Increased levels of circulating acetylcholine receptor (AChR)-reactiveIL-10-secreting cells are characteristic for myasthenia gravis (MG)

Y.-M. HUANG, P. KIVISAKK, V. OZENCI, R. PIRSKANEN* & H. LINK Division of Neurology, Unit ofNeuroimmunology, Karolinska Institutet, Huddinge University Hospital and*Division of Clinical Neurosciences, Karolinska Institutet,

Karolinska Hospital, Stockholm, Sweden

(Accepted for publication 11 August 1999)

SUMMARY

Antibodies to the nicotinic AChR are pivotal in the immunopathogenesis of MG. Cytokines produced byT-helper cells are important regulators of humoral immune responses. IL-10 is considered an anti-inflammatory cytokine, but it promotes B cell activation and worsens experimental autoimmune MG inLewis rats, an experimental model of MG. To study IL-10 and, as a control, interferon-gamma (IFN-g)in MG, we used an enzyme-linked immunospot (ELISPOT) assay, thereby assessing numbers of bloodmononuclear cells (MNC) secreting IL-10 and IFN-g spontaneously and after stimulation with AChR.Low numbers of IL-10-secreting cells were regularly found in peripheral blood from patients with MGas well as in controls with other neurological diseases and healthy subjects. However, only MG patientshad elevated blood levels of AChR-reactive IL-10- and IFN-g-secreting cells. The MG patients showedno responses to the control autoantigen myelin basic protein, underlining the specificity of the IL-10 andIFN-g responses. Immunosuppressive treatment reduced numbers of AChR-reactive IFN-g-secretingcells but increased the numbers of IL-10-secreting cells. The numbers of IL-10-secreting cells tended tobe higher in patients with generalizedversusocular MG, further suggesting that the augmented IL-10responses may be important in the pathogenesis and perpetuation of MG.

Keywords myasthenia gravis IL-10 interferon-gamma ELISPOT autoimmunity

INTRODUCTION

A hallmark of MG is the occurrence of circulating antibodiesagainst the nicotinic AChR in skeletal muscle, disturbingneuromuscular transmission [1]. The production of anti-AChRantibodies in MG is T cell-dependent and mediated by auto-reactive CD4þ T helper (Th) cells [2,3]. Cytokines produced byactivated T cells are potent regulators of immune responses,skewing the immune system towards cell-mediated or humoralresponses (reviewed in [4,5]). It has previously been demon-strated that patients with MG have increased numbers of circu-lating AChR-reactive Th1-like cells secreting interferon-gamma(IFN-g) [6,7].

IL-10 was originally described as a murine Th2 cytokine,inhibiting cytokine synthesis by Th1 cells [8]. The production ofhuman IL-10 is, as later studies have shown, not restricted toTh2 cells since Th0 and Th1 cells, B cells and macrophages havealso been shown to express IL-10. Nevertheless, many of the

effects of IL-10 are similar to, or overlapping with, the functionsof Th2 cytokines and there seems to be a close correlationbetween the induction of Th2-like responses and the expressionof IL-10 (reviewed in [9]).

Interestingly, IL-10 mediates a range of both immuno-suppressive and immunostimulatory effects in mice and humans[10]. Since IL-10 is an important growth factor for B cells,augmenting B cell proliferation and differentiation into antibody-producing cells as well as inducing isotype switching in B cells[11,12], a role of IL-10 in MG could be hypothesized. We havepreviously shown that patients with MG have elevated numbers ofT cells expressing IL-10 mRNA upon stimulation with AChRcompared with control patients, probably reflecting enhancedIL-10 production [13]. These data must however be interpretedcautiously, since cytokine mRNA expression may not necessarilyparallel protein secretion.

In an attempt to define further the involvement of IL-10 in MG,we adopted enzyme-linked immunospot (ELISPOT) assays toenumerate mononuclear cells (MNC) secreting IL-10 and IFN-g

spontaneously and after stimulation with AChR in patients withMG and controls.

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Correspondence: Dr Pia Kivisa¨kk, Department of Neurology, HuddingeUniversity Hospital, 141 86 Huddinge, Sweden.

