Myasthenia gravis: recognition of a human autoantigen at the molecular level

The symptoms of myasthenia gravis are primarily or exclusively due to an autoimmune response against the muscle nicotinic acetylcholine receptor (AChR) and this has been the object of intensive investigations for almost 20 years. A detailed picture at the molecular level of the interaction of this autoantigen with the key elements involved in the autoimmune response, such as anti-AChR antibodies, the T-cell receptor and restricting major histocompatibility complex molecules, is now emerging for both human myasthenia gravis and its experimental model, experimental autoimmune myasthenia gravis. Here, Maria Pia Protti and colleagues focus on the molecular interactions occurring in human myasthenia gravis and summarize recent information on pathogenic mechanisms of the autoimmune response, and the structure of epitopes recognized by B cells and CD4⁺T cells of myasthenic patients on the AChR molecule.     

Neuronal acetylcholine receptor alpha9-subunit: a possible central nervous system autoantigen

Nicotinic acetylcholine receptor (AChR) is a membrane glycoprotein composed of five subunits. Muscle AChR is consist of two alpha1 and one each beta, delta, and epsilon subunits, whereas the neuronal AChR molecules are made up of various combinations of alpha (alpha2-alpha10) and beta (beta1-beta4) subunits. Myasthenia gravis (MG) develops as a result of an autoimmune attack against muscular AChR. While the prevailing symptom is muscle weakness, very rarely MG patients may develop additional central nervous system (CNS) symptoms. The majority of the anti-AChR antibodies responsible from disease induction is directed against alpha1 subunit of AChR. There is considerable identity between muscular alpha1 and neuronal alpha9 subunits. Preliminary studies showed antibodies reactive with the CNS antigens in the serum samples of mice with experimental autoimmune myasthenia gravis (EAMG). Also, alpha9 was present in the CNS in widespread locations and the binding pattern of anti-alpha9 antibody was reminiscent of that of serum samples of some of the mice with EAMG. Serum anti-AChR antibodies of myasthenic patients might be cross-reacting with CNS AChR subunits and thus inducing CNS symptoms. Neuronal AChR alpha9-subunit might be a major target antigen in this process.     

Experimental autoimmune myasthenia gravis and CD5+ B-lymphocyte expression

Myasthenia gravis is one of the typical organ specific autoimmune disease and the CD5+ B-lymphocytes are known to be associated with the secretion of autoimmune antibodies. The authors performed the study to establish an animal model of experimental autoimmune myasthenia gravis (EAMG) by immunizing the nicotinic acetylcholine receptor (AChR) and to understand CD5+ B-lymphocyte changes in peripheral blood of EAMGs. Lewis rats weighing 150-200 g were injected subcutaneously three times with 50 microg AChR purified from the electric organ of Torpedo marmorata and Freund's adjuvant. The EAMG induction was assessed by evaluating clinical manifestations. The CD5+ B-lymphocyte was double stained using monoclonal PE conjugated anti-CD5+ and FITC conjugated anti-rat CD45R antibodies and calculated using a fluorescence-activated cell sorter (FACS). In three out of ten Lewis rats injected with purified AChR, the EAMG models were established. The animals showed definite clinical weakness responded to neostigmine; they had difficulty in climbing the slope, or easily fell down from a vertical cage. The range of CD5+ B-lymphocytes of peripheral blood in the EAMG models was 10.2%-17.5%, which was higher than in controls. In conclusion, the EAMG models were successfully established and the CD5+ B-lymphocyte expression in peripheral blood increased in EAMGs. This provided indirect evidence of the autoimmune pathomechanism of human myasthenia gravis.     

IL-12 is involved in the induction of experimental autoimmune myasthenia gravis,an antibody- mediated disease

