Effect of complement and its regulation on myasthenia gravis pathogenesis

Myasthenia gravis (MG) is primarily caused by antibodies directed towards the skeletal muscle acetylcholine receptor, leading to muscle weakness. Although these antibodies may induce compromise of neuromuscular transmission by blocking acetylcholine receptor function or antigenic modulation, the predominant mechanism of injury to the neuromuscular junction is complement-mediated lysis of the postsynaptic membrane. The vast majority of data to support the role of complement derives from experimentally acquired MG (EAMG). In this article, we review studies that demonstrate the central role of complement in EAMG and MG pathogenesis along with the emerging role of complement in T- and B-cell function, as well as the potential for complement inhibitor-based therapy to treat human MG.     

Antibody effector mechanisms in myasthenia gravis—Pathogenesis at the neuromuscular junction

Myasthenia gravis (MG) is an autoimmune disorder caused by autoantibodies that are either directed to the muscle nicotinic acetylcholine receptor (AChR) or to the muscle-specific tyrosine kinase (MuSK). These autoantibodies define two distinct subforms of the disease—AChR-MG and MuSK-MG. Both AChR and MuSK are expressed on the postsynaptic membrane of the neuromuscular junction (NMJ), which is a highly specialized region of the muscle dedicated to receive and process signals from the motor nerve. Autoantibody binding to proteins of the postsynaptic membrane leads to impaired neuromuscular transmission and muscle weakness. Pro-inflammatory antibodies of the human IgG1 and IgG3 subclass modulate the AChR, cause complement activation, and attract lymphocytes; together acting to decrease levels of the AChR and AChR-associated proteins and to reduce postsynaptic folding. In patients with anti-MuSK antibodies, there is no evidence of loss of junctional folds and no apparent loss of AChR density. Anti-MuSK antibodies are predominantly of the IgG4 isotype, which functionally differs from other IgG subclasses in its anti-inflammatory activity. Moreover, IgG4 undergoes a posttranslational modification termed Fab arm exchange that prevents cross-linking of antigens. These findings suggest that MuSK-MG may be different in etiological and pathological mechanisms from AChR-MG. The effector functions of IgG subclasses on synapse structure and function are discussed in this review.     

Acetylcholine Receptor-Induced Experimental Myasthenia Gravis: What Have We Learned from Animal Models After Three Decades?

Myasthenia gravis (MG) is an autoimmune disease caused by an immunological response against the acetylcholine receptor (AChR) at the neuromuscular junction. Anti-AChR antibodies induce degradation of the receptor, activation of complement cascade and destruction of the post-synaptic membrane, resulting in a functional reduction of AChR availability. The pathophysiological role of autoantibodies (auto-Abs) and T helper lymphocytes has been studied in the experimental autoimmune MG (EAMG) models. EAMG models have been employed to investigate the factors involved in the development of MG and to suggest new therapies aimed to preventing or modulating the ongoing disease. EAMG can be induced in susceptible mouse and rat strains, which develop clinical symptoms such as muscular weakness and fatigability, mimicking the human disease. Two major types of EAMG can be induced, passive and active EAMG. Passive transfer MG models, involving the injection of auto-Abs, are helpful for studying the role of complement molecules and their regulatory proteins, which can prevent neuromuscular junction degradation. Active models, induced by immunization, are employed for the analysis of antigen-specific immune responses and their modulation in order to improve disease progression. In this review, we will concentrate on the main pathogenic mechanisms of MG, focusing on recent findings on EAMG experimental models.     

