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.     

'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.     

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.      

Modulation of anti-acetylcholine receptor antibody specificities and of experimental autoimmune myasthenia gravis by synthetic peptides.

Synthetic peptides corresponding to selected sequences from the nicotinic acetylcholine receptor (AChR) were employed to identify possible antigenic determinants within the receptor which can modulate the anti-AChR response and experimental autoimmune myasthenia gravis (EAMG). Immunization of rabbits with peptides T alpha 73-89, T alpha 351-368, T delta 354-367 and H alpha 351-368, prior to AChR inoculation, affected the course of EAMG in six out of eight rabbits. These six protected rabbits survived three inoculations of AChR and survived for at least five months after the third injection with AChR, whereas control rabbits died following one or two injections of AChR. The survival of peptide-preimmunized rabbits injected with AChR seemed to correlate with the antibody specificities in immunoblots. Following AChR inoculation there was a shift in reactivity, from a subunit-restricted response, to reactivity with all subunits of the receptor. This shift was delayed in protected rabbits. This may indicate that the reactivity with the entire Torpedo receptor molecule represents a loss of tolerance to AChR which culminates in the autoimmune disease, EAMG.     

Disruption of the IL-1beta gene diminishes acetylcholine receptor-induced immune responses in a murine model of myasthenia gravis

Human autoimmune myasthenia gravis (MG) is associated with the IL-1beta TaqI RFLP allele 2. Individuals positive for this allele have high levels of inducible IL-1beta in their peripheral blood. Here, we have characterized MG induction and the immune response elicited by Torpedo acetylcholine receptor (AChR) immunization in wild-type and IL-1beta deficient (-/-) mice. Compared with wild-type mice, IL-1beta-/- mice were relatively resistant to induction of clinical experimental autoimmune myasthenia gravis (EAMG). Draining lymph node cells from IL-1beta-/- mice showed poor proliferative capacity upon AChR stimulation in vitro. Both Th1 (IFN-gamma, IL-2) and Th2 (IL-4) cytokine responses were reduced and levels of serum anti-AChR antibodies decreased in IL-1beta-/- mice compared to wild-type mice. Taken together, these results reveal a critical role for IL-1beta in the induction of MG in mice, and support a role for IL-1beta in the pathogenesis of MG in man.     

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.     

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。     

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.     

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.     

Tumor necrosis factor receptor-1 is critically involved in the development of experimental autoimmune myasthenia gravis

Tumor necrosis factor receptor-1 (TNFR1, CD120a) has been implicated in the pathogenesis of several experimental models of T cell-mediated autoimmune disorders, but its role in antibody-mediated autoimmune diseases has not been addressed. Experimental autoimmune myasthenia gravis (EAMG), an autoantibody-mediated T cell-dependent neuromuscular disorder, represents an animal model for myasthenia gravis in human. To investigate the role of TNFR1 in the pathogenesis of EAMG, TNFR1(-/-) and wild-type mice were immunized with TORPEDO: acetylcholine receptor (AChR) in complete Freund's adjuvant. TNFR1(-/-) mice failed to develop EAMG. Lymphoid cells from TNFR1(-/-) mice produced low amounts of T(h)1 (IFN-gamma, IL-2 and IL-12)-type cytokines, but elevated levels of T(h)2 (IL-4 and IL-10)-type cytokines compared with lymphoid cells of wild-type mice. Accordingly, the levels of anti-AChR IgG2 antibodies were severely reduced and the level of anti-AChR IgG1 antibodies were moderately reduced. Co-injection of recombinant mouse IL-12 with AChR in adjuvant restored T cell responses to AChR and promoted development of EAMG in TNFR1(-/-) mice. These results demonstrate that the TNF/TNFR1 system is required for the development of EAMG. The lack of a functional TNF/TNFR1 system can, at least in part, be substituted by IL-12 at the stage of initial priming with AChR and adjuvant.     

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.     

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.     

Anti-TNF-alpha antibodies suppress the development of experimental autoimmune myasthenia gravis

To understand the role of TNF-alpha in the induction of experimental autoimmune myasthenia gravis (EAMG) and detect a possible effect of anti-TNF-alpha antibodies in the treatment of EAMG, anti-TNF-alpha antibodies were administrated intraperitoneally to Lewis rats twice per week for 5 weeks from the day of immunization with Torpedo AChR and complete Freund's adjuvant (CFA). Administration of anti-TNF-alpha antibodies resulted in lower incidence of EAMG, and in delayed onset and only mild muscle weakness compared with control EAMG rats. These mild clinical signs were accompanied by lower AChR-specific lymphocyte proliferation, down-regulated IFN-gamma and IL-10, and up-regulated TGF-beta. The lower levels of anti-AChR IgG, Ig2a and IgG2b and decreased anti-AChR IgG affinity were found in rats treated with anti-TNF-alpha antibodies. These results demonstrate that anti-TNF-alpha antibodies can suppress the induction and development of EAMG.     

