Plasma from myasthenia gravis patients reduces acetylcholine receptor agonist-induced Na flux into TE671 cell line

Plasma from myasthenia gravis patients was tested for its ability to inhibit agonist-induced 22Na+ influx into the TE671 cell line that expresses human acetylcholine receptors. Reduced 22Na+ influx correlated weakly with the total anti-acetylcholine receptor antibody level in the plasma, and was also related to the presence of antibody directed against the agonist binding site, as detected by inhibition of 125I-alpha-bungarotoxin binding. However, in some cases there was inhibition of 22Na+ flux without evident anti-alpha-bungarotoxin binding site antibody. We conclude that in most patients antibodies that interfere with 22Na+ influx do so by blocking the agonist binding site. However, in some cases antibodies may be directed at the Na+ ion channel or some important functional determinant.     

A muscle acetylcholine receptor is expressed in the human cerebellar medulloblastoma cell line TE671

The human neuromedulloblastoma cell line TE671 is shown by single-channel recordings to express nicotinic acetylcholine receptors (AChRs) that are blocked by alpha-bungarotoxin (alpha Bgt). These AChRs do not react with antisera to the alpha Bgt-binding protein of brain or with monoclonal antibodies (mAbs) to brain nicotinic AChRs that do not bind alpha Bgt. TE671 AChRs do react with autoantibodies to muscle AChRs from myasthenia gravis patients and with mAbs to muscle AChRs, including mAbs specific for extrajunctional AChRs. AChRs. AChRs purified from TE671 cells are composed of 4 kinds of subunits corresponding to those of muscle AChR. Sequences of cDNAs for the ACh-binding alpha subunit and the delta subunit of this AChR further identify it as muscle AChR. Expression of TE671 AChR can be up-regulated by nicotine and dexamethasone, and down-regulated by forskolin.     

Modulation of acetylcholine receptor function in TE671 (rhabdomyosarcoma) cells by non-AChR ligands: possible relevance to seronegative myasthenia gravis

The acetylcholine receptor (AChR) is the main target antigen in myasthenia gravis (MG), but about 15% of patients with typical, immunologically mediated MG do not have detectable anti-AChR antibodies. Previous studies showed that plasma from these ‘seronegative’ patients (SNMG) reduced AChR function in the human AChR-expressing TE671 cell line, and it was proposed that SNMG plasmas may act indirectly via phosphorylation of AChR. We show here that substances such as the β₂-adrenergic agonist, salbutamol, calcitonin-gene-related-peptide (CGRP), and cholera toxin, that increase intracellular cAMP via binding to specific cell-surface receptors, reduced AChR function in TE671 cells. Moreover, non-specific activation of cell surface proteins by lectins achieved similar results. These observations lead us to hypothesise that SNMG immunoglobulins act in TE671 cells by cross-linking of specific cell surface antigen(s) resulting in generation of intracellular cAMP and/or other second messengers. The role of such antibodies at the neuromuscular junction in vivo could be reduction in AChR function by desensitization and/or damage to the postsynaptic membrane following complement activation.     

Purified IgG from seropositive and seronegative patients with myasthenia gravis reversibly blocks currents through nicotinic acetylcholine receptor channels

The mechanism of block of nicotinic acetylcholine receptor (nAChR) channels by purified antibodies from patients with myasthenia gravis (MG) was investigated by using an ultrafast system for solution exchange at outside-out patches. IgG of MG patients and controls was purified by using protein A–Sepharose columns. Probes from 9 seropositive MG patients and 3 seronegative MG patients were tested. As a preparation, cultured mouse myotubes expressing the embryonic-type nAChR channels were used. Twenty-millisecond pulses of 1.0 mM ACh were applied repetitively to outside-out patches. Outside-out patches were preexposed with IgG in concentrations between 0.1 and 200 mg/L during application of ACh pulses. The peak current amplitude was reduced to values between 6% and 71% of control for the 9 seropositive and 3 seronegative MG patients. The block was concentration dependent and fully reversible after washout of antibodies. Incubation with IgG from different control patients did not reduce the peak current amplitude. In addition, our findings with purified IgG from seronegative MG patients support the idea of the immunopathogenesis of this disorder and may allow the development of a diagnostic test for seronegative MG patients.     

