion of 2×106/ml. A solid-phase ELISPOT assay was adopted [13].Nitrocellulose-bottomed microtitre plates (Microtiter-HAM plates;Millipore Co., Bedford, MA) were coated with 100ml per well at15mg/ml of rat interferon-gamma (IFN-g) capture antibody DB1(Innogenetics, Genth, Belgium). Aliquots of 200ml of cell suspen-sion containing 4×105 mononuclear cells (MNC) were added toindividual wells in triplicate, followed by antigen (AChR, MBP),or mitogen (concanavalin A (Con A); Sigma) in 10-ml aliquots to afinal concentration of 10mg/ml (AChR, MBP), or 5mg/ml (Con A).The wells were emptied after 48 h of culture. Secreted and boundIFN-g was visualized by sequential application of rabbit polyclo-nal anti-rat IFN-g antibody (Innogenetics), biotinylated anti-rabbitIgG and avidin-biotin peroxidase complex (ABC; Dakopatts).After peroxidase staining, the red-brown spots which correspondedto the cells that had secreted IFN-g were enumerated in a dissectionmicroscope. To calculate the numbers of T cells responding to aparticular antigen or mitogen, numbers of spots in culture without

antigen (usually 1·5–2·4 per 105 MNC in this study) were sub-tracted from values obtained after antigen challenge. The data wereexpressed as numbers of spots per 105 cultured MNC.

Lymphocyte proliferation responsesBriefly, 200-ml aliquots of MNC suspensions with a cell density of2×106/ml were applied in triplicate into round-bottomed wells of96-well microtitre plates (Nunc, Copenhagen, Denmark). Ten-microlitre aliquots of AChR, MBP or Con A were added intoappropriate wells at final concentrations of 10mg/ml (AChR, MBP)or 5mg/ml (Con A). After 60 h of incubation, the cells werelabelled for an additional 12 h with 10-ml aliquots containing1mCi of 3H-methylthymidine (specific activity 42 Ci/mmol; Amer-sham, Aylesbury, UK). Cells were harvested onto glassfibre filtersand thymidine incorporation was measured. The results wereexpressed as stimulation index (SI).

Determination of AChR-specific IgG isotype antibodiesMicotitre plates (Costar, Cambridge, MA) were coated with AChRat 5mg/ml in a volume of 100ml/well and incubated at 48C over-night. After washing with PBS containing 0·05% Tween 20, non-specific binding was blocked with 1% bovine serum albumin(BSA). After washing, serum samples diluted 1:1000 were overlaidand incubated for 2 h at room temperature, followed by biotiny-lated mouse anti-rat IgG2a (1:2000; Ams, Frankfurt, Germany) orIgG1 (1:2000; Ams) and incubated for 2 h at room temperature.After washing and further incubation with avindin-biotin-alkalinephosphatase complex (Vector, Burlingame, CA), reaction wasvisualized after incubation withp-nitrophenyl phosphate (Sigma)solution and read at 405 nm.

Detection of relative affinity of serum anti-AChR IgG antibodiesThe relative affinity of anti-AChR IgG antibodies was determinedby ELISA using thiocyanate elution [14]. Briefly, microtitre plates(Costar) were coated with AChR and uncoated sites were blockedwith 10% fetal calf serum (FCS; GIBCO, Paisley, UK). Dilutedserum with a predetermined amount of anti-AChR antibodies wasadded and incubated. Then, appropriate quantities of potassiumthiocyanate (KSCN) were added in duplicate and incubated,followed by biotinylated goat anti-rat IgG and alkaline phospha-tase-labelled avidin D. The colour was developed withp-nitrophe-nyl phosphate and expressed as absorbance at 405 nm. The relativeaffinity is expressed as affinity index, equal to the molarity ofKSCN resulting in 50% of the absorbance obtained in the absenceof KSCN.

