Neuromuscular transmission in experimental autoimmune myasthenia gravis (EAMG)

Chronic experimental autoimmune myasthenia gravis (EAMG) was induced in rats by immunization with acetylcholine receptor (AChR) purified from the electroplax of Torpedo californica. 35–40 days after immunization, serum anti-AChR antibody titers were about 40 nM. At this stage, electrophysiology was performed on isolated M. omohyoideus muscle-preparations from myasthenic and from normal (control) rats.For the study of the equilibrium interaction between acetylcholine (ACh) and AChR, dose-response curves were obtained by quantitative ionophoretic application of ACh to voltage-clamped end-plates. Analysis of dose-response curves yielded the following parameters: maximum end-plate conductance per unit surfaceg _max (EAMG)=10.3±1.1 nS/m²,g _max (normal)=20.2±1.8 nS/m²; apparent dissociation constant K (EAMG)=96±5 M, K (normal)=58±6 M; Hill-coefficient n_H (EAMG)=2.3±0.1, n_H (normal)=2.3±0.1. Single channel properties were derived from an analysis of ACh-induced end-plate current noise: the mean single channel conductance was (EAMG)=29.1±2.2 pS, (normal)=27.6±1.8 pS and the mean channel life-time (EAMG)=1.39±0.09 ms, (normal)=1.32±0.08 ms (T=22°C).The electrophysiological data are interpreted as follows: (1) At myasthenic end-plates there is a 50–60% reduction of functioning AChR (decrease ofg _max). A total number of about 2×10⁶ (1×10⁶) channels per end-plate was calculated for control (myasthenic) rats. (2) The affinity of AChR for ACh is reduced and/or there is an impediment of the conformational change from the closed- to the open-channel configuration (increase of K). (3) Single channel properties are essentially unaffected.This work was supported by the Deutsche Forschungsgemeinschaft SFB 38, project N and We 667/6     

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.     

重肌灵片在重症肌无力小鼠模型T细胞活化中的作用

AchR免疫C57BL/6小鼠建立EAMG小鼠模型,观察重肌灵片在自身免疫性重症肌无力小鼠T细胞活化过程中的作用。结果显示重肌灵片抑制AChR诱导的EAMG小鼠淋巴细胞增殖、DTH反应并降低血清中抗AChR抗体;抑制CD28和CTLA-4的表达、增强CD80和CD86的表达,但对CD40和CD40L的表达无显著影响;并降低Th细胞中表达IFN-γ的阳性细胞数、升高表达IL-4的阳性细胞数。提示重肌灵片显著抑制EAMG小鼠T细胞的活化,其抑制作用可能是通过调节细胞表面协同刺激分子的表达,及抑制Th细胞向Th1极化、促进其向Th2极化实现的。     

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.     

Treatment of passively transferred experimental autoimmune myasthenia gravis using papain

Antibody-mediated acetylcholine receptor (AChR) loss at the neuromuscular junction, the main cause of the symptoms of myasthenia gravis, is induced by bivalent or multivalent antibodies. Passive transfer of experimental autoimmune myasthenia gravis (EAMG) can be induced very efficiently in rats by administration of intact MoAbs directed against the main immunogenic region (MIR) of the AChR, but not by their monovalent Fab fragments. We tested whether papain, which has been used therapeutically in autoimmune and other diseases, is capable of preventing EAMG by in vivo cleavage of the circulating anti-AChR antibodies into Fab fragments. EAMG was induced in 4-week-old female Lewis rats by i.p. injection of anti-MIR mAb35. A total of 0.75 mg of papain was given as one or three injections 3-7 h after MoAb injection. The mAb35 + papain-treated animals developed mild weakness during the first 30 h and subsequently recovered, while all animals that received only mAb35 developed severe myasthenic symptoms and died within 24-30 h. Animals treated only with papain showed no apparent side effects for up to 2 months. Serum anti-AChR levels in mAb35 + papain-treated rats decreased within a few hours, whereas in non-papain-treated rats they remained high for at least 30 h. Muscle AChR in mAb35 + papain-treated animals was partially protected from antibody-mediated degradation. These results show that treatment of rats with papain can prevent passively transferred EAMG without any apparent harm to the animals, and suggest a potential therapeutic use for proteolytic enzymes in myasthenia gravis.     