Antibody-secreting cells to acetylcholine receptor and to presynaptic membrane receptor in seronegative myasthenia gravis

Peripheral blood and bone marrow from seronegative and seropositive myasthenics were evaluated for antibody-secreting cells (ASC). Cells secreting antibody to acetylcholine receptor (AchR) and to presynaptic membrane receptor (prsmR) were counted using an immunospot assay. Immunoglobulin G (IgG) anti-AchR ASC were present in peripheral blood lymphocytes (PBL) from nine of 13 seronegative and nine of 12 seropositive myasthenics and in bone marrow lymphocytes (BML) from nine of 13 seronegative and eight of 12 seropositive myasthenics. The mean number of IgG anti-AchR ASC was lower for seronegative than for seropositive patients (P < 0.01 for PBL and P < 0.0001 for BML). In seropositive patients the mean number of IgG anti-AchR ASC was higher for BML than for PBL (P < 0.01); in seronegative patients it was not. IgG anti-prsmR ASC were detected in PBL from four of eight seronegative and six of eight seropositive myasthenics and in BML from three of eight seronegative and five of eight seropositive patients. The mean number of IgG-anti-prsmR ASC did not differ between seronegative and seropositive patients for PBL but for BML the value was higher for seropositive than for seronegative patients (P < 0.01). We conclude that seronegative myasthenia gravis is an autoimmune disease and that ASC to AchR and to prsmR are present both in the blood and the bone marrow in seronegative patients as in seropositive ones. A major difference between the groups lies in the significantly greater number of ASC found in the bone marrow in the seropositive cohort.     

Cellular response to human acetylcholine receptor in patients with myasthenia gravis

A preparation of human skeletal muscle acetylcholine receptor (AchR) was used in vitro as an antigen to stimulate lymphocytes from patients with myasthenia gravis (MG). Clinical data obtained from the patients included duration and severity of disease; history of steroid treatment or prior thymectomy; and the presence of thymoma. Lymphocytes from patients with MG showed a significantly higher response to human AchR antigen than did lymphocytes from control subjects. Previous studies of cellular response to AchR have used receptor prepared from eel or ray electric organs. By stimulating lymphocytes from MG patients with a preparation of human AchR, we have come one step closer to documenting a possible contribution of a cellular immune response to the pathogenesis of MG.     

T cell recognition of muscle acetylcholine receptor in ocular myasthenia gravis

We examined the proliferative response of blood CD4(+) cells to muscle acetylcholine receptor (AChR) subunits and the epitope repertoire of the epsilon and gamma subunits, in ocular myasthenia gravis (oMG) patients and healthy subjects. oMG patients seldom recognized all subunits. The frequency and intensity of recognition was the same for all subunits, irrespective of the disease duration. The responses in oMG were lower than in generalized myasthenia gravis. Healthy subjects had frequent, low responses to one or more subunits. oMG patients recognized several epitopes on the gamma and epsilon subunits, that partially overlapped those recognized in gMG. The subunits and epitopes recognized by individual oMG patients changed over time. Thus, oMG patients have minimal and unstable sensitization of anti-AChR CD4(+) cells, in agreement with their low and inconsistent synthesis of anti-AChR antibody.     

