Overexpression of IFN-induced protein 10 and its receptor CXCR3 in myasthenia gravis

Myasthenia gravis (MG) and its animal model, experimental autoimmune MG (EAMG), are autoimmune disorders in which the acetylcholine receptor (AChR) is the major autoantigen. Microarray technology was used to identify new potential drug targets for treatment of myasthenia that would reduce the need for the currently used nonspecific immunosuppression. The chemokine IFN-gamma-inducible protein 10 (IP-10; CXCL10), a CXC chemokine, and its receptor, CXCR3, were found to be overexpressed in lymph node cells of EAMG rats. Quantitative real-time PCR confirmed these findings and revealed up-regulated mRNA levels of another chemoattractant that activates CXCR3, monokine induced by IFN-gamma (Mig; CXCL9). TNF-alpha and IL-1beta, which act synergistically with IFN-gamma to induce IP-10, were also up-regulated. These up-regulations were observed in immune response effector cells, namely, lymph node cells, and in the target organ of the autoimmune attack, the muscle of myasthenic rats, and were significantly reduced after suppression of EAMG by mucosal tolerance induction with an AChR fragment. The relevance of IP-10/CXCR3 signaling in myasthenia was validated by similar observations in MG patients. A significant increase in IP-10 and CXCR3 mRNA levels in both thymus and muscle was observed in myasthenic patients compared with age-matched controls. CXCR3 expression in PBMC of MG patients was markedly increased in CD4(+), but not in CD8(+), T cells or in CD19(+) B cells. Our results demonstrate a positive association of IP-10/CXCR3 signaling with the pathogenesis of EAMG in rats as well as in human MG patients.     

Experimental models of myasthenia gravis: lessons in autoimmunity and progress toward better forms of treatment

The nicotinic acetylcholine receptor (AChR) is a large membrane protein found in muscle cells. It is involved in the transformation of acetylcholine packets into a membrane depolarization, which thereby leads to a muscle twitch. This large, complex molecule is the target of the autoimmune attack in myasthenia gravis, and much has been learned in the past decade about myasthenia by the induction of autoimmunity to AChR in experimental animals. Experimental autoimmune myasthenia gravis (EAMG) has been produced in a variety of animals by immunization with AChR or AChR-like material, or by the passive transfer of anti-AChR antibodies or lymphocytes from afflicted animals into normal animals. EAMG is a remarkably faithful model of human myasthenia and has provided much information about how the immune response to AChR progresses and how weakness and damage to the neuromuscular junction ensure. EAMG has also allowed the development of a number of revolutionary forms of treatment in which only the abnormal response to AChR is restrained, and other necessary immune functions are left intact. These advances in treatment are not far from being tested in human myasthenia gravis. The experience gained in applying these concepts in EAMG and human myasthenia will be helpful in developing similar forms of treatment for other autoimmune diseases.     

Recombinant human acetylcholine receptor alpha-subunit induces chronic experimental autoimmune myasthenia gravis

A synthetic gene encoding the 210 N-terminal residues of the alpha-subunit of the nicotinic acetylcholine receptor (AChR) of human skeletal muscle was cloned into an inducible expression plasmid to produce a fusion protein in high yield in Escherichia coli. Like native human AChR, the recombinant human alpha 1-210 protein induced AChR-binding, AChR-modulating, and AChR-blocking autoantibodies in rats when injected once intradermally as an emulsion in CFA, with Bordetella pertussis vaccine as supplementary adjuvant. The minimum dose of recombinant protein required to induce biochemical signs of experimental autoimmune myasthenia gravis (EAMG) with 100% incidence was 2.2 micrograms. With 6.6 to 22 micrograms, serum levels of autoantibodies were persistent, and clinically apparent EAMG lasted more than a month. Clinical, electrophysiological, and biochemical indices of EAMG induced by doses of 66 micrograms or more were more uniformly severe and persistent, with 33% fatality. Rats receiving a control extract of E. coli containing plasmid without the alpha 1-210 codon insert, with adjuvants, did not develop autoantibodies or signs of EAMG. This highly reproducible new model of EAMG induced by a recombinant human autoantigen should be valuable for testing Ag-specific immunotherapeutic strategies that might be applicable to treating acquired myasthenia gravis in humans.     