IL-12 has been shown to be involved in the pathogenesis of Th1-mediated autoimmune diseases, but its role in antibody-mediated autoimmune pathologies is still unclear. We investigated the effects of exogenous and endogenous IL-12 in experimental autoimmune myasthenia gravis (EAMG). EAMG is an animal model for myasthenia gravis, a T cell-dependent, autoantibody-mediated disorder of neuromuscular transmission caused by antibodies to the muscle nicotinic acetylcholine receptor (AChR). Administration of IL-12 with Torpedo AChR (ToAChR) to C57BL/6 (B6) mice resulted in increased ToAChR-specific IFN-γ production and increased anti-ToAChR IgG2a serum antibodies compared with B6 mice primed with ToAChR alone. These changes were associated with earlier and greater neurophysiological evidence of EAMG in the IL-12-treated mice, and reduced numbers of AChR. By contrast, when IL-12-deficient mice were immunized with ToAChR, ToAChR-specific Th1 cells and anti-ToAChR IgG2a serum antibodies were reduced compared to ToAChR-primed normal B6 mice, and the IL-12-deficient mice showed almost no neurophysiological evidence of EAMG and less reduction in AChR. These results indicate an important role of IL-12 in the induction of an antibody-mediated autoimmune disease, suggest that Th1-dependent complement-fixing IgG2a anti-AChR antibodies are involved in the pathogenesis of EAMG, and help to account for the lack of correlation between anti-AChR levels and clinical disease seen in many earlier studies.     

Autoantibodies to ganglionic acetylcholine receptors in autoimmune autonomic neuropathies

BACKGROUND: Idiopathic autonomic neuropathy is a severe, subacute disorder with a presumed autoimmune basis. It is indistinguishable from the subacute autonomic neuropathy that may accompany lung cancer or other tumors. Autoantibodies specific for nicotinic acetylcholine receptors in the autonomic ganglia are potentially pathogenic and may serve as serologic markers of various forms of autoimmune autonomic neuropathy. METHODS: We tested serum from 157 patients with a variety of types of dysautonomia. Immunoprecipitation assays with iodine-125-labeled epibatidine and solubilized human neuroblastoma acetylcholine receptors were used to detect autoantibodies that bound to or blocked ganglionic receptors. RESULTS: Ganglionic-receptor-binding antibodies were found in 19 of 46 patients with idiopathic or paraneoplastic autonomic neuropathy (41 percent), in 6 of 67 patients with postural tachycardia syndrome, idiopathic gastrointestinal dysmotility, or diabetic autonomic neuropathy (9 percent), and in none of 44 patients with other autonomic disorders. High levels of the binding antibodies correlated with more severe autonomic dysfunction (including the presence of tonic pupils). Levels of these antibodies decreased in patients who had clinical improvement. All seven patients with ganglionic-receptor-blocking antibodies had ganglionic-receptor-binding antibodies and had idiopathic or paraneoplastic autonomic neuropathy. CONCLUSIONS: Seropositivity for antibodies that bind to or block ganglionic acetylcholine receptors identifies patients with various forms of autoimmune autonomic neuropathy and distinguishes these disorders from other types of dysautonomia. The positive correlation between high levels of ganglionic-receptor antibodies and the severity of autonomic dysfunction suggests that the antibodies have a pathogenic role in these types of neuropathy.     

Structural characterization of the main immunogenic region of the Torpedo acetylcholine receptor

To develop antigen-specific immunotherapies for autoimmune diseases, knowledge of the molecular structure of targeted immunological hotspots will guide the production of reagents to inhibit and halt production of antigen specific attack agents. To this end we have identified three noncontiguous segments of the Torpedo nicotinic acetylcholine receptor (AChR) α-subunit that contribute to the conformationally sensitive immunological hotspot on the AChR termed the main immunogenic region (MIR): α(1–12), α(65–79), and α(110–115). This region is the target of greater than 50% of the anti-AChR Abs in serum from patients with myasthenia gravis (MG) and animals with experimental autoimmune myasthenia gravis (EAMG). Many monoclonal antibodies (mAbs) raised in one species against an electric organ AChR cross react with the neuromuscular AChR MIR in several species. Probing the Torpedo AChR α-subunit with mAb 132A, a disease inducing anti-MIR mAb raised against the Torpedo AChR, we have determined that two of the three MIR segments, α(1–12) and α(65–79), form a complex providing the signature components recognized by mAb 132A. These two segments straddle a third, α(110–115), that seems not to contribute specific side chains for 132A recognition, but is necessary for optimum antibody binding. This third segment appears to form a foundation upon which the three-dimensional 132A epitope is anchored.     

