T-lymphocytes in experimental autoimmune myasthenia gravis. Isolation of T-helper cell lines

The role of T-lymphocytes in Experimental Autoimmune Myasthenia Gravis (EAMG) was investigated. We generated highly purified, acetylcholine receptor (AChR)-specific T-cell populations and subsequently characterized these cell lines with respect to their membrane phenotype and their function. Using a series of mouse monoclonal antibodies directed against rat lymphocyte surface differentiation antigens, the vast majority of line cells was shown to express a leucocyte common antigen, a T-common antigen and a T-helper antigen. Small subpopulations were Ia or T suppressor antigen-positive. Adaptive transfer to sublethally irradiated, thymectomized recipients revealed that 1 X 10(6) AChR-specific line cells could cooperate effectively with 10 X 10(6) AChR-primed, complement (C3) receptor-bearing (B-cell enriched) spleen cells in the production of anti-AChR autoantibodies. Recipients of B-cells along with relevant line cells developed an acute myasthenic syndrome 6-7 days after cell transfer. Electron-microscopical examination revealed the typical features of "acute phase" EAMG with heavy mononuclear infiltration. There was, however, no evidence antibody-independent cytotoxic activity exerted by AChR-specific line cells.     

Content and release of acetylcholinesterase in skeletal muscle of rats with experimental autoimmune myasthenia gravis

The effects of experimental autoimmune myasthenia gravis (EAMG) on acetylcholinesterase (AChE) were investigated in diaphragms of adult female Lewis rats. Both total AChE activity per muscle and release of enzyme activity during a 3-h incubation in vitro were measured. Two groups of myasthenic animals were used. Acute EAMG was induced by intravenous injection 48 h earlier with a syngeneic monoclonal autoantibody against the nicotinic acetylcholine receptor (AChR) of rat skeletal muscle; age- and weight-matched controls received a monoclonal anti-AChR antibody nonreactive with mammalian muscle. Chronic EAMG was induced by immunization 4 weeks earlier with AChR purified from Torpedo electroplax; controls received only adjuvants. When preparations from rats with acute or chronic EAMG were compared with the appropriate controls, no statistically significant differences in content or release of AChE activity were detected. Neither was there any change in the relative amounts of the various molecular forms of AChE in samples from animals with chronic EAMG. We conclude that the structural and functional changes arising in EAMG are highly specific for the acetylcholine receptor and associated elements of the neuromuscular junction, but have little impact on the biology of AChE.     

In vivo and in vitro studies of the prevention of proteolipid apoprotein-induced murine experimental allergic encephalomyelitis by monoclonal antibody against L3T4

The suppressive effect of anti-L3T4 monoclonal antibody (mAb) on murine experimental allergic encephalomyelitis (EAE) induced by sensitization with proteolipid apoprotein (PLP) was examined in vivo and in vitro. This mAb inhibited the antigen-specific proliferation of the encephalitogenic T cell lines but did not block the mitogen-mediated response. Serial injections of the mAb during the pre-effector phase of EAE markedly suppressed the development and relapse of the disease but this treatment initiated after appearance of clinical signs was not effective. In treated animals, L3T4+ T cells in the spleen were profoundly decreased and the antigen-specific proliferative response of spleen cells was completely suppressed. Moreover, adoptive transfer of spleen cells from the treated mice induced resistance against EAE induction in the recipients. However, no obvious evidence for antigen-specific suppressor cells was found in vitro in the L3T4- populations of spleen cells from treated mice.     