E-mail: Pia.Kivisakk@neurotec.ki.se

PATIENTS AND METHODS

PatientsPeripheral blood was obtained from 23 patients (11 women) withMG, aged between 23 and 83 years (mean 54 years). The diagnosisof MG was based on the presence of characteristic fluctuatingmuscular fatiguability, the results from laboratory tests thatincluded determinations of serum anti-AChR antibody concentra-tions and single fibre electromyography, and the response totreatment with cholinesterase inhibitors. Anti-AChR antibodieswere detected in serum from 17 patients. The onset of MGbefore the age of 40 years was documented in 10 patients. Fivepatients had ocular MG, 13 patients had mild generalized MG andfour patients had severe generalized disease according to theclassification of Osserman & Genkins [14]. One patient was inremission without any medication. Fourteen patients had under-gone thymectomy. Ten patients had thymus hyperplasia, twopatients thymoma, while the remaining two patients had anormal appearing thymus. Seven patients were treated with immu-nosuppressive agents at the time of sampling (azathioprine orcyclosporin A (CsA)).

In parallel, blood was collected from 19 control patients (sixwomen) with other neurological diseases (OND), mainly acutecerebrovascular disorders. Their age was 46–78 years (mean64 years). Thirteen healthy volunteers (10 women, age range25–58 years, mean 35 years) consisting of staff from thedepartment were also included.

Antigen preparationAChR was purified from electroplax tissue ofTorpedo californica(Pacific Biomarine, Venice, CA) by affinity chromatography on ana-cobratoxin-agarose resin (Sigma, St Louis, MO) [15]. Myelinbasic protein (MBP) was prepared from human brain white matter[16]. Purity of MBP was assessed by SDS–PAGE, which showed asingle band migrating at approximately 22 kD.

Detection of IL-10- and IFN-g-secreting MNC by ELISPOTThe ELISPOT assay described by Czerkinskyet al. [17] wasadopted to detect and enumerate cells secreting IL-10 as well asIFN-g [6]. MNC were separated from peripheral blood by densitygradient centrifugation on Lymphoprep (Nycomed, Oslo,Norway). The cells from the interface were collected, washedtwice with Dulbecco’s modified Eagle’s medium (Life Technolo-gies, Paisley, UK) supplemented with antibiotics, 5% fetal calfserum (Life Technologies), 1% MEM amino acids (Life Tech-nologies) and 1%L-glutamine (Life Technologies) and adjusted toa concentration of 1×106cells/ml. Cell viability as measured bytrypan blue exclusion always exceeded 95%.

Microtitre plates with nitrocellulose bottoms (Multiscreen-HAplates; Millipore, Molsheim, France) were coated overnight at 48Cwith an anti-human IL-10 (9-D7) or IFN-g (1-D1K) MoAb(Mabtech, Stockholm, Sweden) diluted in filtered PBS pH 7·4 toa concentration of 10mg/ml. After removal of coating solutions bysuction through the nitrocellulose membranes and subsequentwashing in PBS, 200-ml aliquots containing 2×105 MNC wereapplied to individual wells in duplicates. To evaluate antigen-reactive cells, AChR or MBP were added in 10-ml aliquots to a finalconcentration of 10mg/ml. This antigen concentration was found inpreliminary experiments to give high numbers of spots in casessupposed to be positive. To enumerate cells spontaneously secret-ing IL-10 or IFN-g, wells without any antigen were prepared in

parallel. Plates were incubated at 378C for 24 h (IL-10) or 48 h(IFN-g) in humidified air containing 5% CO2, then emptied andwashed. One hundred microlitres of a biotinylated anti-human IL-10 (12G8) or IFN-g (7-B6-1) MoAb (Mabtech) diluted in PBS to aconcentration of 1mg/ml were added for 2 h at room temperature.Wells were washed and streptavidin-alkaline phosphatase (Mab-tech) diluted 1:1000 was added for 1 h at room temperature. Forstaining, BCIP-NBT (Life Technologies) diluted in Tris-bufferpH 9·5 was used. Blue immunospots, each considered to representa cell secreting IL-10 or IFN-g, were counted under a dissectionmicroscope.

Statistical analysisKruskal–Wallis non-parametricANOVA test was used for multiplecomparisons. The non-parametric Mann–Whitney test was usedfor two-group comparisons and Wilcoxon signed rank test whencomparing paired samples from the same individual. ReportedPvalues are two-tailed andP<0·05 was considered statisticallysignificant.

RESULTS

Blood MNC secreting IL-10 and IFN-g without antigenstimulationBlood MNC spontaneously secreting IL-10 upon short-term cul-ture without antigen were found in all the patients with MG. Mostof them also showed IFN-g-secreting cells. Such cells werehowever also detected in the two groups of controls, and levelsof IL-10- or IFN-g-secreting MNC did not differ significantly inMG compared with OND or healthy individuals.