Modulation of the anti-acetylcholine receptor response and experimental autoimmune myasthenia gravis by recombinant fragments of the acetylcholine receptor

Myasthenia gravis (MG) is a neuromuscular disorder of man caused by a humoral response to the acetylcholine receptor (AChR). Most of the antibodies in MG and in experimental autoimmune myasthenia gravis (EAMG) are directed to the extracellular portion of the AChR α subunit, and within it, primarily to the main immunogenic region (MIR). We have cloned and expressed recombinant fragments, corresponding to the entire extracellular domain of the AChR α subunit (Hα1 – 210), and to portions of it that encompass either the MIR (Hα1 – 121) or the ligand binding site of AChR (Hα122 – 210), and studied their ability to interfere with the immunopathological anti-AChR response in vitro and in vivo. All fragments were expressed as fusion proteins with glutathione S-transferase. Fragments Hα1 – 121 and Hα1 – 210 protected AChR in TE671 cells against accelerated degradation induced by the anti-MIR monoclonal antibody (mAb)198 in a dose-dependent manner. Moreover, these fragments had a similar effect on the antigenic modulation of AChR by other anti-MIR mAb and by polyclonal rat anti-AChR antibodies. Fragments Hα1 – 121 and Hα1 – 210 were also able to modulate in vivo muscle AChR loss and development of clinical symptoms of EAMG, passively transferred to rats by mAb 198. Fragment Hα122 – 210 did not have such a protective activity. Our results suggest that the appropriate recombinant fragments of the human AChR may be employed in the future for antigen-specific therapy of myasthenia.     

Prevention and therapy with electrolectin of experimental autoimmune myasthenia gravis in rabbits

Electrolectin (EL), an endogenous β-D -galactoside-binding lectin from Electrophorus electricus, was found to have a prophylactic and therapeutic action on the experimental autoimmune myasthenia gravis (EAMG) in rabbits. EAMG is an autoimmune disease induced by immunization with the purified acetylcholine receptor protein (AChR) and is considered to be a good model for the human disease myasthenia gravis. Simultaneous immunization with AChR and EL completely prevented the onset of myasthenic symptoms. This preventive effect was accompanied by a decrease in the recognition of AChR by anti-AChR antibodies. The administration of EL to myasthenic rabbits led, in most cases, to a complete recovery which was not accompanied by any significant change in the level of circulating anti-AChR antibodies. No evidence for an action of EL at the muscular level could be obtained. EL, however, was found to bind to rabbit lymphocytes and to stimulate their mitosis. These results suggest that EL produces its effects on EAMG by acting at the level of the immune system. It is proposed that EL may play a role in the immunological regulation of the response to self-antigen, which could be one of the biological functions of this animal lectin.     

Treatment of passively transferred experimental autoimmune myasthenia gravis using papain

Antibody-mediated acetylcholine receptor (AChR) loss at the neuromuscular junction, the main cause of the symptoms of myasthenia gravis, is induced by bivalent or multivalent antibodies. Passive transfer of experimental autoimmune myasthenia gravis (EAMG) can be induced very efficiently in rats by administration of intact MoAbs directed against the main immunogenic region (MIR) of the AChR, but not by their monovalent Fab fragments. We tested whether papain, which has been used therapeutically in autoimmune and other diseases, is capable of preventing EAMG by in vivo cleavage of the circulating anti-AChR antibodies into Fab fragments. EAMG was induced in 4-week-old female Lewis rats by i.p. injection of anti-MIR mAb35. A total of 0.75 mg of papain was given as one or three injections 3-7 h after MoAb injection. The mAb35 + papain-treated animals developed mild weakness during the first 30 h and subsequently recovered, while all animals that received only mAb35 developed severe myasthenic symptoms and died within 24-30 h. Animals treated only with papain showed no apparent side effects for up to 2 months. Serum anti-AChR levels in mAb35 + papain-treated rats decreased within a few hours, whereas in non-papain-treated rats they remained high for at least 30 h. Muscle AChR in mAb35 + papain-treated animals was partially protected from antibody-mediated degradation. These results show that treatment of rats with papain can prevent passively transferred EAMG without any apparent harm to the animals, and suggest a potential therapeutic use for proteolytic enzymes in myasthenia gravis.     