抗乙酰胆碱受体单克隆抗体A7诱导实验性自身免疫性重症肌无力作用

目的探讨抗乙酰胆碱受体单克隆抗体A7(抗AChRmAbA7)对实验性自身免疫性重症肌无力(EAMG)的作用。方法经Lewis大鼠腹腔注射5mL20倍浓缩的含抗AChRmAbA7培养上清液,建立EAMG被动转输模型。对照组经腹腔注射同体积的PBS。每8h监测体重和临床症状评分。48h后处死实验动物。采用放射免疫法检测AChR,并计算出AChR损失率。结果抗AChRmAbA7诱导的EAMG模型AChR损失率高达243%~452%,而且AChR损失率与临床症状评分相关(r=074,P<001)。抗AChRmAbA7攻击位点在终板的AChR上。结论抗AChRmAbA7可用于诱导EAMG模型及有关研究。     

A form of acute experimental autoimmune myasthenia gravis in mice occurring in absence of detectable circulating anti-acetylcholine receptor antibodies: Acute experimental myasthenia in mice

A form of acute experimental autoimmune myasthenia gravis (EAMG) appears within 10 days after immunization of mice with rat acetylcholine receptor (AcChR) in complete adjuvant. This acute phase of EAMG differs from the chronic form reported earlier in the absence of detectable circulating anti-AcChR antibodies and by electrophysiologic signs of neuromuscular blockade which are not reversed by edrophonium injection. This form of acute EAMG occurs similarly in animals whose humoral response has been markedly reduced by pretreatment with cyclophosphamide. These data indicate that the pathogenesis of this acute form might differ from that of chronic EAMG and that it could involve mechanisms of cell-mediated immunity.     

Induction of the morphologic changes of both acute and chronic experimental myasthenia by monoclonal antibody directed against acetylcholine receptor

Summary To investigate pathogenic mechanisms in experimental autoimmune myasthenia gravis (EAMG) and myasthenia gravis (MG), we studied the acute and chronic effects in rats of injection of rat monoclonal antibodies (MCABs) directed against the acetylcholine receptor (AChR). Animals were severely weak 12 h after a single injection, at which time macrophages were found invading endplate regions of muscle and cholinesterase-stained regions were separted from the underlying muscle fibers. Ultrastructural studies showed findings identical to the acute phase of EAMG: degenerating postsynaptic membranes and invasion and phagocytosis of endplate regions by macrophages. Animals receiving sublethal doses of MCAB recovered clinically by 4–5 days after injection. Recovery was accompanied by a progressive decrease in the number of macrophages associated with endplates and reapposition to the myofibers of the cholinesterasestained regions. Animals injected once, or repeatedly over several months, remained clinically and electromyographically normal after recovery from the initial episode of weakness, but their endplate ultrastructure was highly simplified with blunted or absent synaptic folds and shallow or absent secondary synaptic clefts. These studies demonstrate that anti-AChR MCABs can induce the changes of both acute and chronic EAMG. There is good correlation between the inflammatory changes and the acute clinical disease but poor correlation between morphological and clinical parameters in the chronic syndrome. The latter observation suggests that severe ultrastructural changes, similar to those seen in chronic EAMG and MG, cannot account, at least in rats, for the clinical and electrophysiologic abnormalities of MG.Supported in part by grants from the Muscular Dystrophy Association and the National Institutes of Health (NS15462, A19268)     

Passive transfer of experimental autoimmune myasthenia gravis by monoclonal antibodies to the main immunogenic region of the acetylcholine receptor

Experimental autoimmune myasthenia gravis (EAMG) was passively transferred to rats by injecting monoclonal antibodies (mAbs) directed at the main immunogenic region (MIR) of the nicotinic acetylcholine receptor (AChR). The MIR is located on the extracellular part of the AChR alpha-subunit. All four mAbs directed at the MIR which were tested were very efficient in inducing EAMG: within 2 days the rats became moribund or very weak and their muscle AChR content decreased to about 50% of normal. These mAbs are of two different IgG subclasses (IgG1 and IgG2a) and derived from rats immunized with AChR from either fish electric organs or mammalian muscles. One mAb directed at the extracellular side of the beta-subunit did not cause AChR loss or induce symptoms of EAMG. mAbs to the cytoplasmic side were, as expected, ineffective.     

