Acetylcholine receptor antibody-producing cells in thymus and lymph nodes in myasthenia gravis

Eleven patients with myasthenia gravis (5 with thymoma) were studied and spontaneous production of antibody to acetylcholine receptor (AChR) in vitro was found by thymus cells in 10 (and in all 5 with thymoma) and by lymph node cells in 8 (and in 3 with thymoma). The rate of AChR antibody production by thymus cells was greater than that by lymph node cells (10.7 +/- 11.6 and 1.4 +/- 1.5 fmol/10(6) cells/week, respectively, mean +/- SD, P less than 0.05 by paired t test), although the B-cell population was always smaller in the thymus than in the lymph nodes (9.5 +/- 9.4 and 31 +/- 12.7%, P less than 0.001), suggesting the principal role of the thymus in AChR antibody production. It is suggested that lymph nodes can be one of the main sites of AChR antibody production in myasthenia gravis but the antibody-producing cells may originate in the thymus.     

Acetylcholine receptor antibody in patients with myasthenia gravis

The determination of acetylcholine receptor antibody (AChR Ab) titer by an enzyme-linked immunosorbent assay (ELISA) in patients with myasthenia gravis was introduced. The optimal conditions were determined by chequerboard determination. The specificity was confirmed by inhibition tests. The sensitivity is 9 p mole. The comparison of AChR Ab titers among 49 myasthenic patients, 19 non-myasthenic neurological patients and 20 healthy blood donors has shown that it is a highly sensitive, specific, reproducible, rapid, simple and inexpensive method for determining AChR Ab and that it is highly valuable for the diagnosis of myasthenia gravis.     

The thymus in myasthenia gravis. Changes typical for the human disease are absent in experimental autoimmune myasthenia gravis of the Lewis rat.

In human myasthenia gravis (MG) formation of autoantibodies against acetylcholine receptor (AChR) is commonly associated with thymic changes termed lymphofollicular hyperplasia (LFH). To learn whether the thymic lesions of human MG are primary changes in the autoimmune pathogenesis, or rather secondary events caused by peripheral autoimmunization, the authors compared the pathologic changes of MG thymuses with the thymuses of Lewis rats with experimental autoimmune myasthenia gravis (EAMG). EAMG was induced either actively by immunization with AChR, or transferred passively with monoclonal antibodies (mAb) binding to AChR. The clinical diagnosis of EAMG was confirmed by electromyography. Germinal centers, which are typical for human MG thymuses, were not detectable in the thymus of EAMG rats. Scattered B cells were seen as normal components of the thymic medulla. In EAMG their number was not augmented, nor were they accumulated focally. The perivascular spaces (PVS) were not distended and the amount of reticulin was not increased. Thymic myoid cells were identified in EAMG as well as in control thymuses; their cellular microenvironment was inconspicuous. Both in normal and in EAMG thymuses, a subpopulation of myoid cells expressed the main immunogenic region of the AChR. Heavily affected rats showed a severe cortical involution, but no specific changes of the medulla. The fact that none of the thymic lesions characteristic for human MG was found in EAMG is compatible with the concept that the thymic changes in MG are primary events in the autoimmune pathogenesis of this disease.     

Immunogenetics of experimental autoimmune myasthenia gravis

Myasthenia gravis (MG) is an autoimmune neuromuscular disease manifested by muscle weakness and fatiguability. The primary pathology in MG is antibody and complement-mediated destruction of muscle acetylcholine receptor (AChR). Like other autoimmune diseases, MG is associated with certain HLA antigens, particularly HLA-B8 and DR3 in Caucasians. Also, certain GM antigens and complotypes are associated with MG. Therefore, it is crucial to study the immunogenetic aspect of MG in animal models to evaluate disease etiopathogenesis and eventual strategy for specific therapy. In the introduction of this review article, I focus on the association of HLA and GM antigens in MG and emphasize the mouse model of experimental autoimmune myasthenia gravis (EAMG) as an ideal model to study the immunogenetic aspect of MG. The following sections deal with the role of (1) major histocompatibility complex (MHC), (2) immune response gene, (3) the IA molecule, (4) the Igl locus, (5) the complement genes, and (6) non-MHC genes on EAMG pathogenesis. The review concludes with future immunogenetic analysis and eventual strategy for specific therapy from an immunogeneticist's point of view.     

