Myasthenia gravis as a prototype autoimmune receptor disease

Myasthenia gravis (MG) is an organ-specific autoimmune disease in which autoantibodies against nicotinic acetylcholine receptors (AChR) at the postsynaptic membrane cause loss of functional AChR and disturbed neuromuscular transmission. The immunopathogenic mechanisms responsible for loss of functional AChR include antigenic modulation by anti-AChR antibodies, complement-mediated focal lysis of the postsynaptic membrane, and direct interference with binding of acetylcholine to the AChR or with ion channel function. The loss of AChR and subsequent defective neuromuscular transmission is accompanied by increased expression of the different AChR subunit genes, suggesting a role for the target organ itself in determining susceptibility and severity of disease. Experimental autoimmune myasthenia gravis (EAMG) is an animal model for the disease MG, and is very suitable to study the immunopathogenic mechanisms leading to AChR loss and the response of the AChR to this attack. In this article the current concepts of the structure and function of the AChR and the immunopathological mechanisms in MG and EAMG are reviewed.     

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.     

The role of muscle-specific tyrosine kinase (MuSK) and mystery of MuSK myasthenia gravis

MuSK myasthenia gravis is a rare, severe autoimmune disease of the neuromuscular junction, only identified in 2001, with unclear pathogenic mechanisms. In this review we describe the clinical aspects that distinguish MuSK MG from AChR MG, review what is known about the role of MuSK in the development and function of the neuromuscular junction, and discuss the data that address how the antibodies to MuSK lead to neuromuscular transmission failure.     

大鼠经鼻腔乙酰胆碱受体耐受诱导的试验研究

用乙酰胆碱受体（ＡＣｈＲ）加完全弗氏佐剂（ＣＦＡ）免疫大鼠前，鼻腔给予ＡＣｈＲ可有效地预防实验性自身免疫性重症肌无力（ＥＡＭＣ）。本文结果表明，与非耐受对照组比，鼻腔ＡＣｈＲ耐受组大鼠免疫后，国窝、腹股沟淋巴结（ＰＩＬＮ）的单个核细胞（ＭＮＣ）ＡＣｈＲ特异的淋巴细胞增殖反应受到明显抑　制，鼻腔耐受组ＰＩＬＮ中ＡＣｈＲ特异的ＣＤ４~＋／ＣＤ８~－克隆增生也明显受抑制，差异均有显著性。此外，鼻腔耐受组ＡＣｈＲ特异性皮肤迟发型过敏反应（ＤＴＨ）也显著降低。提示鼻腔ＡＣｈＲ耐受后引起的特异性Ｔ淋巴细胞反应的低调可能是临床肌无力抑制的基础。     

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.     

Acetylcholine receptor antibody characteristics in myasthenia gravis. III. Patients with low anti-AChR antibody levels.

Twenty-five myasthenia gravis (MG) patients whose anti-acetylcholine receptor (anti-AChR) titres against denervated human leg AChR fell in the range of less than 0.1-2.0 nM were tested against AChR preparations from normal human leg muscle, and from human extra-ocular muscle, and the results compared with those of sera with titres greater than 2.0 nM. Seven sera failed to react appreciably with any of the AChR. Many of the remaining 18 low titre sera reacted better with normal leg and ocular AChR than with denervated AChR. In contrast, sera from patients with high anti-AChR titres generally reacted better with denervated, AChR, and as a group this was significantly different from the low titre patients. Sera reacting better with ocular AChR are not restricted to patients with purely ocular symptoms. The results indicate a subgroup of myasthenia gravis patients who have low titres, tend to be male and have relatively mild disease. The aetiology of the autoimmune disorder in this subgroup may differ from that in other MG patients.     

T细胞共刺激分子在重症肌无力发病机制中的作用

重症肌无力（MG）是由乙酰胆碱受体抗体介导的细胞免疫依赖性和补体参与的神经肌接头部位的自身免疫性疾病,免疫应答异常是其发病本质。近年研究发现CD28：CTLA4／87和CD154／CD40是最重要的共刺激分子,对T和B淋巴细胞的增殖、活化、抗体和细胞因子的分泌均具有重要作用,其对重症肌无力的发生和发展也具有十分关键的作用。     

The thymus in myasthenia gravis. Changes typical for the human disease are absent in experimental autoimmune myasthenia gravis of the Lewis rat.