Experimental autoimmune myasthenia gravis in the rat: A preliminary report

In chronic experimental myasthenia gravis (EAMG) in rat, the decrement of electrical and mechanical responses evoked by maximal repetitive (3–167/sec) stimuli to the nerve was greater in the slow-twitch soleus (SOL) than in the fast-twitch extensor digitorum longus (EDL) muscle. Excitation-contraction coupling was impaired in moderate to severe EAMG, as evidenced by a diminished staircase phenomenon and by diminished posttetanic potentiation (PTP) of twitch tension in two EDLs. The only morphologic abnormality observed was an increase in length of the endplates in the EDLs of those animals that had had an acute phase of EAMG. The latter had more than a 90% reduction in the amplitude of the action potential and in the twitch tension of the EDL when stimuli were applied to the nerve. Stimuli applied directly to the muscle evoked a tetanic tension that was one-third of normal. The staircase and PTP were normal. Necrosis occurred in the endplate and in the adjacent segment of muscle fiber; outside the endplate region, the muscle fiber was normal.     

Acetylcholine receptor-reactive antibody induces nitric oxide production by a rat skeletal muscle cell line: influence of cytokine environment

The monoclonal Lewis rat skeletal muscle cell line, LE1, responded to the acetylcholine receptor (AChR)-reactive antibody mAb35 by up-regulating levels of mRNA for inducible nitric oxide synthase (iNOS/NOS-II), followed by levels of NO. Interferon-gamma (IFN-γ) and interleukin-1 (IL-1) were also each capable of inducing iNOS message, and synergistically with mAb35. Finally, myocyte-derived NO was implicated as a possible source of immunomodulation in experimental autoimmune myasthenia gravis (EAMG), as shown by the ability of the culture fluids from IFN-γ-activated LE1 cells to inhibit the proliferation of AChR-reactive T cells.     

Lymphocyte responsiveness to acetylcholine receptor in rats with experimental autoimmune myasthenia gravis

We examined the time course of lymphocyte responsiveness to acetylcholine receptor (AChR) in rats with experimental autoimmune myasthenia gravis (EAMG). Rats were immunized with purified torpedo AChR. At intervals of one to eight weeks later, lymphocytes from the lymph nodes and spleen were cultured with purified torpedo AChR and rat muscle extract containing AChR. Lymphocyte responsiveness (stimulation index) was determined from uptake of ³H-labeled thymidine by the cultured cells. The response of lymphocytes to torpedo antigen began earlier and rose more rapidly than that to the homologous (rat) antigen. Lymph node cells responded more promptly than spleen lymphocytes. The stimulation indexes peaked at four to six weeks while antibodies to both antigens continued to rise. Delineation of this pattern of lymphocyte responsiveness sheds further light on the pathogenesis of the autoimmune response in EAMG and will be useful in the future design of immunotherapeutic strategies.     

Myocytes respond in vivo to an antibody reactive with the acetylcholine receptor by upregulating interleukin-15: an interferon-gamma activator with the potential to influence the severity and course of experimental myasthenia gravis

The monoclonal antibody, mAb35, which binds the main immunogenic region of the post-junctional muscle receptor for acetylcholine (AChR), results in contractile dysfunction and symptoms of experimental myasthenia gravis (EAMG). As described below, exposure to mAb35 also results in the production by muscle of increased levels of the interferon-gamma (IFN-gamma)-activating cytokine, interleukin-15. This effect is accompanied by the increased trafficking of leukocytes through muscle, some that produce IFN-gamma. These observations may be relevant to the induction of disease symptoms since numerous reports from other investigators indicate that IFN-gamma may play a pivotal role in this disease process.     

Anti-tumor necrosis factor therapy abrogates autoimmune demyelination.

