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.     

Pathogenesis of myasthenia gravis. Acetylcholine receptor-related antigenic determinants in tumor-free thymuses and thymic epithelial tumors.

The authors describe an immunohistologic study of acetylcholine receptor (AChR)-related antigenic determinants in tumor-free thymuses of myasthenia gravis (MG) patients (13 cases) and nonmyasthenic controls (10 cases) and in thymic epithelial tumors of patients with MG (8 cases) and without MG (6 cases). Monoclonal antibodies (MAbs) to the cytoplasmic part and to the extracellular main immunogenic region (MIR) of the alpha subunit of AChRs were used. Their intrathymic binding sites were defined by double-immunostaining, and compared with alpha-bungarotoxin (alpha-Bgt) labeling demonstrated by fluorescence microscopy. Tumor-free thymuses of MG patients and control patients contained cytoplasmic AChR epitopes and alpha-Bgt binding sites on myoid cells and some epithelial cells. Only myoid cells also expressed extracellular MIR epitopes, suggesting that they bear complete AChRs, and are important targets for the autoimmune attack in tumor-free MG thymus. Evidence that AChR-related antigenic determinants of epithelial cells are also significant for MG is provided by our findings in thymic epithelial tumors. All eight tumors with MG but only two out of six tumors without MG showed cytoplasmic AChR epitopes and alpha-Bgt binding sites on neoplastic epithelial cells. Myoid cells and MIR epitopes did not occur in the neoplasms, but in some tumor-free thymic remnants beside thymomas. It is assumed that nonneoplastic and neoplastic thymic epithelial cells contain only incomplete AChRs or AChR-like molecules. The different expression of AChR epitopes in thymic epithelial tumors and tumor-free thymuses might explain some of the heterogeneous region specificities of anti-AChR antibodies in sera of MG patients with and without thymoma.     

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.      

Pathogenesis of myasthenia gravis

 Various studies over the last 25 years in Man and animal models have revealed many steps in the pathogenesis of myasthenia gravis (MG) which is now considered the classical organ specific, autoantibody mediated and T cell dependent human autoimmune disease. Though not a disease entity, MG is associated with pathological alterations of the thymus in about 80% of cases. These are described here with reference to distinct models of autoimmunization against the acetylcholine receptor (AChR). In MG with thymitis, intrathymic production of AChR-specific autoantibodies is the result of a classical antigen-driven immune reaction that occurs completely inside the thymus and probably involves AChR on myoid cells as the triggering (myasthenogenic) antigen. Genetic factors contribute essentially to the pathogenesis of this form of MG. In thymoma-associated MG genetic factors are probably of marginal significance. Neither intratumour autoantibody production nor T cell activation seem to occur and the AChR is not the myasthenogenic antigen. Instead, abnormal neurofilaments that share epitopes with the AChR and other autoantigen targets in paraneoplastic MG are expressed in thymomas and may trigger autoantigen-specific, non-tolerogenic T cell selection by molecular mimicry. These data support the hypothesis that initial steps in the pathogenesis of most MG cases take place within abnormal thymic microenvironments, be they inflammatory or neoplastic. Where these initial steps occur in MG cases without thymic pathology is not known. Likewise, the factors involved in the initial triggering of MG remain enigmatic in all MG subtypes. Received: 16 September 1996 / Accepted: 28 October 1996     

'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.     

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.     

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.     

Acetylcholine receptor and thymus in experimental autoimmune myasthenia gravis and experimental myositis.

This study was attempted to obtain information about biological properties of junctional acetylcholine receptor (AChR) and extrajunctional AChR, and about nerve influences on muscles AChRs under the pathological conditions of experimental myasthenia and myositis. Experimental autoimmune myasthenia gravis (EAMG) was induced in Wistar rats by immunizations with AChR purified from the electric organ of Narke Japonica without using Freund's complete adjuvant experimental myositis by immunization with rat muscle extract depleted of AChR. Thirty-five days after the initial immunization, unilateral dissection of the ischiadic nerve was performed in all immunized rats. Contents of AChR in both hind limb muscles were measured by double immunoprecipitation assay method 15 days after the experimental denervation. In the control animals the amount of AChR extractable from innervated muscles was 2.7 +/- 0.5 (mean +/- s.d.) pmole/g muscle and increased about 10-fold 15 days after the denervation (30 +/- 7.9). In rats with EAMG, AChR contents was reduced in both denervated (1.1 +/- 1.0) and innervated muscles (1.3 +/- 0.9). In experimental myositis, the increase of muscle AChR was impaired in denervated muscles (2.4 +/- 0.6), but AChR contents was not reduced in innervated muscles (2.7 +/- 0.9). These results suggest that nerves may influence AChR metabolism, keeping numbers of AChR constant even in inflammatory condition. In addition, germinal centre formation in thymic medulla was detected in EAMG rats.     

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.     