Overexpression of rapsyn in rat muscle increases acetylcholine receptor levels in chronic experimental autoimmune myasthenia gravis

The primary autoantigen in myasthenia gravis, the acetylcholine receptor (AChR), is clustered and anchored in the postsynaptic membrane of the neuromuscular junction by rapsyn. Previously, we found that overexpression of rapsyn by cDNA transfection protects AChRs in rat muscles from antibody-mediated loss in passive transfer experimental autoimmune myasthenia gravis (EAMG). Here, we determined whether rapsyn overexpression can reduce or even reverse AChR loss in muscles that are already damaged by chronic EAMG, which mimics the human disease. Active immunization against purified AChR was performed in female Lewis rats. Rapsyn overexpression resulted in an increase in total muscle membrane AChR levels, with some AChR at neuromuscular junctions but much of it in extrasynaptic membrane regions. At the ultrastructural level, most endplates in rapsyn-treated chronic EAMG muscles showed increased damage to the postsynaptic membrane. Although rapsyn overexpression stabilized AChRs in intact or mildly damaged endplates, the rapsyn-induced increase of membrane AChR enhanced autoantibody binding and membrane damage in severe ongoing disease. Thus, these results show the complexity of synaptic stabilization of AChR during the autoantibody attack. They also indicate that the expression of receptor-associated proteins may determine the severity of autoimmune diseases caused by anti-receptor antibodies.     

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.      

Complement and cytokine based therapeutic strategies in myasthenia gravis

Myasthenia gravis (MG) is a T cell–dependent and antibody-mediated disease in which the target antigen is the skeletal muscle acetylcholine receptor (AChR). In the last few decades, several immunological factors involved in MG pathogenesis have been discovered mostly by studies utilizing the experimental autoimmune myasthenia gravis (EAMG) model. Nevertheless, MG patients are still treated with non-specific global immunosuppression that is associated with severe chronic side effects. Due to the high heterogeneity of AChR epitopes and antibody responses involved in MG pathogenesis, the specific treatment of MG symptoms have to be achieved by inhibiting the complement factors and cytokines involved in anti-AChR immunity. EAMG studies have clearly shown that inhibition of the classical and common complement pathways effectively and specifically diminish the neuromuscular junction destruction induced by anti-AChR antibodies. The inborn or acquired deficiencies of IL-6, TNF-α and TNF receptor functions are associated with the lowest EAMG incidences. Th17-type immunity has recently emerged as an important contributor of EAMG pathogenesis. Overall, these results suggest that inhibition of the complement cascade and the cytokine networks alone or in combination might aid in development of future treatment models that would reduce MG symptoms with highest efficacy and lowest side effect profile.     

Complement membrane attack is required for endplate damage and clinical disease in passive experimental myasthenia gravis in Lewis rats

Myasthenia gravis (MG) is a debilitating and potentially fatal neuromuscular disease characterized by the generation of autoantibodies reactive with nicotinic acetylcholine receptors (AChR) that cause loss of AChR from the neuromuscular endplate with resultant failure of neuromuscular transmission. A role for complement (C) in the pathology of human MG has been suggested based upon identification of C activation products in plasma and deposited at the endplate in MG. In the rat model, experimental autoimmune MG (EAMG), C depletion or inhibition restricts clinical disease, further implicating C in pathology. The mechanisms by which C activation drives pathology in MG and EAMG are unclear. Here we provide further evidence implicating C and specifically the membrane attack complex (MAC) in the Lewis rat passive EAMG model of MG. Rats deficient in C6, an essential component of the MAC, were resistant to disease induction and endplate destruction was reduced markedly compared to C6-sufficient controls. After reconstitution with C6, disease severity and endplate destruction in the C6-deficient rats was equivalent to that in controls. The data confirm the essential role of the MAC in the destruction of the endplate in EAMG and raise the prospect of specific MAC inhibition as an alternative therapy in MG patients resistant to conventional treatments.     