In human myasthenia gravis (MG) formation of autoantibodies against acetylcholine receptor (AChR) is commonly associated with thymic changes termed lymphofollicular hyperplasia (LFH). To learn whether the thymic lesions of human MG are primary changes in the autoimmune pathogenesis, or rather secondary events caused by peripheral autoimmunization, the authors compared the pathologic changes of MG thymuses with the thymuses of Lewis rats with experimental autoimmune myasthenia gravis (EAMG). EAMG was induced either actively by immunization with AChR, or transferred passively with monoclonal antibodies (mAb) binding to AChR. The clinical diagnosis of EAMG was confirmed by electromyography. Germinal centers, which are typical for human MG thymuses, were not detectable in the thymus of EAMG rats. Scattered B cells were seen as normal components of the thymic medulla. In EAMG their number was not augmented, nor were they accumulated focally. The perivascular spaces (PVS) were not distended and the amount of reticulin was not increased. Thymic myoid cells were identified in EAMG as well as in control thymuses; their cellular microenvironment was inconspicuous. Both in normal and in EAMG thymuses, a subpopulation of myoid cells expressed the main immunogenic region of the AChR. Heavily affected rats showed a severe cortical involution, but no specific changes of the medulla. The fact that none of the thymic lesions characteristic for human MG was found in EAMG is compatible with the concept that the thymic changes in MG are primary events in the autoimmune pathogenesis of this disease.     

Development of myasthenia gravis after interferon alpha therapy

Interferon (IFN) alpha is now used in the treatment of some malignant diseases and chronic viral hepatitis. There have been several reports of development of autoantibodies and autoimmune diseases or the deterioration of preexisting disorders in patients under treatment. We enclose a case of myasthenia gravis (MG) which developed after six weeks of treatment as fluctuating bilateral ptosis, intermittent diplopia, and mild weakness of limb and neck muscles. A test dose of edrophonium chloride was administered, resulting in improved muscle strength. Elevated anti acetylcholine receptor (AChR) antibody titer was found. Single fiber electromyography showed an increased jitter from extensor digitorum communis, frequently accompanied by transmission blocking. Repetitive electric 3 Hz stimulation of the abductor pollicis brevis muscle, revealed an abnormal decrement of 28% in compound motor action potential. Myasthenia gravis was diagnosed and the patient was given pyridostigmine, immunoglobulines and prednisone with benefit. Six months latter he developed an acute myasthenic crisis with severe respiratory failure and high anti AChR antibody titer. IFN-alpha can induce MG or simply manifests a preexisting subclinical disease, but otherwise its therapeutic efficacy in MG has been shown in experimental and clinical studies. Autoimmune mechanisms, as the release of different cytokines as IFN, by immunocompetent cells, may be involved in the pathogenesis of both MG and chronic active hepatitis. Autoantibody production against postsynaptic membrane structures by IFN-alpha could be the underlying pathophysiology.     

Monoclonal Antibodies to Acetylcholine Receptor Secreted by Human X Human Hybridomas Derived from Lymphocytes of a Patient with Myasthenia Gravis

Peripheral blood lymphocytes of a patient with myasthenia gravis (MG) were fused to the non-secreting human lymphoblastoid line HuNSI to produce human x human hybridomas that secrete monoclonal antibodies (mAb) to acetylcholine receptor (AChR). Screening of hybridomas for antibody production involved an enzyme-linked immunosorbent (ELISA) assay with AChR from Torpedo californica (TAChR). 25 of 302 wells tested (8.3%) were positive for anti-AChR antibody production and have been stable in their secretion of mAb for eleven months. Nine lines have been studied in detail. All produced IgM mAb, and most had greater activity against membrane-bound TAChR, than against solubilized TAChR. For anti-AChR clones, the mAb concentration in culture supernatants ranged from 2 to 33 ug/ml. Saturation curves of binding to TAChR performed on 4 lines demonstrated dissociation constants (Kds) estimated to range from 0.1-1.0 nM. The patient whose lymphocytes were used in this study had a serum anti-AChR antibody concentration of 243nM against human AChR and 15nM against AChR from T. californica. The results demonstrate the feasibility of producing stable human x human hybridomas secreting mAb to the autoantigen from the peripheral blood of patients with organ-specific autoimmune diseases. The mAb produced here may prove to be useful in analyzing, and possibly treating, the autoimmune phenomena in MG.     