Immunological similarities between an experimental autoimmune myasthenia gravis model and human myasthenia gravis

The induction of experimental autoimmune myasthenia gravis (EAMG) in rabbits after immunization with an acetylcholine (ACh) conjugate was found to possess immunological similarities with human myasthenia gravis. Anti-ACh antibodies, present in human sera, recognized the antigenic determinant, glutarylcholine, used to raise anti-ACh antibodies in rabbits. Identification of anti-anti-ACh antibodies in MG patients enabled us to test for recognition of the anti-ACh antibodies present in rabbit sera. The reverse, the recognition of rabbit auto-anti-anti-ACh antibodies by human anti-ACh antibodies was also tested and found to be specific.     

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。     

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。     

Rapsyn对正常及实验性自身免疫性重症肌无力小鼠乙酰胆碱受体的作用

目的: 探讨突触后膜乙酰胆碱受体缔合蛋白(rapsyn)对正常小鼠及实验性自身免疫性重症肌无力(EAMG)小鼠肌肉组织内乙酰胆碱受体(AChR)的作用。方法: 将扩增的pcDNA-rapsyn质粒注入小鼠左后肢,右后肢相同部位注射等量生理盐水,2周后将实验小鼠随机分为E组和C组,E组经腹腔注射AchR单克隆抗体35 (mAb35)诱导EAMG动物模型,C组经腹腔注射相同剂量的生理盐水,模型诱导48 h后处死实验动物分离双侧后肢肌肉,注射pcDNA-rapsyn质粒的左后肢肌肉分别为LE组和LC组,注射生理盐水的右后肢肌肉分别为RE组和RC组。免疫荧光染色法观察突触后膜AChR与运动终板的结合情况,RT-PCR和Western blotting法检测AChRα mRNA和蛋白表达情况。结果: 结果表明,LC组与RC组相比AChRα蛋白表达量增多(P<0.05),LE组与RE组相比AChRα蛋白表达量增多(P<0.05); LC组与RC组相比AChRα mRNA表达变化无统计学意义(P>0.05),LE组与RE组相比AChRα mRNA表达有明显降低(P<0.01)。结论: 在肌肉组织内上调rapsyn蛋白的表达对正常及EAMG小鼠AChR受体发挥保护性作用。     

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.     

Acetylcholine receptor antibody characteristics in myasthenia gravis. II. Patients with penicillamine-induced myasthenia or idiopathic myasthenia of recent onset.

Anti-acetylcholine receptor (anti-AChR) antibody characteristics including light chain, IgG subclass, avidity for denervated human acetylcholine receptor and reaction with various human and mammalian AChR preparations were examined in 11 patients who developed myasthenia during penicillamine treatment of rheumatoid arthritis. Results were compared with those already reported in 35 patients with generalized idiopathic myasthenia gravis (MG). We found significant differences in the avidity and the light chain of the anti-AChR. However, anti-AChR characteristics in 12 patients with recent onset (less than 4 months'' duration) idiopathic MG did not differ significantly from those in patients with penicillamine-induced MG. In the patients with generalized MG a trend was found towards higher percentage of kappa light chain and higher anti-AChR avidity with duration of disease. Anti-acetylcholine receptor antibodies in penicillamine-induced myasthenia gravis therefore appear to be similar to those of idiopathic myasthenia gravis of recent onset.     

Transplacental transmission of experimental autoimmune myasthenia gravis. A morphological study

Using neonates born from experimental autoimmune myasthenic rabbits, the authors demonstrated antibodies to acetylcholine receptor (AChR) in the newborn sera. By radioimmunoassay, antibody titers of 1-day-old neonates were roughly one seventh to one ninth of the mothers. At 8 weeks postpartum, the antibody was no longer detectable. Ultrastructural observations of the intercostal muscles of the neonates revealed two types of changes. The first type was degenerative alterations in the postsynaptic membrane. The second type of change, which was morphometrically analyzed, was immaturity of postsynaptic membrane structure with underdeveloped secondary synaptic clefts. After 28 days postpartum, these changes were not visible, thereby indicating that the process is reversible as the antibody titer decreases. These results suggested the possibility that the antibody to AChR, transferred transplacentally, arrested the development of postsynaptic structure, although reversible, by blocking of the receptor sites in the end-plate.     