Author Index to Volume 32

Myasthenia gravis is a spontaneously occurring autoimmune disease in which antibodies and lymphocytes are specifically reactive with nicotinic ACh receptors of skeletal muscle. Antibodies reactive with junctional receptors of human muscle are found in 90% of patients with myasthenia gravis and not at all in other diseases. Their capacity to cross the placenta suggests their involvement in the pathogenesis of neonatal myasthenia. The role of the thymus in myasthenia gravis remains a mystery, but it has recently been established that the thymus contains nicotinic ACh receptors and that anti-receptor antibodies are present in myasthenic thymuses.

Antibodies of myasthenic patients detect only partial cross reactivity between ACh receptors of different species. However, greater antibody binding is observed with receptors isolated from denervated rat muscle than with receptors from normal rat muscle. This suggests that extrajunctional and junctional ACh receptors might express different antigenic determinants. Although human antibodies bind minimally to ACh receptors of the electric organs of eels and marine rays, lymphocyte reactivity to electric eel receptors is found in high incidence in myasthenic patients. This suggests that electric organ and mammalian muscle ACh receptors may share more lymphocyte-defined than serologically-defined antigenic determinants.

Both cellular and Numeral immune responses to ACh receptors can be induced experimentally. Sufficient antigenic homology exists between receptors of different species that electric organ receptors are capable of inducing in mammals experimental autoimmune myasthenia gravis. Syn-geneic muscle receptor also is immunogenic in rats. Induction of both myasthenia and antibodies to ACh receptor requires participation of thymus-derived lymphocytes. The majority of ACh receptors in myasthenic rat muscle exist complexed with antibody, but antibody is not bound directly to the receptor's ACh-binding site. Anti-receptor antibodies in vitro are capable of impairing the electrophysiological function of ACh receptors with minimal blocking of the ACh-binding site and in the absence of complement. Thus, myasthenia gravis and its experimental model provide unique biological tools for studying the structure, function and pathology of cell membrane receptors.     

Pathogenic autoimmunity to affinity-purified mouse acetylcholine receptor induced without adjuvant in BALB/c mice

Myasthenia gravis (MG) and experimental autoimmune myasthenia gravis (EAMG) are antibody-mediated disorders in which anti-acetylcholine receptor (anti-AChR) antibodies cause loss of muscle AChR and subsequent weakness. Many species are susceptible to induction of EAMG with purified xenogeneic AChR in adjuvant, but injection of Torpedo AChR without adjuvants can also induce evidence of EAMG. To see whether pathogenic autoimmunity could be induced in mice by isolated mouse AChR we injected BALB/c mice with several doses (1 pmole; about 0.1 ug) of affinity-purified AChR (from the BC3H1 cell line but thought to be identical with denervated mouse muscle) intraperitoneally, without adjuvant, over a period of 10-22 weeks. Some of the mice became ill and died. High levels of serum anti-mouse AChR, directed mainly towards the main immunogenic region, were found and, in the survivors, correlated with loss of muscle AChR. Thus BALB/c mice can mount an autoimmune response to minute amounts of mouse AChR, without the use of adjuvants, and this response is very similar to that found in MG. This novel finding has implications regarding the etiology of the human disease.     

Myasthenia gravis with thymoma is not associated with an increased incidence of non-muscle autoimmune disorders.

Three groups of thymectomized patients with myasthenia gravis (MG) were selected for study, 16 with thymoma, 16 with thymic atrophy and 32 with follicular hyperplasia of the thymus. All 16 patients with thymoma, 15/16 with thymus atrophy and 30/32 with follicular hyperplasia had AChR antibodies. Non-receptor muscle (CA) antibodies were found in sera of 15/16 patients with thymoma, 3/16 with thymus atrophy and in none of the sera from patients with follicular hyperplasia. There were 2 patients with thymoma and polymyositis, but none of the thymoma patients had rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) or other autoimmune disorders. Among the 32 patients with follicular hyperplasia of the thymus were 2 with SLE, 2 with RA and 1 with juvenile diabetes mellitus. In this study, there was an increased incidence of non-muscle autoimmune disorders among MG patients with follicular hyperplasia of the thymus but not among MG patients with thymoma.     