Presentation of endogenous acetylcholine receptor epitope by an MHC class II-transfected human muscle cell line to a specific CD4 T cell clone from a myasthenia gravis patient

Muscle or thymic myoid cells, if induced to express MHC class II in addition to endogenous acetylcholine receptor (AChR), might present epitopes derived from the AChR to specific CD4⁺ T cells. These T cells could in turn initiate or maintain the anti-AChR response that is responsible for AChR loss in myasthenia gravis (MG). We transfected the AChR⁺ TE671 (rhabdyyosarcomyosarcoma) cells with HLA-DR4 and co-cultured them with the DR4-restricted, CD4⁺ T cell clone (PM-A1; raised from a hyperplastic thymus of an MG patient and previously shown to recognise all forms of the AChR that contain the sequence α144–156). Significant T cell activation, demonstrated both by ³H-thymidine incorporation and by lysis of the TE671 cells, was found in the presence of added α144–156 and, more importantly, in the absence of exogenous antigen. These results show that MHC class II-expressing muscle or other AChR-expressing cells could present endogenous AChR to pathogenic T cells. This process may be important in the aetiology of MG.     

Anti-acetylcholine receptor (AChR) antibodies measurement in myasthenia gravis: The use of cell line TE671 as a source of AChR antigen

Acetylcholine receptor (AChR) from the human rhabdomyosarcoma cell line TE671 was compared with that of human ischaemic muscle AChR as a source of the antigen for the diagnosis of myasthenia gravis (MG). The sera, which were anti-TE671 3ell AChR antibody-negative, all came from patients with low anti-human muscle AChR antibody titers. None of the sera that were seronegative as a result of the human muscle AChR RIA became positive with TE671 cell AChR. The overall sensitivity was 7% less using TE671 cell AChR. The lower sensitivity was observed irrespective of the clinical form of MG. It also appeared from this study that epitopes specific to the junctional isoform of human AChR are essential for the detection of low antibody titers, which accounts for this feature, since TE671 cells only express the extrajunctional isoform of AChR in the surface membrane. Accordingly, AChR from cell line TE671 cannot replace human muscle AChR in the conventional diagnostic immunoprecipitation RIA. There are, however, many other useful implications of AChR from cell line TE671.     

Mechanism of block of nicotinic acetylcholine receptor channels by purified IgG from seropositive patients with myasthenia gravis

OBJECTIVE: To clarify the mechanism of block of nicotinic receptor channels by myasthenic antibodies. BACKGROUND: Nicotinic acetylcholine receptor (nAChR) channel currents are functionally blocked by purified immunoglobulin G (IgG) of patients with myasthenia gravis (MG). METHODS: The molecular mechanism of block of IgG fractions containing antibodies to nAChR channels was tested with the patch-clamp technique in combination with a system for ultrafast solution exchange. For the experiments, outside-out patches from cultured mouse myotubes that express embryonic-type nAChR channels at their surface were used. RESULTS: Incubation of outside-out patches with purified IgG from four myasthenic patients blocked nAChR channel currents activated by the application of 1.0 mM ACh reversibly. The peak current amplitude and the time course of block of nAChR channels decreased with increasing concentrations of IgG. The block became at least partly irreversible if incubation time of outside-out patches exceeded 2 minutes. For the block of nAChR channel currents with a-bungarotoxin, a similar mechanism of block was found. CONCLUSIONS: The reversibility of functional block of nAChR channel currents by myasthenic IgG depended strongly on the incubation time of the receptors with antibodies. Interaction of myasthenic antibodies with nicotinic receptors may proceed in several stages from a low-affinity reversible to a high-affinity irreversible binding.     

Antibodies in sera from patients with myasthenia gravis do not bind to nicotinic acetylcholine receptors from human brain

Nicotinic acetylcholine receptors (AChRs) from brains of chickens and rats have recently been purified and characterized (Whiting and Lindstrom, Biochemistry, 25 (1986) 2082-2093; J. Neurosci., 6 (1986) 3061-3069; Proc. Natl. Acad. Sci. U.S.A., 84 (1987) 595-599). Using both antisera and monoclonal antibodies prepared to AChRs from rat brain, we have demonstrated the existence of a homologous AChR in human brain. Here we report that antibodies to muscle AChRs in the sera of patients with myasthenia gravis (MG) do not bind to AChRs from human brain. Similarly, there was no binding of sera from patients with Guillain-Barré, amyotrophic lateral sclerosis, multiple sclerosis, or Lambert-Eaton myasthenic syndrome. Additionally, no binding of any of these sera to the alpha-bungarotoxin (alpha-Bgt) binding protein from human brain could be detected. This data is consistent with other data using antibodies to AChRs from muscle and nerve in demonstrating that the AChR in brain is antigenically distinct from the AChR in skeletal muscle AChR, and, together with the lack of central neurological symptoms in MG, suggests that the low concentrations of anti-AChR antibodies in the cerebrospinal fluid of MG patients do not bind to AChRs in brain.     