Microenvironments in the normal thymus and the thymus in myasthenia gravis.

The disposition of epithelial cells and extracellular matrix, in the thymus of 8 cases of myasthenia gravis (MG) and in controls (over a wide age range) was studied. In the controls, the subcapsular epithelium was strongly Leu-7-positive in the fetus, negative in childhood, and positive again in adults. Another antibody, RFD4, also labeled the subcapsular epithelium in childhood and adults, but not fetal samples. The samples from MG cases showed the same staining pattern as adult control samples. The medullary epithelium was also RFD4+, and at all ages. The most striking changes in the advanced cases of MG were the unusual arrangement and hypertrophic appearance of medullary epithelial cell areas, separated by laminin-positive basement membranes from the alternating multiple bands of peripheral lymph-node-like areas. The latter had regions resembling the paracortex of lymph nodes as well as germinal centers (GCs). The T-cell zones contained heavy deposits of fibronectin. These T-cell zones were unique to the thymus in MG and were absent in the two normal thymic samples with isolated GCs. In MG the laminin-containing basement membrane, which separated the medullary epithelial and peripheral lymph-node-like areas, was fenestrated at circumscribed points closest to the GCs, thus apparently permitting communication among the medullary epithelium, the T-cell zones, the GCs and the associated antigen-presenting cells. Large numbers of interdigitating cells and some lymphocytes of cortical thymocyte phenotype were also found at these special sites, where opportunities for autosensitization may persist in MG.     

Blocking of IL-6 suppresses experimental autoimmune myasthenia gravis

Suppressive regulatory T cells (Treg) and pathogenic T helper 17 (Th17) cells are two lymphocyte subsets with opposing activities in autoimmune diseases. The proinflammatory cytokine IL-6 is a potent factor in switching immune responses in vivo from the induction of Treg to pathogenic Th17 cells. We studied the Treg and Th17 cell compartments in experimental autoimmune myasthenia gravis (EAMG) and healthy control rats in order to assess whether the equilibrium between Treg and Th17 cells is perturbed in the disease. We found that Th17 cell-related genes are upregulated and Treg-related genes are downregulated in EAMG. The shift in favor of Th17 cells in EAMG could be reversed by antibodies to IL-6. Administration of anti-IL-6 antibodies to myasthenic rats suppressed EAMG when treatment started at the acute or at the chronic phase of disease. Suppression of EAMG by anti-IL-6 antibodies was accompanied by a decrease in the overall rat anti-AChR antibody titer and by a reduced number of B cells as compared with control treatment. Administration of anti-IL-6 antibodies led to down-regulation of several Th17 related genes including IL-17, IL-17R, IL-23R and IL-21 but did not affect the number of Treg cells in the lymph nodes. These data identify IL-6 as an important target for modulation of autoimmune responses.     

The effect of thalidomide on experimental autoimmune myasthenia gravis

Thalidomide is reported to have immunosuppressive and anti-inflammatory effects which have led to its use in the treatment of a number of immune-mediated disorders including leprosy, prurigo, discoid lupus, and Behcet's disease. In addition, thalidomide has recently been used to prevent immunological rejection phenomena following skin and bone-marrow grafts. The immune responses in these conditions are thought to be cell-mediated. However, little is known about the effectiveness of thalidomide in suppressing antibody-mediated immune responses. In the present study, we have examined the effect of thalidomide in a model antibody-mediated autoimmune disorder--experimental autoimmune myasthenia gravis (EAMG). To induce EAMG, Lewis rats were immunized with acetylcholine receptor (AChR) purified from the electric organ of Torpedo californicus. Groups of rats were treated daily, either with thalidomide in excess of doses reported to prevent graft-versus-host (GVH) disease in bone-marrow-transplanted rats, or with control treatments. Our results show that thalidomide failed to inhibit AChR antibody production despite good absorption and high blood levels of the drug. This suggests that thalidomide is not likely to be generally useful in the treatment of antibody-mediated autoimmune conditions. However the selective effect of thalidomide in suppressing certain presumably cellular immune responses, while sparing antibody production, is inherently interesting, and merits further study.     

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