PATHOGENICITY AND SEQUENCE ANALYSIS OF A MOUSE MONOCLONAL ANTIBODY AGAINST HUMAN ACETYLCHOLINE RECEPTOR IN MYASTHENIA GRAVIS

通过对乙酰胆碱受体（ＡＣｈＲ）自身抗体分子结构以及与致病性关系的研究探讨重症肌无力（ＭＧ）及其动物模型——实验性自身免疫性重症肌无力（ＥＡＭＧ）的发病机理。ＡＣｈＲ抗体被动转移至大鼠后诱导出明显的ＥＡＭＧ。全身肌肉ＡＣｈＲ损失率和体重减轻率达４７．２±１５．３％和１３．４±２．２％。这株ＡＣｈＲ抗体的重链可变区基因由小鼠Ｑ５２胚系基因编码，其同源性为９４．８％，将这株抗体的重链和轻链可变区、尤其是互补决定区（ＣＤＲ）的核苷酸和氨基酸序列与其他致病性ＡＣｈＲ抗体比较发现，能诱导ＭＧ和ＥＡＭＧ的致病性ＡＣｈＲ抗体的结构并不是完全一致的。     

'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.     

Antibodies to muscle and ganglionic acetylcholine receptors (AchR) in celiac disease

Background: About 2.5% of patients with idiopathic peripheral neuropathy or idiopathic dysautonomia have underlying celiac disease (CD). Antibodies to ganglioside have been reported in CD patients with neuropathy. No data are so far available on the presence in CD of acetylcholine receptor (AChR) antibodies. Muscle AChR antibodies are found in patients with myasthenia gravis, and ganglionic AChR antibodies in patients with autoimmune autonomic neuropathy.

Objective: To determine the frequency of AChR antibodies in CD patients and assess possible correlations with neurological manifestations.

Methods: Seventy CD patients (16 M, 54 F, mean age 36 years) underwent neurological and electrophysiological evaluation. AChR antibodies were detected with radioimmunoprecipitation assay. Sera from 15 age-matched patients with systemic lupus erythematosus (SLE) and 10 with Sjogren syndrome were studied as controls.

Results: None of our CD patients complained of autonomic symptoms or fatigable weakness. Borderline titres (0.03–0.05 nmol/l) of ganglionic AChR antibodies were present in 4 patients, one affected with type I diabetes and one with subclinical neuropathy. Three of the 4 patients underwent cardiovascular autonomic function tests, which showed no abnormalities. Low levels of ganglionic AChR antibodies (0.05–0.10 nmol/l) were found in 2 SLE control patients, one of whom had a severe sicca complex. Muscle AChR antibodies (>1.0 nmol/l) were found in two CD patient and one control patient with SLE. Neither had symptoms or signs of myasthenia gravis.

Discussion and conclusions: CD is occasionally associated with neurologic disease, and with antibody reactivity to neuronal antigens. None of our CD patients had autonomic failure or significant levels of ganglionic AChR antibodies. Two CD patient and one control with SLE had muscle AChR antibodies without clinical evidence of myasthenia. The presence of antibodies in CD and in SLE patients may reflect a non-specific autoimmune response in these patients or may indicate subclinical autoimmune autonomic and neuromuscular involvement.     

Acetylcholine receptor antibody characteristics in myasthenia gravis. II. Patients with penicillamine-induced myasthenia or idiopathic myasthenia of recent onset.