Anti-acetylcholine receptor antibodies block bungarotoxin binding to native human acetylcholine receptor on the surface of TE671 cells

We assayed sera from 20 myasthenics of various clinical stages and anti-acetylcholine receptor (AChR) antibody levels for their ability to affect bungarotoxin (BGT) binding to native human AChR on the surface of TE671 cells. Thirty-five percent of sera blocked BGT binding to the AChR, some at a dilution of up to 1:1000. The 7 patients whose sera blocked toxin binding were all generalized myasthenics with particularly severe disease, 6 of whom had had myasthenic crisis at some point in their course. No ocular myasthenics had blocking antibody. Blockade of toxin binding by myasthenic antibody to TE671 cells resembled blockade produced by unlabeled toxin in being irreversible with washing. There was little correlation between ability to block toxin binding and amplitude of the AChR binding antibody. These data are consistent with the hypothesis that patients with more aggressive generalized myasthenia preferentially have anti-AChR antibody that blocks toxin binding.     

Demonstration of a functional motilin receptor in TE671 cells from human cerebellum

BACKGROUND: Our laboratory has described the presence of motilin receptors in the rabbit cerebellum. We discovered its presence in the human TE671 cell line, which is of cerebellar origin. METHODS: Cytosolic Ca(2+) fluxes were monitored on a confocal microscope in cells loaded with Indo-1 and stimulated with motilin under various conditions. Binding studies were performed with 125I-[Nle(13)]porcine motilin. Using primers, PCR for the motilin receptor was performed. RESULTS: Cells responded to motilin after 45+/-20 s. At different concentrations of motilin (10(-8), 10(-7), 10(-6.5), 10(-6) and 10(-5) M) the percentage of responding cells was 0+/-0, 0.6+/-1.5, 4.9+/-4.7, 21.7+/-15 and 35.7+/-12, respectively. The response was blocked by the motilin antagonists [Phe(3), Nle(13)]po-motilin (0.8+/-1.8%) and GM-109 (0.0+/-0.0%) and mimicked by the agonist ABT-229 (23.6+/-15%). After stimulation with motilin, ABT-229 or [Phe(3),Leu(13)]po-motilin, but not with the antagonist GM-109, cells were desensitized. The response to motilin persisted in Ca(2+)-free solution (22.8+/-14.7%), was not affected by nifedipine (44+/-11%) but was abolished by incubation with thapsigargin (0+/-0%). Neither ryanodine, nor a previous stimulation with caffeine (0+/-0%) in Ca(2+)-free Krebs, nor both could block the response to motilin (28, 32.0+/-5.7, 41.3+/-6.1%, respectively). Binding studies revealed two binding sites for motilin, with a pK(d) of 8.9+/-0.05 and 6.11+/-0.61 (n=4). There were 100 times more low than high affinity receptors per cell. The presence of receptor mRNA was confirmed by PCR. CONCLUSION: Functional motilin receptors are present in TE671 cells. The response requires intracellular IP(3)-sensitive Ca(2+) stores. These cells may serve as a model of the central motilin receptor.     