Proteasome inhibition with bortezomib depletes plasma cells and autoantibodies in experimental autoimmune myasthenia gravis

Bortezomib, an inhibitor of proteasomes, has been reported to reduce autoantibody titers and to improve clinical condition in mice suffering from lupus-like disease. Bortezomib depletes both short- and long-lived plasma cells; the latter normally survive the standard immunosuppressant treatments targeting T and B cells. These findings encouraged us to test whether bortezomib is effective for alleviating the symptoms in the experimental autoimmune myasthenia gravis (EAMG) model for myasthenia gravis, a disease that is characterized by autoantibodies against the acetylcholine receptor (AChR) of skeletal muscle. Lewis rats were immunized with saline (control, n = 36) or Torpedo AChR (EAMG, n = 54) in CFA in the first week of an experimental period of 8 wk. After immunization, rats received twice a week s.c. injections of bortezomib (0.2 mg/kg in saline) or saline injections. Bortezomib induced apoptosis in bone marrow cells and reduced the amount of plasma cells in the bone marrow by up to 81%. In the EAMG animals, bortezomib efficiently reduced the rise of anti-AChR autoantibody titers, prevented ultrastructural damage of the postsynaptic membrane, improved neuromuscular transmission, and decreased myasthenic symptoms. This study thus underscores the potential of the therapeutic use of proteasome inhibitors to target plasma cells in Ab-mediated autoimmune diseases.     

Mice with IFN-gamma receptor deficiency are less susceptible to experimental autoimmune myasthenia gravis

IFN-gamma can either adversely or beneficially affect certain experimental autoimmune diseases. To study the role of IFN-gamma in the autoantibody-mediated experimental autoimmune myasthenia gravis (EAMG), an animal model of myasthenia gravis in humans, IFN-gammaR-deficient (IFN-gammaR-/-) mutant C57BL/6 mice and congenic wild-type mice were immunized with Torpedo acetylcholine receptor (AChR) plus CFA. IFN-gammaR-/- mice exhibited significantly lower incidence and severity of muscle weakness, lower anti-AChR IgG Ab levels, and lower Ab affinity to AChR compared with wild-type mice. Passive transfer of serum from IFN-gammaR-/- mice induced less muscular weakness compared with serum from wild-type mice. In contrast, numbers of lymph node cells secreting IFN-gamma and of those expressing IFN-gamma mRNA were strongly augmented in the IFN-gammaR-/- mice, reflecting a failure of negative feedback circuits. Cytokine studies by in situ hybridization revealed lower levels of lymphoid cells expressing AChR-reactive IL-1beta and TNF-alpha mRNA in AChR + CFA-immunized IFN-gammaR-/- mice compared with wild-type mice. No differences were found for AChR-reactive cells expressing IL-4, IL-10, or TGF-beta mRNA. These results indicate that IFN-gamma promotes systemic humoral responses in EAMG by up-regulating the production and the affinity of anti-AChR autoantibodies, thereby contributing to susceptibility to EAMG in C57BL/6-type mice.     

Role of tolerogen conformation in induction of oral tolerance in experimental autoimmune myasthenia gravis

We recently demonstrated that oral or nasal administration of recombinant fragments of the acetylcholine receptor (AChR) prevents the induction of experimental autoimmune myasthenia gravis (EAMG) and suppresses ongoing EAMG in rats. We have now studied the role of spatial conformation of these recombinant fragments in determining their tolerogenicity. Two fragments corresponding to the extracellular domain of the human AChR alpha-subunit and differing in conformation were tested: Halpha1-205 expressed with no fusion partner and Halpha1-210 fused to thioredoxin (Trx), and designated Trx-Halpha1-210. The conformational similarity of the fragments to intact AChR was assessed by their reactivity with alpha-bungarotoxin and with anti-AChR mAbs, specific for conformation-dependent epitopes. Oral administration of the more native fragment, Trx-Halpha1-210, at the acute phase of disease led to exacerbation of EAMG, accompanied by an elevation of AChR-specific humoral and cellular reactivity, increased levels of Th1-type cytokines (IL-2, IL-12), decreased levels of Th2 (IL-10)- or Th3 (TGF-beta)-type cytokines, and higher expression of costimulatory factors (CD28, CTLA4, B7-1, B7-2, CD40L, and CD40). On the other hand, oral administration of the less native fragments Halpha1-205 or denatured Trx-Halpha1-210 suppressed ongoing EAMG and led to opposite changes in the immunological parameters. It thus seems that native conformation of AChR-derived fragments renders them immunogenic and immunopathogenic and therefore not suitable for treatment of myasthenia gravis. Conformation of tolerogens should therefore be given careful attention when considering oral tolerance for treatment of autoimmune diseases.     