Dehydroepiandrosterone therapy ameliorates experimental autoimmune myasthenia gravis in Lewis rats

To detect a possible effect of dehydroepiandrosterone (DHEA) in the pathogenesis of experimental autoimmune myasthenia gravis (EAMG), DHEA (0.5 mg/rat) was administrated intraperitoneally to Lewis rats every other day from day 4 postimmunization (p.i.) to day 35 p.i. with Torpedo acetylcholine receptor (AChR) and Freund's complete adjuvant. Rats treated with DHEA had a lower clinical score (mean clinic score, 2 versus 0.5 on day 37 p.i.) and a lower body weight loss (mean body weight, 169 versus 142 g on day 37 p.i.) compared with control EAMG rats. DHEA treatment decreased serum anti-AChR IgG and IgG2b antibody titers on days 7, 14, and 21 p.i. and inhibited the levels of anti-AChR IgG antibody secreting cells (60%), accompanied by decreased IL-4 (33%) and augmented TGF-1-positive cells (41%) among lymph node mononuclear cells. These results obtained from EAMG in Lewis rats further encourage us to study DHEA treatment in human MG.     

SIN-1降低重症肌无力大鼠的自身免疫反应

用一氧化氮 (NO)供体 3 吗啉 斯德酮亚胺 (SIN 1)干预乙酰胆碱受体 (AChR)致敏的实验性自身免疫性重症肌无力 (EAMG)大鼠模型。并进行临床评分、体重测量、免疫学指标及细胞凋亡检测。结果显示在大鼠致敏后 0～ 7天和 16～ 2 5天给药后 ,SIN 1组大鼠临床症状明显减轻伴发病延迟 ,血清IgG含量始终明显低于对照组 ;致敏后第10天 ,SIN 1组大鼠脾单个核细胞AChR反应性IFN γ分泌性细胞数 (6 38± 1 0 6 )比对照组 (8 2 5± 1 6 7)明显减少(P <0 0 5 ) ,外周血AChR反应性凋亡细胞含量 (8 5 9± 1 35 )与对照组 (5 0 5± 0 6 3)相比明显增多 (P <0 0 1)。实验证明SIN 1可以促使AChR反应性MNC凋亡增加 ,下调自身免疫反应 ,在EAMG的发病和疾病进展中起保护作用。     

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.     

雷帕霉素通过下调Th17细胞/调节性T细胞比值减轻实验性自身免疫性重症肌无力大鼠症状

目的 初步研究雷帕霉素(RAPA)对实验性自身免疫性重症肌无力(EAMG)大鼠的治疗作用,并研究相关免疫机制.方法 应用鼠源性乙酰胆碱受体α亚基97～116肽段(R97-116)免疫Lewis大鼠,建立EAMG动物模型,随机分为完全Freund佐剂对照组(CFA组)、EAMG模型对照组、RAPA处理组[1 mg/(kg...     

Age- and sex-related resistance to chronic experimental autoimmune myasthenia gravis (EAMG) in Brown Norway rats

The influence of age and sex on the induction of chronic EAMG was analysed. Aged male rats, immunized with Torpedo acetylcholine receptor (tAChR), showed no clinical signs of disease or AChR loss. Immunization of young male Brown Norway (BN) rats resulted in both clinical signs of disease and 65% AChR loss. In contrast, both young and aged female BN rats showed comparable AChR loss (58% and 50%, respectively), although aged female rats did not develop clinical signs of disease. Differences in antibody titres, isotype distribution, fine specificity or complement activation could not account for the observed resistance. These results suggest that resistance against EAMG in aged rats is due to resistance of the AChR against antibody-mediated degradation, or to mechanisms able to compensate for AChR loss.     

