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Human autoimmune myasthenia gravis (MG) is associated with the IL-1beta TaqI RFLP allele 2. Individuals positive for this allele have high levels of inducible IL-1beta in their peripheral blood. Here, we have characterized MG induction and the immune response elicited by Torpedo acetylcholine receptor (AChR) immunization in wild-type and IL-1beta deficient (-/-) mice. Compared with wild-type mice, IL-1beta-/- mice were relatively resistant to induction of clinical experimental autoimmune myasthenia gravis (EAMG). Draining lymph node cells from IL-1beta-/- mice showed poor proliferative capacity upon AChR stimulation in vitro. Both Th1 (IFN-gamma, IL-2) and Th2 (IL-4) cytokine responses were reduced and levels of serum anti-AChR antibodies decreased in IL-1beta-/- mice compared to wild-type mice. Taken together, these results reveal a critical role for IL-1beta in the induction of MG in mice, and support a role for IL-1beta in the pathogenesis of MG in man.

Myasthenia gravis (MG) is an autoimmune disease characterised by serum anti-acetylcholine receptor (AChR) antibodies and several pathogenic mechanisms for the action of these antibodies have been elucidated. In this study, we have analysed a possible additional mechanism for these antibodies, namely antibody-dependent cell-mediated cytotoxicity (ADCC). Using as target cells a cell line expressing AChR, we could show an increased ADCC mediated by sera from MG patients. Sera with AChR antibodies induced a higher cytotoxicity than sera from patients without these antibodies or healthy individuals. Sera from MG patients with thymoma induced a higher cytotoxic effect than sera from other patients. There was a strong positive correlation between the concentration of AChR antibodies and cytotoxicity mediated by purified IgG fractions from thymoma patients. In addition, there was a higher cytotoxicity mediated by sera from thymoma patients with extended dinucleotide repeats, (AT)n repeats, in the CTLA-4 gene. ADCC mediated by AChR antibodies may thus be another possible pathogenic mechanism that could operate in MG patients, especially in patients with thymoma.

An idiotypic network involving T and B cells bearing complementary structures has been suggested to be important for the regulation of immune response in healthy and disease situations. A previous study by the authors has demonstrated the presence of a relatively higher concentration of anti-idiotypic antibodies than of idiotypic in early myasthenia gravis (MG), suggesting that the development of an anti-idiotypic immunity is important in early MG. The present study was conducted to examine the cellular components of the idiotypic network in the same situation. T and B cells reactive to acetylcholine receptor (AChR) or to a disease-related idiotype and to an anti-idiotype were analysed in seven patients with early MG at various times after the start of the disease. The results show that a significant increase in the number of idiotype-reactive interferon-gamma-secreting T cells and a shift from AChR-reactive to idiotype- and/or anti-idiotype-reactive T cells in the patients at 6 month follow-up were noted. Such changes seem to correlate to a clinical improvement in the patients. The enhanced anti-idiotypic T-cell response and the clinical improvement in the patients may speak in favour of a role for the anti-idiotypic immunity in controlling the autoimmune response in MG, i.e., down-regulating autoantibody-producing B cells and idiotypic (AChR-specific) T cells. Thus, an immune intervention towards the enhancement of the anti-idiotypic immunity in patients might be a rewarding approach. Further studies with regard to cell interactions and immune regulations in the network are warranted.

Patients with myasthenia gravis have a high prevalence of acetylcholine receptor-specific T lymphocytes in the peripheral blood. Our earlier study shows that these T lymphocytes are stimulated to secrete interferon (IFN)-gamma and interleukin (IL)-2 in response to the autoantigen. Such stimulated T cells may be subdivided into different subsets according to the pattern of cytokine production. In the present study we have investigated the subpopulations of cells by analyzing their IL-4, IFN-gamma and IL-2 secretion pattern. Autoantigen-stimulated IL-4 secretion was found in 55% of the patients, IFN-gamma secretion in 86% and IL-2 secretion in 72%. T lymphocytes from all patients who responded with increased IL-2 secretion also showed increased IFN-gamma secretion. Stimulated IL-4 secretion was detected both in the presence and absence of stimulated IFN-gamma secretion. Depletion of monocytes/macrophages from peripheral blood mononuclear cell preparation and treatment of the cells with a mouse anti-human HLA-DR antibody abolished the secretion of IFN-gamma and IL-4. There were positive correlations between the numbers of IFN-gamma- and IL-2-secreting T cells and the numbers of B cells secreting antibodies against the acetylcholine receptor. Our results show that acetylcholine receptor-stimulated T lymphocytes secrete IL-4, IFN-gamma and/or IL-2. This T cell response is major histocompatibility complex (MHC) class II-restricted and monocyte/macrophage-dependent. Our study indicates that both Th1/Th2 or Th0 subpopulations of the T lymphocytes are involved in the autoimmune response in myasthenia gravis.