Breakdown of tolerance to a self-peptide of acetylcholine receptor alpha-subunit induces experimental myasthenia gravis in rats

Experimental autoimmune myasthenia gravis (EAMG), a model for human myasthenia (MG), is routinely induced in susceptible rat strains by a single immunization with Torpedo acetylcholine receptor (TAChR). TAChR immunization induces anti-AChR Abs that cross-react with self AChR, activate the complement cascade, and promote degradation of the postsynaptic membrane of the neuromuscular junction. In parallel, TAChR-specific T cells are induced, and their specific immunodominant epitope has been mapped to the sequence 97-116 of the AChR alpha subunit. A proliferative T cell response against the corresponding rat sequence (R97-116) was also found in TAChR-immunized rats. To test whether the rat (self) sequence can be pathogenic, we immunized Lewis rats with R97-116 or T97-116 peptides and evaluated clinical, neurophysiological, and immunological parameters. Clinical signs of the disease were noted only in R97-116-immunized animals and were confirmed by electrophysiological signs of impaired neuromuscular transmission. All animals produced Abs against the immunizing peptide, but anti-rat AChR Abs were observed only in animals immunized with the rat peptide. These findings suggested that EAMG in rats can be induced by a single peptide of the self AChR, that this sequence is recognized by T cells and Abs, and that breakdown of tolerance to a self epitope might be an initiating event in the pathogenesis of rat EAMG and MG.     

Suppression of development of experimental autoimmune myasthenia gravis with isogeneic monoclonal anti-idiotopic antibody

We have made use of isogeneic anti-idiotopic (anti-Id) monoclonal antibodies (mAb to modify experimental autoimmune myasthenia gravis (EAMG) in Lewis rats. High-avidity anti-Id mAb HC-4A (Kd = 0.1 nM) and HC-29 (Kd = 0.1 nM) were produced against an anti-acetylcholine receptor (anti-AChR) Lewis-rat mAb 132A (Kd = 0.34 nM) that is capable of inducing passive-transfer EAMG. mAb HC-4A and HC-29 define separate framework Id cross-reactive with anti-AChR mAb recognizing different AChR epitopes. Animals were preinjected i.p. with either anti-Id mAb or with control mAb and then were actively immunized 2 wk later with purified AChR. All animals had elevated total serum anti-AChR antibody titers, despite the absence of weakness or decremental electromyographic findings. Animals preinjected with control mAb developed serum anti-AChR titers of 1.34 +/- 0.29 microM (mean +/- SEM) and reduced muscle AChR content to 30 percent of normal. Animals injected with 0.5 mg/kg of either anti-Id had significantly lower serum anti-AChR titers, 0.55 +/- 0.1, p less than 0.05, and normal muscle AChR content. Both the 132A Id and the anti-Id complementary to 132A were detected in the serum of all of the animals preinjected with this dose of either anti-Id HC-29 or HC-4A, whereas both were detected in a much smaller percentage of the animals receiving control mAb. These results show that pretreatment with anti-Id not only perturbs this Id-anti-Id network, but also suppresses the overall polyclonal anti-AChR response with resultant protection of actively immunized animals from EAMG.     

Cathepsin S is required for murine autoimmune myasthenia gravis pathogenesis

Because presentation of acetylcholine receptor (AChR) peptides to T cells is critical to the development of myasthenia gravis, we examined the role of cathepsin S (Cat S) in experimental autoimmune myasthenia gravis (EAMG) induced by AChR immunization. Compared with wild type, Cat S null mice were markedly resistant to the development of EAMG, and showed reduced T and B cell responses to AChR. Cat S null mice immunized with immunodominant AChR peptides showed weak responses, indicating failed peptide presentation accounted for autoimmune resistance. A Cat S inhibitor suppressed in vitro IFN-gamma production by lymph node cells from AChR-immunized, DR3-bearing transgenic mice. Because Cat S null mice are not severely immunocompromised, Cat S inhibitors could be tested for their therapeutic potential in EAMG.     