To define a role for the cytokine tumor necrosis factor (TNF) in immune-mediated demyelination, the effect of anti-TNF antibody was investigated with a form of experimental autoimmune encephalomyelitis (EAE) in SJL/J mice induced by the adoptive transfer of myelin basic protein-(MBP)-sensitized T lymphocytes, an animal model of the human disease multiple sclerosis (MS). In three separate experiments, no mouse sensitized for EAE and then treated with anti-TNF by intraperitoneal injection developed signs of central nervous system (CNS) disease. Examination of CNS tissue from anti-TNF-treated animals showed no pathological changes. CNS tissue from control animals demonstrated extensive inflammatory cell infiltration and demyelination. To test whether anti-TNF therapy was inhibitory to encephalitogenic cells, preincubation of MBP-sensitized T lymphocytes with anti-TNF in vitro prior to injection into recipient mice was performed, and resulted in no diminution of their ability to transfer EAE. In addition, spleen cells from anti-TNF-treated mice were capable of serial transfer of EAE, similar to spleen cells from control animals. However, spleen cells from anti-TNF-treated mice did not produce TNF on stimulation with MBP or concanavalin A. This study showed that anti-TNF antibody can inhibit effectively the development of EAE by interfering with the effector, rather than the induction, phase of the disease. Anticytokine therapy may have important applications in the development of new therapeutic strategies for MS.     

激发型CD40单克隆抗体对实验性自身免疫性重症肌无力耐受的影响

目的 探讨激发型CD40单克隆抗体(CD40mAb)对未成熟树突状细胞(iDC)诱导实验性自身免疫性重症肌无力(EAMG)耐受的影响. 方法 20只Lewis大鼠按照随机数字表法分为正常组、EAMG对照组、耐受组和CD40mAb组,每组5只.耐受组和CD40mAb组每只大鼠于背部皮下分4点分别注射总量为1 mL的乙酰胆碱受体(AChR)+iDC,其中CD40mAb组每只大鼠在接种iDC的同时和次日分别给予CD40mAb腹腔注射,0.5mg/次.EAMG对照组注射1 mL无血清培养基.正常组不做任何处理.3周后用AChR和完全弗氏佐剂(CFA)免疫,观察免疫7周后重症肌无力(MG)相关指标的改变. 结果 耐受组大鼠和正常组大鼠一样,用AChR和CFA免疫后,MG相关指标无明显改变;而CD40mAb组大鼠和EAMG对照组一样,产生了明显的MG症状,重复电刺激出现明显衰减,血清AChRab滴度明显增高,神经肌肉接头呈现典型的MG样改变. 结论 激发型CD40mAb可阻止AChR负载的iDC诱导EAMG耐受,提示iDC功能异常可能与MG异常免疫反应的启动密切相关.

Abstract：

Objective To investigate the effect of agonist CD40 monoclonal antibody (CD40mAb) on the tolerance of experimental autoimmune myasthenia gravis (EAMG) induced by immature dendritic cells (iDCs). Methods Lewis rats were equally randomized into normal group,EAMG group, tolerance group and CD40mAb treatment group (n=5). Rats in the tolerance group and CD40mAb treatment group were vaccinated with AChR pulsed iDCs; and rats in the CD40mAb treatment group were intraperitoneally injected CD40mAb at a dosage of 0.5 mg once when performing the vaccination and on the 2rd d of vaccination. One mL serum-free medium was given to the rats in the EAMG group; normal group did not receive any treatment. Three weeks after that, rats in the above 4 groups were immunized with AChR and complete Freund's adjuvant (CFA). Seven weeks after the immunization, the corresponding indexes of MG were observed: behavioral assessment was performed and electromyogram was employed to detect the repetitive nerve stimulation on these rats; enzyme-linked immunosorbent assay (ELISA) was used to determine the level of AChRab; the pathological changes of neuromuscular junction were also detected. Results Just as the rats in the normal group, the rats in the tolerance group did not have significant changes in any of the corresponding indexes of MG after being immunized with AChR and CFA. In contrast, rats in both EAMG group and CD40mAb treatment group showed typical changes in the corresponding indexes of MG: their electromyogram wave amplitude obviously attenuated; the level of serum AChRab increased and neuromuscular junction appeared as a typical damage of MG. Conclusion Agonist CD40mAb could abrogate the tolerance of EAMG induced by AChR pulsed iDCs, suggesting that the dysfunction of DCs is related to the priming of abnormal immune of MG.     