Detection of functional ability of anti-AChR antibodies in muscleculture systemCultures of limb muscles of 1–3-day-old Lewis rats were preparedas described [14]. Degradation of AChR was measured by anindirect method that depends on the specific labelling of thereceptors with125I-labelleda-BT (Amersham) and the release ofradioactive material derived from degraded receptors into theculture medium. In order to test the effect of the antibody onreceptor degradation in the cultures, the usual culture medium wasreplaced with 1 ml minimum essential medium (MEM; GIBCO), towhich 0·1 ml (2 mg/ml) of either control immunoglobulin orimmunoglobulin from control EAMG rats was added, and EAMGrats were treated with AChR 600mg/rat. The immunoglobulins wereprepared [15] from pools of sera from control EAMG rats, andfrom EAMG rats treated with AChR 600mg/rat. Immunoglobulin

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prepared from normal Lewis rats served as control. The radio-activity released into the medium, representing degraded receptors,was determined at intervals of up to 40 h, and degradation rateswere calculated as described [16].

Detection of IFN-g, tumour necrosis factor-alpha, IL-4, IL-10 andtransforming growth factor-beta mRNA-expressing cells byin situhybridizationIn situ hybridization (ISH) was performed to detect cytokinemRNA expression in MNC as described [6]. Aliquots of 200mlof cell suspensions from PILN containing 4×105 MNC were addedinto round-bottomed microtitre plates (Nunc) in triplicate. Separatecultures received no antigen, 10ml aliquots of Con A (50mg/ml),AChR (100mg/ml), MBP (100mg/ml). After culture for 24 h, cellswere washed, counted and dried onto restricted areas of electricallycharged glass slides (ProbeOn slides; Fisher Scientific, Pittsburgh,PA). Oligonucleotide probes (Scandinavian Gene Synthesiz AB,Koping, Sweden) were labelled using35S-deoxyadenosine-50-a-(thio)-triphosphate with terminal deoxynucleotidyl transferase(Amersham). The oligonucleotide sequences were obtained fromGenBank using the MacVector System (Table 1). Control slideswere hybridized with the same total amount of a sense probe withnucleotide sequence for exon 4 of rat IFN-g. Cells were hybridizedwith 106 ct/min of labelled probe per 100ml of hybridizationmixture. After emulsion autoradiography, development and fixa-tion, the coded slides were examined by dark field microscopy forpositive cells containing>15 grains per cell in a star-like distribu-tion. In many positive cells, the grains were so heavily accumu-lated within and around the cells that it was not possible to countevery single grain. In cells judged negative, the numbers of grainswere mostly 0–2 per cell, and the grains were scattered randomlyover the cells and not distributed in a star-like fashion. There weretherefore no difficulties in differentiating between positive andnegative cells. A control probe of the sense sequence for rat IFN-g

exon 4 was used in parallel with the cytokine probes, producing auniformly weak background signal without revealing any positivecells. The number of cells used for ISH was not equal to thenumbers that were ultimately detected on the slides. To compensate

for cell losses, the total number of cells on the slides was counted.With the help of a microscope grid as a measuring unit, the radius(r) of the surface area (A) covered by cells was determined. TheareaA was calculated by the formulaA¼ p× r2. Cells were usuallycounted in two grids at the periphery and one grid at the centre ofthe surface covered by cells. In case of uneven distribution, cells inadditional grids were counted. The mean value of the number ofthe cells per grid was determined and multiplied byA. Variationbetween duplicates was< 10%. Results are expressed as numbersof labelled cells per 105 MNC.

Statistical analysisDifferences between pairs of groups were tested by Student’st-test.Differences between four groups were tested by one-factor analysisof variance (ANOVA). Mann–Whitney’sU-test was used to comparethe maximal severity of clinical signs. The level of significancewas set toa ¼ 0·05 or 0·01. All tests were two-sided.