Spectrotypic analysis of antibodies to acetylcholine receptors in experimental autoimmune myasthenia gravis

We have studied the isoelectric focusing pattern of antibodies expressed in rats with experimental autoimmune myasthenia gravis (EAMG) induced by immunization with acetylcholine receptors (AChR) purified from Torpedo californica. Sera or tissue eluates were obtained at intervals in the course of disease and subjected to isoelectric focusing. Subsequently, the focused antibodies were detected by autoradiography of gels labelled with 125I-alpha-bungarotoxin conjugated AChR. Reverse electrofocusing was used to separate complexes of antibody and AChR formed in vivo, thereby allowing detection of the full spectrotype (banding pattern). As little as 1.1 X 10(-12) moles of monoclonal antibodies (MoAbs) to AChR yielded distinct bands of radiolabelled antigen binding by this technique. The anti-AChR MoAbs studied showed a multitude of bands localized in neutral to alkaline position. The clonotypes expressed in late post-immunization sera were compared to early sera. The spectrotypes of immunized Lewis and Brown Norway rats were not identical. In early sera most of the isoelectric focusing bands were specific for T. californica AChR, whereas in late sera further expansion of the repertoire produced bands that reacted with rat muscle AChR as well. The focused bands that bound rat AChR also bound T. californica AChR. The anti-AChR antibodies eluted from muscles of rats with EAMG showed similar binding patterns to anti-receptor antibodies in rats' sera. These results indicate that the antibody specificities detected in serum are the same specificities which are effective in binding to muscle AChR in vivo. Minor specificities of serum anti-receptor antibodies are not disproportionally represented in the antibodies actually bound at the neuromuscular junction in EAMG.     

In vitro and in vivo actions of acetylcholine receptor educated suppressor T cell lines in murine experimental autoimmune myasthenia gravis.

Immunization of C57Bl/6 mice with Torpedo acetylcholine receptor (AChR) leads to EAMG, experimental autoimmune myasthenia gravis, with characteristic clinical, electrophysiological, and immune features. Present in the lymphoid organs of mice with EAMG are AChR specific suppressor T cells: these cells can be grown in vitro as T cell lines. These lines are able to suppress the in vitro response to AChR, and can suppress the in vivo development of EAMG.     

Experimental myasthenia gravis in congenic mice. Sequence mapping and H-2 restriction of T helper epitopes on the alpha subunits of Torpedo californica and murine acetylcholine receptors.

Immunization of mice with nicotinic acetylcholine receptor from Torpedo electric organ (TAChR) causes a disease similar to human myasthenia gravis (experimental autoimmune myasthenia gravis, EAMG). Susceptibility to EAMG correlates with the H-2 haplotype. In this study we used overlapping synthetic peptide corresponding to the complete sequences of the alpha subunits from TAChR and murine muscle AChR (MAChR) to map T helper epitopes in congenic murine strains of different H-2 haplotype. C57BL/6 and BALB/B mice (highly susceptible to EAMG) and BALB/c and CB17 mice (less susceptible to EAMG), immunized with TAChR, developed similar anti-TAChR antibody titers and L3T4+ (T helper) cell sensitization. Different sequence segments of the TAChR alpha subunit were recognized by L3T4+ cells from strains of H-2b and H-2d haplotype. The sequence segments recognized by the H-2d strains have the highest predicted propensity to form amphipatic alpha helices, while those recognized by the H-2b strains do not. We investigated whether in EAMG T helper cells cross-react with autologous AChR sequences, and a true break of the tolerance occurs. Overlapping synthetic peptides, corresponding to the complete sequence of MAChR alpha subunit, were used to test L3T4+ cell from mice immunized with TAChR. L3T4+ cell strains did not cross-react with any murine peptide sequence, while L3T4+ cells from H-2d mice were strongly stimulated by the peptide sequence Ma alpha 304-322, which is very similar to the homologous Torpedo peptide.     

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.     

Vaccination with a MHC Class II Peptide Attenuates Cellular and Humoral Responses against t AChR and Suppresses Clinical EAMG

An animal model of myasthenia gravis (MG), termed experimental autoimmune MG (EAMG), can be induced in C57BL/6 (B6, H-2 b ) mice by immunization with Torpedo californica acetylcholine receptor ( t AChR). We have investigated the effect of vaccination with MHC class II peptide I-A &#103 b 62-76 on clinical EAMG and on T cell and antibody (Ab) responses against t AChR. B6 mice were vaccinated with the peptide (25 &#119 g/mouse) four times prior to two injections with t AChR. The incidence of clinical EAMG in vaccinated mice was 14% (3 out of 22 mice) compared to 48% (17 out of 35 mice) in control non-vaccinated or PBS-immunized mice. The T cells of the vaccinated group showed lower proliferative responses to t AChR and to T-cell epitope-containing t AChR &#102 -chain peptides than the T cells of controls. In addition, the Ab responses in the vaccinated group was also lower against t AChR and some of the B-cell epitope-containing t AChR &#102 -chain peptides.     

Autoreactive T cell responses and cytokine patterns reflect resistance to experimental autoimmune myasthenia gravis in Wistar Furth rats

Various mouse and rat strains show different susceptibilities to experimental autoimmune myasthenia gravis (EAMG) that can be induced by immunization with acetylcholine receptor (AChR) and Freund's complete adjuvant, and represents a model for the antibody-mediated myasthenia gravis in humans. We examined AChR-induced B and T cell responses and cytokine mRNA expression to study the mechanisms behind susceptibility to EAMG in Lewis rats and resistance in Wistar Furth (WF) rats. Both strains had similarly elevated concentrations and affinities of serum anti-AChR antibodies, and no difference between the two strains for frequencies of cells in lymphoid organs expressing mRNA of the B cell stimulating cytokine interleukin-4 was found. In contrast, T cell responses to AChR measured by proliferation and by enumeration of interferon-γ-expressing cells at both mRNA and protein level were lower in the resistant WF rats. This strain showed, instead, an up-regulation of the anti-inflammatory transforming growth factor-β. Strain-related differences in the susceptibility to actively induced EAMG are thus related to quantitative differences in distribution between pro-inflammatory and anti-inflammatory cytokines.     

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.