IL-1 receptor antagonist-mediated therapeutic effect in murine myasthenia gravis is associated with suppressed serum proinflammatory cytokines, C3, and anti-acetylcholine receptor IgG1

In myasthenia gravis (MG), TNF and IL-1beta polymorphisms and high serum levels of these proinflammatory cytokines have been observed. Likewise, TNF and IL-1beta are critical for the activation of acetylcholine receptor (AChR)-specific T and B cells and for the development of experimental autoimmune myasthenia gravis (EAMG) induced by AChR immunization. We tested the therapeutic effect of human recombinant IL-1 receptor antagonist (IL-1ra) in C57BL/6 mice with EAMG. Multiple daily injections of 0.01 mg of IL-1ra administered for 2 wk following two AChR immunizations decreased the incidence and severity of clinical EAMG. Furthermore, IL-1ra treatment of mice with ongoing clinical EAMG reduced the clinical symptoms of disease. The IL-1ra-mediated suppression of clinical disease was associated with suppressed serum IFN-gamma, TNF-alpha, IL-1beta, IL-2, IL-6, C3, and anti-AChR IgG1 without influencing total serum IgG. Therefore, IL-1ra could be used as a nonsteroidal drug for the treatment of MG.     

Cellular immune response to acetylcholine receptors in murine experimental autoimmune myasthenia gravis: inhibition with monoclonal anti-I-A antibodies

Gene(s) at the I-A subregion of the murine major histocompatibility complex influence susceptibility to experimental autoimmune myasthenia gravis. C57Bl/6 mice immunized with acetylcholine receptors (AChR) in complete Freund's adjuvant demonstrated cellular and humoral immune responses to AChR. They developed muscle weakness characteristic of myasthenia gravis and demonstrated a reduction in the muscle AChR content. The kinetics of AChR-specific lymphocyte proliferation generally correlate with anti-AChR antibody response. AChR-specific lymphocyte proliferation was also observed in C57Bl/6 splenocytes after secondary immunization with AChR. The in vitro cellular reactivity to AChR in experimental autoimmune myasthenia gravis (EAMG) mice (C57Bl/6) was suppressed by monoclonal anti-I-Ab antibodies directed against private (Ia20) or public (Ia8) specificities, suggesting a critical role for these Ia determinants in the cellular immune response to AChR in murine EAMG.     

Characterization of peripheral blood acetylcholine receptor-binding B cells in experimental myasthenia gravis

In myasthenia gravis (MG), the neuromuscular transmission is impaired by antibodies (Abs) specific for muscle acetylcholine receptor (AChR). Anti-AChR Abs can be detected in the serum of MG patients, although their levels do not correlate with disease severity. In this study, we developed a flow cytometric assay for the detection of peripheral blood AChR-specific B cells to characterize B cell phenotypes associated with experimental autoimmune myasthenia gravis (EAMG). Alexa-conjugated AChR was used as a probe for AChR-specific B cells (B220+Ig+). Mice with EAMG had significantly elevated frequencies of AChR-specific IgG2+ and IgM+ B cells. While the frequencies of IgG2+ B cells and plasma anti-AChR IgG2 levels significantly correlated with the clinical grades of EAMG, the frequencies of IgM+ B cells and plasma anti-AChR IgM levels did not. These results indicate that the frequency of AChR-specific and IgG1+ (mouse IgG2 equivalent) peripheral blood B cells and anti-AChR IgG1 levels could be potential biomarkers for MG disease severity.     