RESULTS

Treatment of EAMG with nasal AChR dose-dependentlyameliorates muscular weakness and loss of muscle AChRControl Lewis rats receiving PBS nasally after immunization withAChRþ FCA developed typical two-phase EAMG, with an earlyphase of mild (þ) or moderately severe (þ þ) muscular weaknessaround 8 days p.i., and a late phase of progressive muscularweakness from about 27 days p.i., and afterwards most rats devel-oped severe muscular weakness and four out of 10 rats died duringthe observation period of 7 weeks. Two weeks after immunizationwhen AChR was administered nasally, rats had already recoveredfrom the early phase of muscular weakness. The onset of late phaseof muscular weakness was not affected by AChR nasal adminis-tration. In rats receiving 60mg/rat and 300mg/rat of AChR nasally,

508 F.-D. Shiet al.


Table 1. The cytokine probes used in the study

Gene bank ComplementaryProbes Exon accession no. to bases References

Rat IFN-g exon 1 M-29315-29317 80–125 [17]exon 2 180–227exon 3 298–345

Rat IL-4 exon 1 X16058 83–130 [18]exon 2 209–256exon 3 270–317exon 4 331–378

Mouse IL-10 exon 1 M37897 79–126 [19]exon 2 134–181exon 3 184–231exon 4 402–449

Human TGF-b exon 1 X02812 1766–1813 [20]exon 2 1953–2000

Rat TNF-a exon 1 00475 913–960 [21]exon 2 2059–2106exon 3 2152–2199exon 4 2316–2363

B B

B

B

B

1

0

Clin

ical

sco

res

10

2

3

4

20 30 40 50

B

B

B B

B

B

B

B

BB

0

Days p.i.

Fig. 1.Effects of nasal administration of acetylcholine receptor (AChR) onongoing EAMG. Groups of 8–10 female Lewis rats were first immunizedwith AChRþ Freund’s complete adjuvant (FCA). Two weeks post-immu-nization (p.i.), rats were treated nasally with either different doses of AChR(total 60, 300 or 600mg/rat) or PBS for 10 consecutive days. Arrowsindicate days 14–24 p.i., when nasal treatment with AChR was conducted.Rats treated with 60 or 300mg/rat of AChR exhibited a similar diseasepattern as control EAMG rats treated nasally with PBS only. Rats treatednasally with 600mg/rat of AChR developed relatively mild muscularweakness in comparison with control EAMG rats (mean clinical score atweek 7 p.i.: 0·93versus3·12;P<0·05).A, PBS;B, AChR 60mg/rat;W,AChR 300mg/rat;K, AChR 600mg/rat.

the muscular weakness was as severe as in control EAMG rats. Incontrast, in rats receiving a high dose of AChR (600mg/rat), fiveout of six rats developed relatively mild muscular weaknesscompared with control EAMG rats (mean clinical score at week7 p.i.: 0·93versus3·12;P<0·05) (Fig. 1). This group of rats alsoshowed less pronounced loss of muscle AChR compared withcontrol EAMG rats (Fig. 2).

Treatment of EAMG with nasal AChR suppresses AChR-specificTh1 responsesAChR-specific Th1 responses reflected by levels of AChR-reactiveIFN-g-secreting cells were also reduced in these rats (P¼ 0·02)compared with control EAMG rats (Fig. 3a). Proliferativeresponses to AChR were suppressed in EAMG rats treated nasallywith AChR 600mg/rat (P<0·05) (Fig. 3b). There were no differ-ences in proliferative responses to AChR or numbers of AChR-reactive IFN-g-secreting cells between rats receiving 60mg/rat or300mg/rat AChR compared with results in control EAMG rats.There were also no differences in T cell proliferative responses ornumbers of IFN-g-secreting cells between control EAMG rats andrats treated with the different doses of AChR nasally in controlexperiments when cells were cultured in the presence of MBP,Con A, or no antigen (data not shown).