Complement associated pathogenic mechanisms in myasthenia gravis

The complement system is profoundly involved in the pathogenesis of acetylcholine receptor (AChR) antibody (Ab) related myasthenia gravis (MG) and its animal model experimental autoimmune myasthenia gravis (EAMG). The most characteristic finding of muscle pathology in both MG and EAMG is the abundance of IgG and complement deposits at the nerve–muscle junction (NMJ), suggesting that AChR-Ab induces muscle weakness by complement pathway activation and consequent membrane attack complex (MAC) formation. This assumption has been supported with EAMG resistance of complement factor C3 knockout (KO), C4 KO and C5 deficient mice and amelioration of EAMG symptoms following treatment with complement inhibitors such as cobra venom factor, soluble complement receptor 1, anti-C1q, anti-C5 and anti-C6 Abs. Moreover, the complement inhibitor decay accelerating factor (DAF) KO mice exhibit increased susceptibility to EAMG. These findings have brought forward improvisation of novel therapy methods based on inhibition of classical and common complement pathways in MG treatment.     

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.     

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.     

Passive transfer of experimental myasthenia gravis via antigenic modulation of acetylcholine receptor.

Antigenic modulation of acetylcholine receptor (AChR) is considered to contribute to the reduction of endplate AChR in myasthenia gravis (MG). Yet, the pathogenic significance of this mechanism is unclear. To investigate the in vivo role of AChR antigenic modulation we examined the ability of bivalent F(ab')2 and monovalent Fab fragments of monoclonal antibody (mAb) 35 to passively transfer experimental autoimmune MG (EAMG) in rats. mAb 35 which binds at the main immunogenic region (MIR) of the AChR causes severe EAMG without being involved in channel function. Compared to the intact mAb, F(ab')2 35 proved to be less potent but still capable of inducing moderate EAMG, whereas Fab 35 were totally ineffective. Furthermore, both intact and F(ab')2 35 induced mild EAMG in complement-depleted rats. These results (a) provide evidence that antigenic modulation of endplate AChR is sufficient to generate passive transfer of EAMG and (b) further support the pathogenic potential of the anti-MIR antibodies in MG.     

Thymic myoid cell turnover in myasthenia gravis patients and in normal controls

 Thymic myoid cells (TMC) are sparse muscle-like cells in the thymic medulla, which are believed to trigger the autoimmune response in myasthenia gravis (MG). Ultrastructural investigations have revealed mature, degenerating, and immature TMC, but the number of TMC in MG patients does not differ from that in controls. We examined the turnover of TMC at the subcellular level, performing an immunocytochemical study with muscle-specific anti-desmin labelling of 10 thymuses derived from MG patients with lymphofollicular hyperplasia and from 8 normal controls. All thymuses examined revealed mature, immature, and degenerating TMC. Mature TMC contained desmin filaments in between Z-discs provided the sarcomeres were arranged in register. Morphological features of degenerating TMC included hypercontracted sarcomeres, cytoplasmic granular debris, chromatin clumping and, occasionally, membrane-bound bodies. Macrophages were not involved in the process. Immature TMC were of small diameter and contained myofilaments not arranged in myofibrils. In an MG thymus, small immature TMC were found clustered with dying TMC. It may be that degeneration of TMC is a stimulus for the generation of new TMC with faster turnover. This mechanism may mean that more antigen is available in MG patients than in normal controls, despite constant TMC numbers Received: 2 July 1997 / Accepted: 20 November 1997     

Myogenesis in thymic transplants in the severe combined immunodeficient mouse model of myasthenia gravis. Differentiation of thymic myoid cells into striated muscle cells.

Thymic myoid cells (TMCs) bearing acetylcholine receptors (AchR) on their surface have a central role in the concept of intrathymic autosensitization in the pathogenesis of myasthenia gravis. In a SCID mouse model of myasthenia gravis, solid pieces of thymuses with lymphofollicular hyperplasia were transplanted into SCID mice. The chimeric mice displayed long-term secretion of anti-AchR antibodies. Here, we traced the fate of TMCs contained in transplanted myasthenia gravis thymuses. Unexpectedly, the number of thymic TMCs in transplanted tissue was slightly higher than in untransplanted thymus. More strikingly, the transplanted TMCs were more highly differentiated than their nontransplanted counterparts. This was demonstrated by a more than 10-fold increase of AchR-main immunogenic region epitopes recognized by monoclonal antibody 198. Some TMCs had even differentiated into striated muscle cells. The abundance of AchRs in human thymic transplants in the SCID mouse model of myasthenia gravis may help to study the mechanisms of autosensitization against the AchR in vivo.     

Treatment of experimental myasthenia gravis with total lymphoid irradiation

Total lymphoid irradiation (TLI) has been reported to be effective in the immunosuppressive treatment of certain human and experimental autoimmune disorders. We have investigated the effects of TLI in Lewis rats with experimental autoimmune myasthenia gravis (EAMG) produced by immunization with purified torpedo acetylcholine receptor (AChR). The radiation is given in 17 divided fractions of 200 rad each, and nonlymphoid tissues are protected by lead shielding. This technique suppresses the immune system, while minimizing side effects, and permits the repopulation of the immune system by the patient's own bone marrow cells. Our results show that TLI treatment completely prevented the primary antibody response to immunization with torpedo AChR, it rapidly abolished the ongoing antibody response in established EAMG, and it suppressed the secondary (anamnestic) response to a boost of AChR. No EAMG animals died during TLI treatment, compared with six control animals that died of EAMG. TLI produces powerful and prompt immunosuppression and may eventually prove useful in the treatment of refractory human myasthenia gravis.     

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.     

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.