Determinant spreading and immune responses to acetylcholine receptors in myasthenia gravis

Summary: In myasthenia gravis (MG), antibodies to the muscle acetylcholine receptor (AChR) cause muscle weakness. Experimental autoimmune myaschenia gravis (EAMG) can be induced by immunisation against purified AChR; the main immunogenic region (MIR) is a conformation-dependent site that includes α67-76, EAMG can also occur after immunisation against extracellular AChR sequences, but this probably involves intramolecular determinant spreading.
In MG patients, thymic hyperplasia and germinal centres are found in about 50%, and thymoma in 10–15%. The heterogeneous, high affinity, IgG anti-AChR antibodies appear to be end-products of germinal centre responses, and react mainly with the MIR or a site on fetal AChR; the latter contains a y subunit and is mainly expressed on myoid cells in the thymic medulla, T cells cloned against recombinant AChR subunits recognise principally two naturally processed epitopes: ɛ201 -219 derived from adult AChR which is expressed in muscle, and sometimes in thymic epithelium, and α 146–160, common to fetal and adult AChR. Since AChR is not normally co-expressed with class II, it is unclear how CD4* responses to AChR a and E subunits are initiated, and how and where these spread to induce antibodies against fetal AChR, Various possibilities, including upregulation of class II on muscle/myoid cells and involvement of CD8+ responses to AChR and other muscle antigens, are discussed.     

The effect of CD3-specific monoclonal antibody on treating experimental autoimmune myasthenia gravis

CD3-specific monoclonal antibody was the first one used for clinical practice in field of transplantation. Recently, renewed interests have elicited in its capacity to prevent autoimmune diabetes by inducing immune tolerance. In this study, we tested whether this antibody can also be used to treat another kind of autoimmune disease myasthenia gravis （MG） and explored the possible mechanisms. MG is caused by an autoimmune damage mediated by antibody- and complement-mediated destruction of AChR at the neuromuscular junction. We found that administration of CD3-specific antibody （Fab）2 to an animal model with experimental autoimmune myasthenia gravis （EAMG） （B6 mice received 3 times of AChR/CFA immunization） could not significantly improve the clinical signs and clinical score. When the possible mechanisms were tested, we found that CD3 antibody treatment slightly down-regulated the T-cell response to AChR, modestly up-regulation the muscle strength. And no significant difference in the titers of IgG2b was found between CD3 antibody treated and control groups. These data indicated that CD3-specific antibody was not suitable for treating MG, an antibody- and complementmediated autoimmune disease, after this disease has been established. The role of CD3-specific antibody in treating this kind of disease remains to be determined. Cellular ＆ Molecular Immunology. 2005;2（6）：461-465.     

An immunodominant site of acetylcholine receptor in experimental myasthenia mapped with T lymphocyte clones and synthetic peptides

Myasthenia gravis (MG) is an autoimmune disease of man caused by antibodies directed against the acetylcholine receptor (AChR). In the experimental model of MG in mice, murine experimental autoimmune myasthenia gravis (EAMG), an anti-AChR immune response is induced by immunization with Torpedo AChR, and anti-AChR antibodies. AChR-sensitized T cells, and neuromuscular dysfunction result. The production of antibodies to AChR is thymus-dependent. In order to define the epitopes of the AChR identified by AChR-specific T cells, we generated T cell populations and T cell hybridoma clones and tested their reactivity to synthetic uniform-sized overlapping peptides representing the entire extracellular portion of the alpha-chain of the AChR. The predominant reactivity of the T cell clones and the parent lines was to a peptide corresponding to residues 146-162 of Torpedo AChR. This data is consistent with a highly limited recognition of AChR determinants in murine EAMG by AChR-specific T cells.     

'Split tolerance' induction by intrathymic injection of acetylcholine receptor in a rat model of autoimmune myasthenia gravis; implications for the design of specific immunotherapies.