Anti-AChR IgG2b and IgG1 antibodies are reciprocally regulatedby altered Th1 functions in EAMG rats nasally treated with AChRSerum antibody isotype profiles can serve as relative indicators forTh subset activation in rats [22,23]. IgG1 and IgG2b were analysedat week 7 p.i. The rats treated with AChR 600mg/rat had lowerlevels of AChR-specific IgG2b than control rats, suggesting thatTh1 functions were suppressed in these AChR-treated rats. Thisgroup of rats also showed a borderline increase of serum IgG1levels compared with control EAMG rats, suggesting the presenceof Th2 functions (Fig. 4).

Functional studies of anti-AChR antibodiesTo characterize further the elevated anti-AChR antibodies result-ing from nasal administration of AChR to treat ongoing EAMG,

we measured the antibody affinity by KSCN-ELISA. The relativeaffinity of serum anti-AChR antibodies in control EAMG ratstreated with PBS only increased gradually throughout the observa-tion period. In rats treated with a high dose of AChR nasally, therelative affinity of serum anti-AChR antibodies was lower than incontrol EAMG rats. Significant differences between the twogroups were noticed at 5 and 7 weeks p.i. (Fig. 5). These resultsindicate that serum anti-AChR antibodies at these time points hadpoor binding capacity and were thus less myasthenic. No suchdifferences were found between rats treated with AChR 60, 300mg/rat or control EAMG rats (data not shown).

We also measured the functional ability of serum AChRantibodies to induce accelerated endocytosis and degradation ofAChR in a muscle cell culture system. The degradation rates (%



0

EAMG rats treated nasally with AChR 600 µg/rat

20 40 60 80Loss of muscle AChR (%)



Control EAMG rats

*

Fig. 2. Loss of muscle acetylcholine receptor (AChR) content. The musclecarcasses from EAMG rats treated nasally with AChR and from controlEAMG rats at week 7 post-immunization (p.i.) were examined for AChRcontent by radioimmunoassay (RIA). The percentage loss of muscle AChRwas calculated as described in Materials and Methods. Data from eight ratsin each group are shown. Symbols refer to mean values, bars to s.e.m.*P<0·05.

5

0

AC

hR

-re

act

ive

IF

N-γ-

secr

eti

ng

ce

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pe

r 1

05 M

NC

(a)

PBS

10

15

0.5

0

Pro

life

rati

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esp

on

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to A

Ch

R (

SI)

1

1.5

2

2.5

3

60 300 600

*

PBS 60 300 600

µg/rat

(b)

*

Fig. 3. (a) Numbers of acetylcholine receptor (AChR)-reactive IFN-g-secreting cells per 105 lymph node mononuclear cells (MNC) from EAMGrats treated nasally with different doses of AChR and from control EAMGrats treated nasally with PBS only. Eight rats in each group were killed7 weeks post-immunization (p.i.). Symbols refer to mean values and bars tos.d. *P<0·05. (b) Proliferative responses to AChR on day 49 p.i. of lymphnode cells from EAMG rats treated nasally with AChR and from controlEAMG rats. Results are expressed as stimulation index (SI). Symbols referto mean values and bars to s.e.m. *P<0·05.

per hour) of control immunoglobulin, immunoglobulin fromEAMG rats receiving high dose AChR and control EAMG ratswere 3·216 0·04, 5·326 0·08, 8·786 0·03, respectively. Thiseffect was therefore less pronounced in the EAMG rats receivinghigh-dose AChR.

Treatment of EAMG by nasal AChR suppresses AChR-reactive Th1but not Th2 and Th3 cytokine mRNA expressionIn rats receiving 600mg/rat AChR nasally, numbers of AChR-reactive IFN-g and tumour necrosis factor-alpha (TNF-a) mRNA-expressing lymph node cells were lower than in control ratsreceiving PBS. No differences were found for AChR-reactiveIL-4, IL-10 and transforming growth factor-beta (TGF-b) mRNA-expressing cells between the two groups. In control experimentswhen lymph node MNC were cultured without antigen or in thepresence of the irrelevant antigen, MBP, or Con A, no differenceswere found for numbers of IFN-g, TNF-a, IL-4, IL-10 or TGF-b

mRNA-expressing cells between the two groups (data not shown).These data imply that nasal administration of AChR in ongoingEAMG actually suppressed antigen-specific Th1 cytokine mRNAexpression, correlating with levels of AChR-reactive IFN-g-secreting cells in the experiments (Fig. 6), while AChR-specificTh2 or Th3 mRNA-expressing cells were not affected.