MG patients treated with immunosuppressive agents(azathioprine or CsA) had lower numbers of blood MNC secretingIFN-g than untreated MG patients (treated, median 4·5/105 MNC;untreated, 18/105 MNC; P< 0·05), while no such differencewas observed for numbers of IL-10-secreting cells (treated,

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Fig. 1. Numbers of IL-10-secreting cells expressed per 105 mononuclearcells (MNC) from patients with MG, patients with other neurologicaldiseases (OND) and healthy volunteers (HC) after cultures in the presenceand absence of AChR or myelin basic protein (MBP).

median 22/105 MNC; untreated, median 21/105 MNC). Whensubgrouping the patients with MG according to clinical variables,there was a trend to lower numbers of IL-10-secreting cells inpatients with ocular MG (median 12·5/105 MNC, n¼ 5) comparedwith patients with generalized MG (median 32/105 MNC, n¼ 17;P¼ 0·07). No differences in numbers of IL-10-secreting cellscould be detected between early onset of symptoms (< 55 years,median 21/105 MNC; n¼ 14)versuslate onset (>55 years, median22/105 MNC; n¼ 9), short duration of MG (< 5 years, median 26·5/105 MNC; n¼ 12) versuslonger duration ($ 5 years, median 22/105 MNC; n¼ 11), or presence of serum anti-AChR antibodies(median 22/105 MNC; n¼ 17) compared with absence of suchantibodies (median 11·5/105 MNC; n¼ 6).

Levels of AChR-reactive IL-10- and IFN-g-secreting blood MNCTo determine the numbers of cells specifically responding toantigen stimulation with secretion of IL-10 or IFN-g, short-termcultures in the presence of AChR or the control antigen MBP wereperformed. Numbers of antigen-reactive cytokine-secreting cellswere calculated by subtraction of the numbers of cells sponta-neously secreting cytokines in parallel cultures without any antigenadded. Using this definition, patients with MG had a mediannumber of 14 AChR-reactive IL-10-secreting cells per 105 bloodMNC, compared with a median of zero AChR-reactive IL-10-secreting cells per 105 blood MNC in both OND and healthyindividuals (Fig. 1). The median number of AChR-reactive IFN-g-secreting cells in patients with MG was 14/105 blood MNC,compared with 2/105 blood MNC in the patients with OND and1/105 blood MNC in healthy individuals (Fig. 2). The differencesbetween MGversusOND and healthy individuals were significantboth for IL-10 (P<0·0001 for both comparisons) and IFN-g

(P<0·001 and P<0·05, respectively). Corresponding dataobtained after culture with MBP showed no differences betweenMG versusOND or healthy individuals, underlining the specificityof the observations when AChR was used as antigen.

Individual patients with MG showed an elevation of eitherAChR-reactive IFN-g- or AChR-reactive IL-10-secreting bloodMNC. The combination of high numbers of AChR-reactive IL-10-and IFN-g-secreting cells was not observed in any patient (Fig. 3).There was an inverse correlation between numbers of AChR-reactive IL-10- and IFN-g-secreting cells in patients with MG(r ¼ – 0·5;P<0·05).

AChR-reactive IL-10- and IFN-g-secreting cells in relation toclinical parametersSeven of the patients with MG were treated with immunosuppres-sive drugs (azathioprine or CsA) at the time of sampling. Immu-nosuppressive treatment reduced the numbers of AChR-reactiveIFN-g-secreting cells (treated, median 2/105 MNC; untreated,median 16/105 MNC; P<0·05). Numbers of AChR-reactive IL-10-secreting cells were on the contrary increased by immunosup-pressive treatment (treated, 47/105 MNC; untreated, 12/105 MNC;P< 0·05). No differences in numbers of IL-10- or IFN-g-secretingAChR-reactive cells were observed when subgrouping the patientsaccording to ocular or generalized MG, short or long duration(<5 years compared with$ 5 years), early or late onset (< 40 yearscompared with$ 40 years) or presence or absence of serum anti-AChR antibodies.

DISCUSSION

This study showed that MG is associated with elevated numbers ofAChR-reactive IL-10- and IFN-g-secreting MNC in peripheralblood. Only low levels of MBP-reactive IL-10- and IFN-g-secret-ing cells were detected in MG, without differences compared withOND or healthy individuals. The data imply that MG is associatedwith elevated levels of specific AChR-reactive blood MNC, andthat both Th1- and Th2-like cells and cytokines participate in thedevelopment of the disease.