Experimental autoimmune myasthenia gravis (EAMG) in the Lewis rat, induced by a single injection of acetylcholine receptor (AChR) protein, is a model used to study human myasthenia gravis (MG). The production of anti-AChR antibodies in the animal model and human MG is T cell-dependent, and AChR-specific T cells have been considered as a potential target for specific immunotherapy. Intrathymic injection of antigens induces antigen-specific tolerance in several T cell-mediated autoimmune models. We examined the effect of intrathymic injection of AChR on T cell responses and the production of antibodies to AChR in EAMG rats. Primed lymph node cells from rats receiving intrathymic injection of AChR exhibited reduced proliferation to AChR with marked suppression of interferon-gamma (IFN-gamma) secretion in the antigen-stimulated culture, compared with those of rats injected with PBS. However, neither anti-Narke AChR nor anti-rat AChR antibody production was suppressed or enhanced in intrathymically AChR-injected animals compared with that of animals injected intrathymically with PBS or perithymically with AChR. This 'split tolerance' may be attributable to the suppression of type-1 T helper cells (Th1). Our results suggest that the suppression of Th1 function alone may not be sufficient for the prevention of antibody-mediated autoimmune diseases.     

B cells produce less IL-10, IL-6 and TNF-α in myasthenia gravis

Abstract

B cells from myasthenia gravis (MG) patients with autoantibodies (Aab) against acetylcholine receptor (AChR), muscle-specific kinase (MuSK) or with no detectable Aab were investigated as cytokine producing cells in this study. B cells were evaluated for memory phenotypes and expressions of IL-10, IL-6 and IL-12A. Induced productions of IL-10, IL-6, IL-12p40, TNF-α and LT from isolated B cells in vitro were measured by immunoassays. MG patients receiving immunosuppressive treatment had higher proportions of memory B cells compared with healthy controls and untreated patients. With CD40 stimulation MG patients produced significantly lower levels of IL-10, IL-6. With CD40 and B cell receptor stimulation of B cells, TNF-α production also decreased in addition to these cytokines. The lower levels of these cytokine productions were not related to treatment. Our results confirm a disturbance of B cell subpopulations in MG subgroups on immunosuppressive treatment. B cell derived IL-10, IL-6 and TNF-α are down-regulated in MG, irrespective of different antibody productions. Ineffective cytokine production by B cells may be a susceptibility factor in dysregulation of autoimmune Aab production.     

Animal models of myasthenia gravis

Myasthenia gravis (MG) is an antibody-mediated, autoimmune neuromuscular disease. Animal models of experimental autoimmune myasthenia gravis (EAMG) can be induced in vertebrates by immunization with Torpedo californica acetylcholine receptors (AChR) in complete Freund's adjuvant. The MHC class II genes influence the cellular and humoral immune response to AChR and are involved in the development of clinical EAMG in mice. A dominant epitope within the AChR alpha146-162 region activates MHC class II-restricted CD4 cells and is involved in the production of pathogenic anti-AChR antibodies by B cells. Neonatal or adult tolerance to this T-cell epitope could prevent EAMG. During an immune response to AChR in vivo, multiple TCR genes are used. The CD28-B7 and CD40L-CD40 interaction is required during the primary immune response to AChR. However, CTLA-4 blockade augmented T- and B-cell immune response to AChR and disease. Cytokines IFN-gamma and IL-12 upregulate, while IFN-alpha downregulates, EAMG pathogenesis. However, the Th2 cytokine IL-4 fails to play a significant role in the development of antibody-mediated EAMG. Systemic or mucosal tolerance to AChR or its dominant peptide(s) has prevented EAMG in an antigen-specific manner. Antigen-specific tolerance and downregulation of pathogenic cytokines could achieve effective therapy of EAMG and probably MG.     

Experimental autoimmune myasthenia gravis induction in B cell-deficient mice

Experimental autoimmune myasthenia gravis (EAMG) is an animal model for human myasthenia gravis (MG). Autoantibody-induced functional loss of nicotinic acetylcholine receptor (AChR) at the postsynaptic membrane is an important pathogenic feature of both MG and EAMG. To evaluate the extent at which the humoral immune response against AChR operates in the pathogenesis of EAMG, we immunized B cell knockout (muMT) and wild- type C57BL/6 mice with AChR and complete Freund's adjuvant. The ability of AChR-primed lymph node cells to proliferate and secrete IFN-gamma in response to AChR and its dominant peptide alpha146-162 were intact in muMT mice as in wild-type mice. Similar amounts of mRNA for IFN-gamma, IL-4 and IL-10 in AChR-reactive lymph node cells were detected in muMT and wild-type mice. However, muMT mice had no detectable anti-AChR antibodies and remained completely free from clinical EAMG. We conclude that B cells are critically required for the genesis of clinical EAMG, but not for AChR-specific T cell priming.