DISCUSSION

Nasal administration of AChR in the Lewis rat before immuniza-tion with AChR in FCA resulted in markedly decreased severity ofclinical EAMG, and suppressed AChR-specific T and B cellresponses when a dose as low as 60mg AChR/rat was used [8].When administering AChR for the treatment of ongoing EAMG,we found that a 10-fold higher amount of AChR (600mg/rat) isrequired to ameliorate muscular weakness of EAMG. These find-ings imply that when an abnormal immune response to AChR isestablished, much higher doses of AChR administered nasally areneeded to arrest the progression of the autoimmune pathologicprocess.

AChR is highly immunogenic. The immune responses toAChR are heterogeneous, involving many myasthenic epitopes([24], reviewed in [25]), associated with determinant spreading[26,27]. In the established EAMG, the broad range of T and B cellspecificities may be more difficult to modulate. In fact, nasal AChRadministration in ongoing EAMG is less effective compared with aprophylactic approach, and much higher doses are required.

High-dose AChR (600mg/rat) resulting in incomplete abroga-tion of EAMG was associated with reduced lymphocyte prolifera-tion and IFN-g secretion in response to AChR, but with aconcomitant augmentation of the production of anti-Torpedoandanti-rat AChR antibodies.

Upon activation by cognate ligand, naive Th cells differentiateinto distinct functional subgroups which are characterized by theirpattern of cytokine secretion [28]. It is likely that Th1- and Th2-type cells also exist in rats [29,30]. AChR-specific rat T cells havebeen shown to secrete Th1 and Th2 cytokines [31]. In the presentstudy, nasal administration of AChR in ongoing EAMG with the

510 F.-D. Shiet al.


0

OD 405

0.1 0.2 0.3 0.4 0.5

EAMG rats treated nasally with AChR

600 µg/rat

EAMG

*

* *

Fig. 4. Serum anti-acetylcholine receptor (AChR) IgG isotype antibodiesmeasured by ELISA. Sera from eight rats were analysed for IgG1 (A) andIgG2b (B) at week 7 post-immunization (p.i.), when anti-AChR antibodyIgG levels were higher in EAMG rats treated with AChR 600mg/rat than incontrol EAMG rats treated with PBS only. Symbols refer to mean valuesand bars to s.e.m. *P<0·05; **P¼ 0·0562.

1

Affi

nity

inde

x

Weeks after immunization

2 4 6 8

2

3

4

B

B

B

B

00

*

*

Fig. 5. The relative affinity of serum anti-acetylcholine receptor (AChR)antibodies measured at 1, 3, 5 and 7 weeks post-immunization (p.i.) bypotassium thiocyanate (KSCN)-ELISA in EAMG rats treated nasally withAChR 600mg/rat (B) and control EAMG rats treated nasally with PBS only(A). Data from eight rats in each group are shown. Symbols refer to meanvalues and bars to s.e.m. *P< 0·05.

10

0Nu

mb

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AC

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e

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pre

ssin

g c

ell

s p

er

10

5 M

NC

IFN-γ

20

30

40

TNF-α IL-4 IL-10 TGF-β

* **

Fig. 6. Numbers of acetylcholine receptor (AChR)-reactive cytokinemRNA-expressing cells per 105 lymph node mononuclear cells (MNC) at7 weeks post-immunization (p.i.) from EAMG rats treated nasally withAChR 600mg/rat (B) and control EAMG rats treated nasally with PBS only(A). Data from eight rats in each group are shown. Symbols refer to meanvalues and bars to s.e.m. *P<0·05; **P<0·01.

current treatment protocol induces selective suppression of Th1function (as reflected by reduced AChR-induced IFN-g mRNAexpression and secretion, and helper function for IgG2bproduction), but not of Th2 cells.