The involvement of IL-10 in MG has been proposed on thebasis of results from the detection of IL-10 mRNA transcripts inMNC. Patients with MG had elevated numbers of AChR-reactiveIL-10 mRNA-expressing blood MNC compared with controls withnon-inflammatory neurological diseases [13]. It can however beargued that mRNA detection does not prove protein production,

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Fig. 2. Numbers of IFN-g-secreting cells expressed per 105 mononuclearcells (MNC) from patients with MG, patients with other neurologicaldiseases (OND) and healthy volunteers (HC) after cultures in the presenceand absence of AChR or myelin basic protein (MBP).

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Numbers of IFN-γ-secreting cells/105 MNCFig. 3. Relation between numbers of mononuclear cells (MNC) fromindividual patients with MG responding to AChR stimulation by IFN-g

and IL-10 secretion. Results are presented as number of cytokine-secretingcells in cultures with AChR added after subtraction of numbers of cytokine-secreting cells in cultures without antigen.

and the secretion of IL-10 has up to now not been demonstratedin MG.

A role for IL-10 in antibody-mediated diseases has previouslybeen suggested. For instance, in NZB/W F1 mice, which sponta-neously develop a severe autoimmune disease resembling systemiclupus erythematosus (SLE) in humans, systemically administeredanti-IL-10 antibodies delayed the onset of symptoms and alsodecreased serum autoantibody levels [18]. In SLE patients, serumIL-10 concentrations correlated with disease activity and with anti-DNA antibody titres [19]. Anti-IL-10 antibodies were shown toreduce immunoglobulin synthesis in cell cultures from SLEpatients [20]. In experimental autoimmune MG (EAMG), ananimal model for MG, s.c. administration of rhIL-10, startingafter immunization, aggravated clinical EAMG and increasedserum levels of anti-AChR antibodies [21]. This disease-provokingrole of IL-10 in antibody-mediated diseases contrasts with theputative immunosuppressive effects often seen in experimentalmodels for autoimmune diseases predominantly associated withTh1-related responses, like experimental autoimmune encephalo-myelitis (EAE), autoimmune diabetes mellitus in non-obese dia-betic (NOD) mice and collagen type II-induced arthritis [22–24].

The physiologic role of IL-10 in autoimmunity is not com-pletely understood. Since IL-10 induces B cell proliferation andimmunoglobulin synthesis [11,12], production of autoantibodiesmight be enhanced. In mice treated with neutralizing anti-IL-10antibodies during development, a depletion of a subpopulation of Bcells, termed Ly-1, has been demonstrated [25]. Ly-1 B cells, andthe human counterpart B1, are of interest in autoimmunity sincethey are the major producers of IgM autoantibodies in mice [26],suggesting that IL-10 could participate in the regulation ofautoantibody production.

MG patients treated with immunosuppressive drugs had lessAChR-reactive IFN-g-secreting cells, while the numbers of AChR-reactive IL-10-secreting cells were higher in these patientscompared with healthy individuals. CsA is known to inhibitintracellular signal transduction in T cells, thus suppressing Tcell activation and production of IL-2 [27]. Recently it has beendemonstrated that CsA modulates the balance of cytokines towardsTh2-dominated responses with increased production of IL-10,rather than exerting a non-specific suppression of all cytokines[28], a finding that is similar to our data. Whether such an up-regulation of IL-10 is beneficial in MG is not known. We observeda tendency for patients with generalized MG to have highernumbers of IL-10-secreting cells compared with patients withocular MG, representing a clinical subgroup of MG with localizedand less severe symptoms [14], arousing the speculation that IL-10is involved in the spreading and progression of symptoms.

In conclusion, we have shown that MG patients have highnumbers of MNC reacting with enhanced secretion of both Th2-related IL-10 and Th1-related IFN-g upon challenge with AChR.The MG patients showed no response to MBP, confirming thespecificity of the IL-10 response. By a combination of B cell-stimulating effects and insufficient suppression of Th1 cells, IL-10might be pivotal for the pathogenesis and perpetuation of MG.

ACKNOWLEDGMENTS

This study was supported by grants from the Swedish Medical ResearchCouncil, the Swedish Association of Neurologically Disabled (NHR) andfunds from Karolinska Institutet. We thank Dr Staffan Paulie (Mabtech,Stockholm, Sweden) for generous gifts of the antibodies 9-D7 and 12G8.

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