Experimental Autoimmune Myasthenia Gravis (EAMG): From immunochemical characterization to therapeutic approaches

Myasthenia Gravis (MG) is an organ-specific autoimmune disease. In high percentage of patients there are autoantibodies to the nicotinic acetylcholine receptor (AChR) that attack AChR on muscle cells at the neuromuscular junction, resulting in muscle weakness. Experimental Autoimmune Myasthenia Gravis (EAMG) is an experimental model disease for MG. EAMG is induced in several animal species by immunization with acetylcholine receptor (AChR), usually isolated from the electric organ of electric fish, which is a rich source for this antigen. Our lab has been involved for several decades in research of AChR and of EAMG. The availability of an experimental autoimmune disease that mimics in many aspects the human disease, provides an excellent model system for elucidating the immunological nature and origin of MG, for studying various existing treatment modalities and for attempting the development of novel treatment approaches. In this review in honor of Michael Sela and Ruth Arnon, we report first on our early pioneering contributions to research on EAMG. These include the induction of EAMG in several animal species, early attempts for antigen-specific treatment for EAMG, elicitation and characterization of monoclonal antibodies and anti-idiotypic antibodies, measuring humoral and cellular AChR-specific immune responses in MG patient and more. In the second part of the review we discuss more recent studies from our lab towards developing and testing novel treatment approaches for myasthenia. These include antigen-dependent treatments aimed at specifically abrogating the humoral and cellular anti-AChR responses, as well as immunomodulatory approaches that could be used either alone, or in conjunction with antigen-specific treatments, or alternatively, serve as steroid-sparing agents.     

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.     

Dendritic cells exposed in vitro to TGF-beta1 ameliorate experimental autoimmune myasthenia gravis

Experimental autoimmune myasthenia gravis (EAMG) is an animal model for human myasthenia gravis (MG), characterized by an autoaggressive T-cell-dependent antibody-mediated immune response directed against the acetylcholine receptor (AChR) of the neuromuscular junction. Dendritic cells (DC) are unique antigen-presenting cells which control T- and B-cell functions and induce immunity or tolerance. Here, we demonstrate that DC exposed to TGF-beta1 in vitro mediate protection against EAMG. Freshly prepared DC from spleen of healthy rats were exposed to TGF-beta1 in vitro for 48 h, and administered subcutaneously to Lewis rats (2 x 10(6)DC/rat) on day 5 post immunization with AChR in Freund's complete adjuvant. Control EAMG rats were injected in parallel with untreated DC (naive DC) or PBS. Lewis rats receiving TGF-beta1-exposed DC developed very mild symptoms of EAMG without loss of body weight compared with control EAMG rats receiving naive DC or PBS. This effect of TGF-beta1-exposed DC was associated with augmented spontaneous and AChR-induced proliferation, IFN-gamma and NO production, and decreased levels of anti-AChR antibody-secreting cells. Autologous DC exposed in vitro to TGF-beta1 could represent a new opportunity for DC-based immunotherapy of antibody-mediated autoimmune diseases.     

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.     

Role for interferon-gamma in rat strains with different susceptibility to experimental autoimmune myasthenia gravis

Experimental autoimmune myasthenia gravis (EAMG) is caused by autoantibodies against the nicotinic acetylcholine receptor (AChR) at the neuromuscular postsynaptic membrane and represents an animal model of myasthenia gravis in human. Recent studies highlighted the roles of TH1 cytokines (IFN-gamma, IL-12), rather than TH2 cytokines (IL-4), in the pathogenesis of EAMG by using homozygous (-/-) knockout mice with an EAMG-susceptible genetic background. To further evaluate a role for IFN-gamma, we injected recombinant rat IFN-gamma (rrIFN-gamma) at the time of immunization with AChR in complete Freund's adjuvant to EAMG-susceptible Lewis rats and EAMG-resistant Wistar Furth (WF) rats. RrIFN-gamma enhanced Lewis rat EAMG. The exacerbated muscular weakness was associated with higher levels of anti-AChR IgG and enhanced TNF-alpha responses. Anti-AChR IgG antibody levels were augmented to a similar extent as in Lewis rats, however, the identical immunization and IFN-gamma injection induced only mild and transient EAMG in WF rats due to the default TH3 phenotype development and inherent low TH1 responses. We conclude that IFN-gamma plays a major role in the pathogenesis of EAMG in the Lewis rat, but fails to break disease resistance in the WF rat.     