Tolerance induction, under certain circumstances, is associatedwith preferential inhibition and/or activation of either of these Tcell subsets, resulting in a dichotomy between cellular and humoralimmune responsiveness, reflecting influence of the reciprocalinhibition between Th1 and Th2 cells which maintains a specificbalance among the two subpopulations [32–34]. Intrathymicinjection of AChR in a rat model of EAMG resulted in thesuppression of Th1 cell functions alone [35]. Tolerization of Tcells, but not B cells, has previously been described after feeding[36,37] and injection of low antigen doses to experimental animals[38]. A gradient of sensitivity to tolerance induction withTh1>Th2>B cells has been proposed [37,39]. This implies thatapproaches that produce specific inhibition of cell-mediated con-ditions like EAE or EAU do not necessarily inhibit an antibody-mediated condition like MG. The manifestation of split tolerance isdependent on the nature of the antigen, route of antigen adminis-tration, and type of adjuvant used [34]. Data from the present studysuggest that the degree of disease suppression is dose-dependent,higher antigen doses may be needed to induce tolerance of Th2cells or B cells.

Besides Th2 cytokines, several lines of evidence implicate Th1cytokines as contributing to the development of humoral immuneresponses in EAMG. Elevated levels of circulating cells secretingIFN-g or expressing IFN-g and TNF-a mRNA in response toAChR could reflect escalated AChR-specific Th1 responses in MGand EAMG [40–43]. Severe MG is associated with augmentedspontaneous TNF-a production in cultures of MNC compared withmild disease [44]. Upon expression of IFN-g within the neuro-muscular junction, EAMG-resistant BALB/c mice exhibited clin-ical weakness and disruption of the neuromuscular junction,accompanied by autoantibody deposition at motor end plates,implying that IFN-g in the milieu of the muscle tissue induceshumoral autoimmunity without circulating anti-AChR antibodies[45]. We and others have recently shown that mice with either IFN-g or IFN-g receptor deficiency are less susceptible to EAMG,associated with lower levels and affinities of anti-AChR antibodies[46,47].

The decreased levels of IFN-g and TNF-a in the present study,on the one hand, may have a serious impact on the balance ofcytokine network (impacts on Th2 cytokines) and alter the amountof anti-AChR antibody. Since the serum antibody levels do notcorrelate with disease severity, either in EAMG or in human MG,differences in binding affinity, fine specificity for AChR epitopes,and isotype may determine the outcome of the disease [48,49].IFN-g and TNF-a direct B cell maturation, IFN-g drives non-immunoglobulin-secreting B cells to active immunoglobulin secre-tion and isotype switches, and affinity maturation [50–53]. There-fore, the decreased levels of IFN-g and TNF-a may contribute tothe production of lower affinity antibodies and the reduced level ofAChR-specific IgG2b, which are probably the only isotype trigger-ing antibody-dependent cell-mediated cytotoxicity (ADCC) in rats[54]. These anti-AChR antibodies have less capacity in inducingAChR degradation, and contribute to the partial suppression ofdisease in rats receiving high-dose AChR.

In conclusion, our results imply that, in contrast to T cell-mediated conditions, the application of a mucosal tolerance strat-egy to an established antibody-mediated autoimmune disease such

as EAMG has major implications for humoral immune responses.These results could provide the basis for designing antigen-specifictherapy in MG.

ACKNOWLEDGMENTS

We thank Anita Gustafsson and Birgitta Jonsson for technical help. Thestudy was supported by grants from the Swedish Medical Research Council,Swedish MS Society (NHR), and Karolinska Institute Research Funds.

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Nasal tolerance in experimental autoimmune myasthenia gravis (EAMG): induction of protective tolerance in primed animals