Anti-acetylcholine receptor (anti-AChR) antibody characteristics including light chain, IgG subclass, avidity for denervated human acetylcholine receptor and reaction with various human and mammalian AChR preparations were examined in 11 patients who developed myasthenia during penicillamine treatment of rheumatoid arthritis. Results were compared with those already reported in 35 patients with generalized idiopathic myasthenia gravis (MG). We found significant differences in the avidity and the light chain of the anti-AChR. However, anti-AChR characteristics in 12 patients with recent onset (less than 4 months'' duration) idiopathic MG did not differ significantly from those in patients with penicillamine-induced MG. In the patients with generalized MG a trend was found towards higher percentage of kappa light chain and higher anti-AChR avidity with duration of disease. Anti-acetylcholine receptor antibodies in penicillamine-induced myasthenia gravis therefore appear to be similar to those of idiopathic myasthenia gravis of recent onset.     

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.     

T helper type 17 cells expand in patients with myasthenia-associated thymoma

Myasthenia gravis (MG) is a prototypical CD4(+) T cell-dependent autoimmune disease mediated by anti-acetylcholine receptor autoantibodies (AChR-Abs). Certain subsets of helper T cells are suggested to be involved in the pathogenesis of MG, including Th1 and regulatory T cells (Treg). However, whether the recently identified Th17 cells play a role in the development of MG and its prognosis is still unknown. Here, we demonstrated that Th17 cells and their associated cytokines are increased, while the Treg cells are decreased in the peripheral blood mononuclear cells (PBMCs) from MG patients with thymomas (TM), but not from those with normal thymus (NT) or thymic hyperplasia (TH). Furthermore, the quantity of Th17 cells correlates with the quantitative myasthenia gravis (QMG) score in patients with TM. We also found a significant positive relationship between the frequency of Th17 cells (%) and the concentration of AChR antibodies in patients with MG. Therefore, the Th17/Treg imbalance in TM may suggest MG with certain pathological subtype, and the increase in Th17 cells may reveal the severity of the disease, which is valuable in the diagnosis and choice of therapeutic strategy for patients with MG.     

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.     

Injection of inactive Bordetella pertussis and complete Freund’s adjuvant with Torpedo californica AChR increases the occurrence of experimental autoimmune myasthenia gravis in C57BL/6 mice

An animal model of myasthenia gravis (MG), termed experimental autoimmune MG (EAMG), is an important tool for investigations of disease mechanisms and/or methods of treatment for this disease. EAMG can be induced in C57BL/6 (B6, H-2^b) mice by 2–3 times injections at 4 weeks intervals with Torpedo californica (t) acetylcholine receptor (AChR) in complete Freund’s adjuvant (CFA). However, the protocol especially with a two-injection schedule occasionally produces a poor incidence of EAMG. We have investigated the efficacy of the additional adjuvant, inactive organisms of Bordetella pertussis (iBP), on the induction with a two-injection schedule. In a group immunized with tAChR in CFA?+?iBP, 76% of mice developed EAMG (average grade in exercise test, 1.02). Whereas, 46% of mice were found EAMG-positive (average grade, 0.73) in a group injected with tAChR/CFA alone. Thus, the combined use of CFA and iBP significantly increased both the occurrence and severity of clinical MG in the immunized mice. This was accompanied by higher antibody (Ab) and T-cell responses to tAChR. The effect on disease occurrence of the iBP use in a three-injection protocol was also described.     

Immunogenetics of experimental autoimmune myasthenia gravis

Myasthenia gravis (MG) is an autoimmune neuromuscular disease manifested by muscle weakness and fatiguability. The primary pathology in MG is antibody and complement-mediated destruction of muscle acetylcholine receptor (AChR). Like other autoimmune diseases, MG is associated with certain HLA antigens, particularly HLA-B8 and DR3 in Caucasians. Also, certain GM antigens and complotypes are associated with MG. Therefore, it is crucial to study the immunogenetic aspect of MG in animal models to evaluate disease etiopathogenesis and eventual strategy for specific therapy. In the introduction of this review article, I focus on the association of HLA and GM antigens in MG and emphasize the mouse model of experimental autoimmune myasthenia gravis (EAMG) as an ideal model to study the immunogenetic aspect of MG. The following sections deal with the role of (1) major histocompatibility complex (MHC), (2) immune response gene, (3) the IA molecule, (4) the Igl locus, (5) the complement genes, and (6) non-MHC genes on EAMG pathogenesis. The review concludes with future immunogenetic analysis and eventual strategy for specific therapy from an immunogeneticist's point of view.