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.     

Recombinant IgG2a Fc (M045) multimers effectively suppress experimental autoimmune myasthenia gravis

Myasthenia gravis (MG) is an autoimmune disorder caused by target-specific pathogenic antibodies directed toward postsynaptic neuromuscular junction (NMJ) proteins, most commonly the skeletal muscle nicotinic acetylcholine receptor (AChR). In MG, high-affinity anti-AChR Abs binding to the NMJ lead to loss of functional AChRs, culminating in neuromuscular transmission failure and myasthenic symptoms. Intravenous immune globulin (IVIg) has broad therapeutic application in the treatment of a range of autoimmune diseases, including MG, although its mechanism of action is not clear. Recently, the anti-inflammatory and anti-autoimmune activities of IVIg have been attributed to the IgG Fc domains. Soluble immune aggregates bearing intact Fc fragments have been shown to be effective treatment for a number of autoimmune disorders in mice, and fully recombinant multimeric Fc molecules have been shown to be effective in treating collagen-induced arthritis, murine immune thrombocytopenic purpura, and experimental inflammatory neuritis. In this study, a murine model of MG (EAMG) was used to study the effectiveness of this novel recombinant polyvalent IgG2a Fc (M045) in treating established myasthenia, with a direct comparison to treatment with IVIg. M045 treatment had profound effects on the clinical course of EAMG, accompanied by down-modulation of pathogenic antibody responses. These effects were associated with reduced B cell activation and T cell proliferative responses to AChR, an expansion in the population of FoxP3⁺ regulatory T cells, and enhanced production of suppressive cytokines, such as IL-10. Treatment was at least as effective as IVIg in suppressing EAMG, even at doses 25–30 fold lower. Multimeric Fc molecules offer the advantages of being recombinant, homogenous, available in unlimited quantity, free of risk from infection and effective at significantly reduced protein loads, and may represent a viable therapeutic alternative to polyclonal IVIg.     

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.     

重症肌无力的免疫耐受治疗

重症肌无力（MG）是累及神经一肌肉接头突触后膜上乙酰胆碱受体（AChR）的自身免疫性疾病,其发病的关键在于自我耐受的破坏,导致自身AChR出现异常的细胞免疫应答和体液免疫应答。这种抗AChR抗体的产生是T细胞依赖性的,故AChR特异性的T细胞就是MG特异性治疗的潜在靶位。随着免疫学的发展,MG免疫耐受治疗有可能会特异性地和有效地防止或阻断MG的发生与进展。     

Treatment of experimental myasthenia gravis with cyclosporin A

Cyclosporin A (CsA) is an immunosuppressive agent that has recently been used to prevent rejection of transplanted tissues. The effects of CsA treatment of rats with experimental autoimmune myasthenia gravis (EAMG), an antibody-mediated autoimmune disorder of acetylcholine receptors (AChRs) at neuromuscular junctions, have been studied. CsA treatment at the time of primary immunization suppressed the antibody responses to AChR virtually completely. Following 12 weeks of CsA, the AChR-immunized rats responded like naive controls to a further challenge of AChR. Treatment of ongoing EAMG resulted in a reduction of AChR antibody by more than 50%. The secondary response to a challenge of AChR was prevented by CsA treatment, but a very large challenge dose in adjuvant partially overwhelmed the effect of CsA. CsA treatment also prevented the loss of AChRs at neuromuscular junctions, as compared with untreated EAMG controls (P less than 0.02). The efficacy of CsA in suppressing ongoing and secondary hetero- and autoimmune responses against AChR in EAMG encourages its ultimate application in autoimmune diseases of man, such as MG. Its usefulness will depend on the ability to determine effective doses of CsA that are well tolerated.     

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。     

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.     

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.