C5 gene influences the development of murine myasthenia gravis

The influence of the C5 gene and C5 deficiency on murine experimental autoimmune myasthenia gravis (EAMG) susceptibility was evaluated. Two co-isogenic strains, B10.D2/nSn (C5 sufficient) and B10.D2/oSn (C5 deficient), which are genetically identical except for the C5 gene locus, were immunized with acetylcholine receptors (AChR) in CFA to induce myasthenia gravis. Both strains had equivalent concentration of serum autoantibodies to muscle AChR and antibodies bound to muscle AChR. C5-sufficient B10.D2/nSn, but not C5-deficient B10.D2/oSn, demonstrated increased incidence of clinical disease and death and lost significant amounts of muscle AChR. Therefore, C5 deficiency in B10.D2/oSn prevented EAMG. C5 gene, which codes for C component C5, may influence EAMG pathogenesis through activation of the terminal lytic C sequence (C5 to C9) required for muscle AChR destruction, which is the primary pathology.     

Inhibition of acute passive transfer experimental autoimmune myasthenia gravis with Fab antibody to complement C6

The role of the lytic complement C5b-9 membrane attack complex (MAC) in acute passive transfer experimental autoimmune myasthenia gravis (EAMG) produced in rats was investigated by in vivo inhibition of MAC formation with anti-C6 Fab. Anti-C6 Fab totally inhibited in vitro serum hemolytic activity, but did not consume or inhibit early complement pathways. Injection of rats with 0.12 mg/ml anti-C6 Fab reduced serum C6 to 8% and inhibited the muscle weakness, electrophysiologic abnormalities and loss of acetylcholine receptor (AChR) associated with acute EAMG. This level of C6 inhibition reduced the total serum complement hemolytic activity to 29% of normal but did not reduce the serum levels of complement components C3, C5, or C7. Treatment of rats with lower amounts of anti-C6 Fab (0.08 mg/ml) also inhibited clinical and electrophysiologic signs of EAMG, however, the lower amount of anti-C6 did not prevent the loss of muscle AChR. Both the higher and the lower amount of anti-C6 Fab inhibited the accumulation of macrophages at muscle motor end-plates. The inhibition by anti-C6 indicates that muscle weakness and electrophysiologic abnormalities associated with EAMG are dependent on the complement MAC, and that muscle weakness results from tissue injury in addition to loss of muscle membrane and AChR.     

Resistance to experimental autoimmune myasthenia gravis in IL-6-deficient mice is associated with reduced germinal center formation and C3 production

To provide direct genetic evidence for a role of IL-6 in experimental autoimmune myasthenia gravis (EAMG), IL-6 gene KO (IL-6(-/-)) mice in the C57BL/6 background were immunized with Torpedo californica acetylcholine receptor (AChR) and evaluated for EAMG. Only 25% of AChR-immunized IL-6(-/-) mice developed clinical EAMG compared to 83% of C57BL/6 (wild-type) mice. A significant reduction in the secondary anti-AChR Ab of IgG, IgG(2b), and IgG(2c), but not the primary or secondary IgM response was observed in AChR-immunized IL-6(-/-) mice, suggesting a possible defect in T cell help and class switching to anti-AChR IgG(2) isotype. The AChR-specific lymphocyte proliferative response, IFN-gamma, and IL-10 production were suppressed in AChR-immunized IL-6(-/-) mice. EAMG resistance in IL-6(-/-) mice was associated with a significant reduction in germinal center formation and decreased serum complement C3 levels. The data provide the first direct genetic evidence for a key role of IL-6 in the autoimmune response to AChR and in EAMG pathogenesis.     

Immune response gene control of lymphocyte proliferation induced by acetylcholine receptor-specific helper factor derived from lymphocytes of myasthenic mice

The role of lymphokines secreted by acetylcholine receptor (AChR)-reactive lymphocytes in the regulation of an autoimmune response to AChR has not been studied in the human or murine model of myasthenia gravis. We investigated whether AChR-immune lymphocytes derived from mice with experimental autoimmune myasthenia gravis (EAMG) can produce an AChR-specific, genetically controlled soluble factor with biologic activity. AChR-reactive lymphocytes of mice with EAMG secreted an AChR-specific helper factor in vitro, which induced proliferation of AChR-immune but not Mycobacterium tuberculosis-immune lymphocytes. Recombinant, I-A mutant, and monoclonal anti-I-A antibody analyses suggest that AChR-specific helper factor-induced lymphocyte proliferation is controlled by an immune response gene at the I-A subregion of the murine major histocompatibility complex, and is mediated by the I-A molecule.