实验性重症肌无力大鼠模型的建立及巨噬细胞在发病机制中的作用探讨

目的探讨早期重症肌无力发病机理中巨噬细胞的作用及巨噬细胞相关的细胞因子和趋化因子的变化。方法用原位杂交探索在EAMG动物模型靶器官肌肉组织中细胞因子和趋化因子mRNA的表达。采用原位杂交和免疫细胞化学染色双染技术,检测在Lewis大鼠EAMG早期,巨噬细胞浸润和程序性细胞凋亡。结果在EAMG早期观察在7和12天的EAMG大鼠肌肉组织中可见TNF-α、IL-1β和IL-6mRNA的表达细胞瞬时爆发,其峰值分别是(8.7±2.2)、(13.1±1.2)和(6.9±2.4)cells/mm2。第15天TNF-αmRNA的表达细胞数量再一次上升,IFN-γ、IL-4、IL-10、TGF-β和溶细胞素的mRNA表达细胞非常低。这些细胞主要在EAMG的早期观察到,范围是1～4cells/mm2。结论(1)EAMG早期大量巨噬细胞浸润,程序性细胞凋亡是浸润的巨噬细胞消除的主要方式;(2)EAMG早期于肌肉组织中细胞因子低水平表达;没有发现C-C趋化因子。     

Cellular mRNA expression of interferon-gamma (IFN-γ), IL-4 and transforming growth factor-beta (TGF-β) in rats nasally tolerized against experimental autoimmune myasthenia gravis (EAMG)

Nasal administration of nicotinic acetylcholine receptor (AChR) to Lewis rats prior to myasthenogenic immunization with AChR plus Freund's complete adjuvant (FCA) resulted in prevention or marked decrease of the severity of EAMG, suppression of AChR-specific B cell responses and of AChR-reactive T cell functions. To examine the involvement of immunoregulatory cytokines and the underlying mechanisms involved in tolerance induction, in situ hybridization with radiolabelled synthetic oligonucleotide probes was adopted to enumerate mononuclear cells (MNC) expressing mRNA for the proinflammatory cytokine IFN-γ, the B cell stimulating IL-4 and the immune response-down-regulating TGF-β. Popliteal and inguinal lymph nodes from EAMG rats contained elevated numbers of AChR-reactive IFN-γ, IL-4 and TGF-β mRNA-expressing cells compared with control rats receiving PBS nasally and injected with FCA only. Nasal tolerance to EAMG was accompanied by decreased numbers of AChR-reactive IFN-γ and IL-4 mRNA-expressing cells, and strong up-regulation of TGF-β mRNA-positive cells in lymphoid organs compared with non-tolerized EAMG control rats. The relative affinity of anti-AChR antibodies was lower, but muscle AChR amounts were higher in nasally tolerized rats compared with non-tolerized EAMG control rats. The results suggest that IFN-γ and IL-4 are central effector molecules in the development of EAMG, and that TGF-β plays an important role in tolerance induction to EAMG.     

Oral tolerance in a murine model of relapsing experimental autoimmune uveoretinitis (EAU): induction of protective tolerance in primed animals

Oral administration of uveitogenic retinal antigens suppresses the expression of EAU induced by a subsequent immunization with these antigens. Effectiveness and mechanisms of oral tolerance in EAU have mainly been studied in the acute, monophasic model in Lewis rats by feeding antigen prior to induction of disease. In this study we investigated the effect of oral tolerance induction in the acute as well as the chronic-relapsing models in the B10.A mouse. In acute murine EAU we could effectively suppress disease by induction of oral tolerance prior to immunization. In the chronic-relapsing EAU, antigen feeding was started only after the animals had recovered from their first attack of uveitis. Under these experimental conditions the subsequent relapse was largely prevented. These experiments demonstrate that oral tolerance may have practical clinical implications in uveitis, which is predominantly a chronic-relapsing condition in humans.     