乙酰胆碱受体诱导实验性自身免疫性重症肌无力模型的建立

目的采用纯化乙酰胆碱受体(acetylcholine receptors,AChR)免疫大鼠和小鼠以建立实验性自身免疫性重症肌无力(experimental autoimmune myasthenia gravis,EAMG)动物模型。方法以亲和层析法从电鳐电器官提取和纯化AChR,并用其免疫接种Lewis大鼠和C57BL/6小鼠,观察接种动物的临床症状、电生理变化以及AChR抗体产生的情况。结果动物模型临床肌无力症状、翻转悬挂时间(小鼠,P<0.05)、重复神经电刺激(repetitive nerve stimulation,RNS)动作电位衰减率、抗体吸光度(P<0.05)及新斯的明试验均为阳性或有统计学意义。结论纯化电器官AChR作为免疫原,成功诱导产生EAMG动物模型。Lewis大鼠和C57BL/6小鼠均对AChR免疫易感而产生EAMG表现,7~8周龄的C57BL/6更易诱导出类似人类MG表现的EAMG。     

Facilitatory effects of 4-aminopyridine on neuromuscular transmission in disease states

The in vitro effects of 4-aminopyridine (4-AP) on neuromuscular transmission were determined by microelectrode techniques in intercostal muscles from patients with myasthenia gravis (MG) and the Eaton-Lambert syndrome (ELS), and in forelimb muscles from rats with experimental autoimmune myasthenia gravis (EAMG). In MG and EAMG, the amplitudes of miniature endplate potentials (MEPPs) and endplate potentials (EPPs) were reduced, and there was increased sensitivity to the blocking action of d-tubocurarine (dTc). In ELS, MEPP amplitude was normal but the average number of acetylcholine quanta released by nerve impulses was reduced, causing subthreshold EPPs. In EAMG muscle, 4-AP produced dose-dependent increases in EPP amplitude and in the duration of indirectly elicited muscle action potentials but no changes in MEPP amplitude and resting membrane potential. 4-AP completely reversed the postsynaptic blockade produced by dTc and EAMG. 4-AP appears to facilitate neuromuscular transmission in EAMG, MG, and ELS by increasing the neurally evoked transmitter release, thus overcoming either the pre- or the postsynaptic neuromuscular blockade.     

In vivo depletion of Lyt-2 cells fails to alter acute and relapsing EAE

The cellular mechanisms of recovery and relapses in experimental allergic encephalomyelitis (EAE) are not known. In order to determine the role of the suppressor/cytotoxic T lymphocytes (Lyt-2+) in EAE we studied the effect of in vivo depletion of this subset using monoclonal antibodies. Intraperitoneal injection of 1 mg of monoclonal antibody 2.43 resulted in rapid depletion of Lyt-2+ cells from lymph node, spleen and blood. Depletion of this subset had no effect on the kinetics of development, severity, and duration of acute EAE. Furthermore, following recovery from acute EAE administration of 2.43 did not result in development of relapses that were different in onset or severity from control animals. These results suggest that T cells of the Lyt-2 phenotype do not play a significant role in the immunoregulation of EAE.     