Antibody to acetylcholine receptor in canine and human myasthenia gravis: differential cross-reactivity with human and rabbit receptor

Anti-acetylcholine receptor (AChR) antibody (ab) was found in the serum of a dog with acute myasthenia gravis (MG) by the use of Cowan 1 strain Staphylococcus aureus to bind radiolabeled anti-AChR ab-AChR immune complexes. Fifteen months later, when the dog was in remission, there was only a very low level of the anti-AChR ab. These observations strengthen the contention that anti-AChR ab is important in the pathophysiology of myasthenia gravis. Higher titers of the canine ab were measured with rabbit than with human AChR, whereas 17 human MG sera, selected to represent a wide range of anti-AChR ab titers, were all more reactive with human AChR. The degree of cross-reactivity of human anti-AChR ab with rabbit AChR varied widely, indicating a heterogeneous population of anti-AChR ab molecules in human myasthenia gravis sera.     

Skeletal muscle antibodies in patients with a thymic tumour but without myasthenia gravis

Sera from 9 patients with a primary thymic tumour but without myasthenia gravis (MG) at the time of thymectomy were examined for skeletal muscle antibodies. Antibodies to a citric acid extract (CA), associated with the presence of a thymic lymphoepithelioma in MG patients, were detected in 3 sera. AChR antibodies were detected in 5 sera, the concentration markedly elevated in 1 of them. Sarcolemmal and cross-striational antibodies were detected in 2 and 3 sera, respectively. Four of the 5 patients with thymic tumours other than a lymphoepithelioma had AChR antibodies, and 3 of them also had CA antibodies.     

Myasthenia gravis patients, but not healthy subjects, recognize epitopes that are unique to the epsilon-subunit of the acetylcholine receptor

Myasthenia gravis (MG) is an autoimmune disease characterized by deficits in neuromuscular transmission due to antibody-mediated damage of the acetylcholine receptor (AChR). We examined the in vitro immune response of peripheral blood mononuclear cells isolated from MG patients (n=38) and healthy nonmyasthenic subjects (n=31) to epitopes on the alpha-, epsilon-, and gamma-chains of the AChR. The epsilon- and gamma-epitopes tested represent regions with little sequence homology to the alpha-chain, and little sequence homology between the epsilon- and gamma-chains. No differences were observed in the immune response of MG patients and healthy subjects to any of the alpha-chain epitopes tested. Serial studies of the immune response to the alpha-peptides suggest that epitope spread does occur over time. Cells from MG patients were stimulated by the epsilon- and gamma-chain peptides, although the response was weaker than that to the alpha-peptides. Cells from healthy subjects showed reactivity to gamma-chain peptides only; none of the healthy subjects responded to the epsilon-chain peptides tested. Differences between the epsilon- and gamma-chains may be important in the development of MG, because only MG patients respond to epitopes that are unique to the epsilon-subunit.     

Rabbit antiserum to a citric acid extract of human skeletal muscle staining thymomas from myasthenia gravis patients

Rabbit antiserum to a citric acid extract of human skeletal muscle (CA) stained both the cell membrane and the cross-striational bands of skeletal muscle cells. The rabbit antiserum also stained the cell membrane of epithelial thymoma cells from myasthenia gravis (MG) patients. Normal and hyperplastic thymus tissue were not stained, apart from scattered myoid thymic cells. Absorption of the antiserum with CA abolished staining of the thymoma, indicating that human skeletal muscle and epithelial thymoma cells possess common antigens.     

In vitro synthesis of antibodies to acetylcholine receptor by Epstein-Barr virus-stimulated B-lymphocytes derived from patients with myasthenia gravis

Peripheral blood lymphocytes and B-cells were obtained from patients with myasthenia gravis and stimulated in vitro with either pokeweed mitogen or Epstein-Barr virus (EBV), respectively. EBV stimulation of B-cells caused a production of antibodies to acetylcholine receptor in 15 of the 25 myasthenia gravis patients: the EBV stimulation of B-cells was more effective in this regard than the pokeweed mitogen stimulation of peripheral blood lymphocytes. The in vitro synthesis of anti-acetylcholine receptor antibodies was found to be positively correlated with both the patients' sera antibody titers and with the disease severity.