BM stromal cells ameliorate experimental autoimmune myasthenia gravis by altering the balance of Th cells through the secretion of IDO

In addition to their capacity to differentiate, BM stromal cells (BMSC) have immunosuppressive qualities that make them strong candidates for use in cell therapy against human autoimmune diseases. We studied the immunoregulatory activities of BMSC on experimental autoimmune myasthenia gravis (EAMG) in vitro and in vivo. Intravenous administration of syngenic BMSC to EAMG‐model rats on the day of their second immunization was effective in ameliorating the pathological features of the disease. In vitro, the proliferative ability of T cells or B cells from EAMG rats was inhibited when they were cocultured with BMSC at proper ratios. This inhibitory effect was at least partially dependent on the secretion of IDO. We also determined that the development of EAMG is accompanied by an imbalance among the Th1, Th2, Th17, and Treg cell subsets, and that this can be corrected by the administration of BMSC, which leads to an increase of Th2 (IL‐4) and Treg (Foxp3) cells, and a reduction of Th1 (IFN‐γ) and Th17 (IL‐17) cells, through an IDO‐dependent mechanism. These results provide further insights into the pathogenesis of MG, EAMG, and other immune‐mediated diseases, and support a potential role for BMSC in their treatment.     

Tissue plasminogen activator involvement in experimental autoimmune myasthenia gravis: Aggravation and therapeutic potential

Tissue plasminogen activator (tPA), a component of the PA/plasmin system, is elevated in inflammatory areas and plays a role in inflammatory neurological disorders. In the present study we explored the involvement of tPA and the potential immunomodulatory activity of tPA in experimental autoimmune myasthenia gravis (EAMG). Mice deficient in tPA (tPA^−/−) present with a markedly more severe disease than wild type EAMG mice. In an attempt to treat EAMG with an 18aa peptide derived from the PA system inhibitor (PAI-1), designed to tether out the endogenous inhibitor, a significant suppression of disease severity was demonstrated. The more severe disease in tPA^−/− mice was accompanied by a higher level of anti-AChR antibodies and increased expression of B-cell markers. In view of the essential role of B-cell activating factor (BAFF) in B-cell maturation, the expression of BAFF family components was tested. An increase in BAFF and BAFF receptor was observed in EAMG tPA^−/− mice, whereas BCMA expression was reduced, consistent with the increased level of pathogenic antibodies and the more severe disease. Given the importance of T regulatory cells (Tregs) in EAMG, they were evaluated and their number was reduced in tPA^−/− mice, in which EAMG was aggravated, whereas following PAI-1dp treatment, Tregs were replenished and the disease was ameliorated. The results show the involvement of tPA in EAMG, implying a protective role for tPA in EAMG pathogenesis. The amelioration of EAMG by PAI-1dp treatment suggests that the PA system may be considered a potential site for therapeutic intervention in neuroimmune diseases.     

Activation of the receptor for advanced glycation end products (RAGE) exacerbates experimental autoimmune myasthenia gravis symptoms

RAGE belongs to immunoglobulin superfamily and serves as a ligand for various immunoregulatory molecules including S100B that has been demonstrated important to T cell mediated autoimmune diseases. In this context, we hypothesized that RAGE could also impact B cell mediated, T cell-dependent autoimmune diseases. This was tested using myasthenia gravis (MG) animal model, EAMG. We show that expression of both RAGE and S100B are increased during EAMG and the interaction between RAGE and S100B affected the Th1/Th2/Th17/Treg cell equilibrium, up-regulate AChR-specific T cell proliferation. Furthermore, addition of S100B in vitro stimulated splenocyte activity linked to COX-2 up-regulation. NS-398, a selective COX-2 inhibitor, effectively diminished S100B mediated activity of AChR-specific antibody secreting splenocytes. These findings suggested that a reciprocal relationship between RAGE and S100B promoted the development of EAMG, highlighting the importance of understanding the mechanisms of EAMG disease as a means of developing new therapies for the treatment of MG.     

Characterization of anti-acetylcholine receptor (AChR) antibodies from mice differing in susceptibility for experimental autoimmune myasthenia gravis (EAMG).

In the murine model for EAMG we investigated the relation between disease susceptibility and fine specificity of anti-AChR antibodies obtained from high susceptible C57Bl/6 and low susceptible BALB/c mice after immunization with Torpedo acetylcholine receptor (tAChR). Anti-AChR MoAbs with fine specificity for the main immunogenic region (MIR), the alpha-bungarotoxin (alpha-BT)/acetylcholine binding sites and other extra- and intracellular epitopes were isolated from both mouse strains. In total, nine out of 38 MoAbs obtained from C57Bl/6 mice were directed against extracellular epitopes on mouse AChR in contrast to only one out of 27 MoAbs from BALB/c mice. A difference in antibody repertoire may underlie the difference in pathogenic response observed between these mouse strains. These results indicate that strain-specific differences in disease susceptibility in murine EAMG may be related to differences in the available repertoire of potential pathogenic antibodies.     