Further electrophysiological studies of neuromuscular transmission in an animal model of myasthenia gravis

The development of experimental autoimmune myasthenia gravis (EAMG) in rats produces a significant reduction in the amplitude of spontaneously occurring miniature end-plate potentials (MEPPs) and impulse-evoked end-plate potentials (EPPs). This junctional abnormality, however, does not impair the ability of the affected fibers to produce propagated muscle action potentials of normal amplitude. Quantal content analysis indicates that in Mg²⁺-blocked EAMG muscle the mean number of acetylcholine (ACh) quanta released per nerve impulse closely approximates the corresponding normal value. At EAMG end-plates, the depolarization produced by either saturating or nonsaturating doses of carbamylcholine was significantly less than that seen at normal end-plates, suggesting a reduced acetylcholine receptor (AChR) content. When the depolarizing response to 250 μm carbamylcholine was examined at various EAMG end-plates with different sizes of MEPPs, there was a direct correlation between the carbamylcholine-induced depolarization and the MEPP amplitude; this correlation, however, was much less pronouced in normal end-plates, which supports the concept that the MEPP size in the receptor-immunized muscle reflects the content of the functional postsynaptic AChR. In control solution, the indirect twitch tension produced in EAMG muscle was normal. The twitch tension in EAMG muscle however, was almost completely abolished by a dose of d-tubocurarine that reduced the tension in normal muscle by only 50%. When 4-aminopyridine, a drug known to increase the quantum content of EPPs, was applied to curarized normal or EAMG muscles, normal muscle contraction was restored and the decremental response to repetitive nerve stimulation was abolished. We conclude that the major defect of neuromuscular transmission in EAMG results from postsynaptic abnormalities at the end-plates, presumably secondary to reduction of the number of functional AChRs. Chronic EAMG in rats is a reasonable model of human myasthenia gravis in which a similar defect of neuromuscular transmission is present.     

Attempts to transfer experimental allergic neuritis with lymphocytes

Attempts have been made to transfer experimental allergic neuritis (EAN) both by the intraneural and the intravenous injection of cells derived from Lewis rats with the disease into naive recipients of the same strain. Lymph node cells obtained 12 and 15 days after inoculation with bovine dorsal root in Freund's complete adjuvant were injected intraneurally. A small number of demyelinated axons were observed, but clinical weakness was not evident. Lymph node cells, lymph node cells cultured with concanavalin A, or cultured spleen cells from animals with EAN were transferred intravenously to normal rats. Uncultured lymph node cells were transferred to X-irradiated animals. There were no clinical or histological differences between these recipients and controls receiving cells from rats inoculated with Freund's adjuvant alone. The findings are discussed in relation to previous reports of attempts to transmit EAN by cell transfer.     

Experimental autoimmune myasthenia gravis.

Injection of animals with purified acetylcholine receptor in complete Freund's adjuvant causes development of antibodies which crossreact with receptors in muscle. The crossreacting antibodies impair neuromuscular transmission. Animals with experimental autoimmune myasthenia gravis (EAMG) are excellent models for studying the complex mechanisms by which the autoimmune response to receptor in myasthenia gravis causes muscle weakness. This review first briefly describes the discovery of EAMG. Then, to provide the necessary perspective, receptor structure and function and properties of anti-receptor antibodies are discussed, followed by a brief review of the pathological mechanisms in EAMG.     

Analysis of lymphocytes in, and host environment of, mice showing conditioned immunosuppression to cyclophosphamide

Mice were subjected to repeated exposures to cyclophosphamide: saccharin (conditioned) or cyclophosphamide:saccharin followed by saccharin only (conditioned:extinguished). Animals in the former group but not the latter subsequently showed diminished IgG antibody-forming cells (AFC) after challenge with sheep red blood cells followed by reexposure to immunologically inert cues (saccharin). When these animals were used as irradiated recipients of syngeneic spleen lymphocytes, reconstituted irradiated conditioned mice showed augmented IgG AFC on transfer of naive spleen cells and reexposure to saccharin. The expected diminished IgG AFC response was seen when cells from conditioned mice were transferred. However, the latter cells gave augmented IgG AFC when transferred to naive irradiated mice. Both of the effects seen with cells from conditioned animals (increased IgG AFC in control recipients; decreased IgG AFC in conditioned mice reexposed to saccharin) were regulated by adoptively transferred T cells in the spleen cell population.