IL‐17‐producing CD4+ T cells contribute to the loss of B‐cell tolerance in experimental autoimmune myasthenia gravis

The role of Th17 cells in the pathogenesis of autoantibody‐mediated diseases is unclear. Here, we assessed the contribution of Th17 cells to the pathogenesis of experimental autoimmune myasthenia gravis (EAMG), which is induced by repetitive immunizations with Torpedo californica acetylcholine receptor (tAChR). We show that a significant fraction of tAChR‐specific CD4⁺ T cells is producing IL‐17. IL‐17^ko mice developed fewer or no EAMG symptoms, although the frequencies of tAChR‐specific CD4⁺ T cells secreting IL‐2, IFN‐γ, or IL‐21, and the percentage of FoxP3⁺ T_reg cells were similar to WT mice. Even though the total anti‐tAChR antibody levels were equal, the complement fixating IgG2b subtype was reduced in IL‐17^ko as compared to WT mice. Most importantly, pathogenic anti‐murine AChR antibodies were significantly lower in IL‐17^ko mice. Furthermore, we confirmed the role of Th17 cells in EAMG pathogenesis by the reconstitution of TCR β/δ^ko mice with WT or IL‐17^ko CD4⁺ T cells. In conclusion, we show that the level of IgG2b and the loss of B‐cell tolerance, which results in pathogenic anti‐murine AChR‐specific antibodies, are dependent on IL‐17 production by CD4⁺ T cells. Thus, we describe here for the first time how Th17 cells are involved in the induction of classical antibody‐mediated autoimmunity.     

Acute and chronic myasthenia gravis in rabbits: Morphological analysis of the neuromuscular junction

Experimental autoimmune myasthenia gravis was induced in rabbits by immunization with acetylcholine receptor obtained from the electric organs of Torpedo marmorata. Some of the animals developed an acute and severe form of the disease, while others developed a mild and chronic form. The morphology of muscle end plates from both groups of myasthenic rabbits was studied by electron microscopic and morphometric analysis. In the acute form the most evident alterations of the end plate were: reduction of the height and the number of the postsynaptic infoldings, where abundant pinocytotic vesicles were common; aggregates of dense granules, not of uniform size and structure, filling the synaptic cleft; the presence of autophagic vacuoles that occupied the enlarged sole plate area.In the chronic form the most consistent finding was the detaching of the entire end plate from the muscle fiber as result of fusion of cytoplasmic vacuoles with profound invaginations of the plasmalemma. The postsynaptic component of the detached end plate underwent a total degradation and was phagocytosed by neutrophils and macrophages. Before the total separation of the end plate from the muscle fiber took place, the nerve endings branched and shifted from their original position. After the separation of the end plate, reinnervation took place in a new intact portion of the muscle fiber.The coexistence in chronic experimental autoimmune myasthenia gravis of the processes of denervation and reinnervation, typical of the human myasthenia gravis makes this experimental model particularly useful in studying the human disease.     

受体相关蛋白对实验性自身免疫性重症肌无力的乙酰胆碱受体的作用

目的：探讨受体相关蛋白(Rapsyn)对实验性自身免疫性重症肌无力(EAMG)动物终板乙酰胆碱受体的作用。方法：用基因工程的方法制备提取pcDNA-Rapsyn，并将其注入动物肌肉左大腿外侧，右大腿相应部位注射相同剂量的空白质粒(Vector)，以方波刺激使其进入细胞膜内，2周后用单克隆抗体35(mAb35)诱导出EAMG模型并进行症状评估，mAb35诱导48小时后取双大腿外侧肌，用放射免疫法检测AChR，并计算出AChR的损失率。结果：在EAMG模型中，被pcDNA-Rapsyn预处理的肌肉AChR损失率明显低于未经处理的肌肉。结论：Rapsyn蛋白对EAMG模型的AChR有保护作用。