The prevalence of T and B cells reactive with the acetylcholine receptor (AChR) of human skeletal muscle was studied in 33 patients with myasthenia gravis (MG), 18 patients with other neurological diseases (OND) or autoimmune disorders (AD) and 27 age- and sex-matched healthy controls. T cell stimulation was estimated by enumerating cells secreting interferon (IFN)-gamma and interleukin (IL)-2 in response to the AChR, whereas B cell reactivity was estimated by enumerating cells secreting IgG antibodies binding to the AChR. AChR-reactive T cells were increased in the peripheral blood of patients with MG as compared to patients with OND, AD and healthy individuals. Of the patients with MG, 29/33 (87.7%) had numbers of IFN-gamma secreting cells higher than the mean +/- 2 SD of the mean of controls as compared to 4/18 (22.2%) of patients with OND or AD and 2/27 (7.4%) of the controls. The mean value of the numbers of AChR-reactive T cells in the patients with MG was 19.6/10(5) PBMC, corresponding to 1/5100 PBMC. Comparable results were obtained also for IL-2-secreting cells. Anti-AChR IgG antibody-secreting cells were detected in the blood of 30/33 (91%) of the patients with MG, 3/18 (16.7%) of the patients with OND or AD and 2/25 (8%) of the controls. The mean value of the antibody-secreting cells in MG was 11.7 cells/10(6) PBMC corresponding to 1/70,400 PBMC in the patients with MG, compared to a mean value of antibody-secreting cells in the patients with OND or AD of 0.33 and controls of 0.16 cells/10(6) PBMC.

The production of autoantibodies to the nicotinic acetylcholine receptor are responsible for many of the neurological symptoms observed in myasthenia gravis. An understanding of the structural organization of the anti-receptor antibodies may help to define the role of these antibodies in the pathogenesis of this disease. The nucleotide sequences of the heavy and light chains of three human monoclonal anti-receptor antibodies isolated from peripheral blood lymphocytes from two patients with myasthenia gravis were analyzed. In addition, the structure of an anti-idiotypic antibody was studied. The VH and VL gene segments used in the anti-receptor antibodies appear to be derived from the same repertoire as gene segments that have been found in other autoantibodies isolated from patients with various autoimmune diseases. The IgM anti-receptor antibodies are direct copies of germline gene segments, while the structures of the IgG anti-receptor antibody and the anti-idiotypic antibody appear to be mutated suggesting that they have undergone antigenic selection.

In myasthenia gravis the production of anti-acetylcholine receptor antibodies is modulated by acetylcholine receptor-specific T cells. Most B- and T-cell epitopes are located on the alpha-subunit of the receptor. In order to map the fine specificity of the antigen-specific T cells in myasthenia gravis, T-cell stimulation in response to 70 hexapeptides was studied in 24 patients and 24 healthy individuals. The hexapeptides overlapped with one amino acid and represented residues 10-84 of the NH2-terminal part of the alpha-subunit of the receptor. The IFN-gamma secretion from single T cells was used to detect T-cell stimulation. A significant difference in the T-cell response to several of the peptides was found between patients and healthy controls. The majority of the hexapeptides induced T-cell stimulation in at least one of the patients. Peptide-induced T-cell stimulation was evident in all but one of the patients. The results indicate that different epitopes and multiple T-cell clones are involved in the T-cell recognition of the acetylcholine receptor.

Auto-antibodies against the acetylcholine receptor of skeletal muscle are considered to be the cause of the neuromuscular dysfunction in myasthenia gravis. However, such auto-antibodies also occur in disease states not accompanied by neuromuscular symptoms. Patients with primary biliary cirrhosis have a high prevalence of different auto-antibodies, including antibodies against the acetylcholine receptor. In primary biliary cirrhosis, these anti-receptor antibodies are predominantly of IgM isotype. The IgM antibodies show a broader reactivity with receptors from other species than antibodies from myasthenic patients. Immunoglobulins from patients with primary biliary cirrhosis bear the same receptor antibody-associated idiotypes, but the repertoire is quantitatively different from that found in myasthenia gravis patients. The IgM receptor antibody activity in a serum from a patient with primary biliary cirrhosis could be inhibited by cardiolipin, poly[dT], poly[I], and ssDNA, whereas this antibody activity in serum from a patient with myasthenia gravis was slightly reduced only by cardiolipin. Generally, IgM antibodies from patients with primary biliary cirrhosis had a broader reactivity with polynucleotides and phospholipids than IgG antibodies and antibodies from patients with myasthenia gravis. These results indicate a difference in the fine specificity between acetylcholine receptor antibodies in primary biliary cirrhosis and in myasthenia gravis.

Antibodies binding to the acetylcholine receptor of human skeletal muscle were found in patients with different kinds of anti-mitochondrial antibodies. In patients with primary biliary cirrhosis (PBC), the antibody activity was found in monomeric and pentameric IgM as well as in IgG, whereas patients with other kinds of anti-mitochondrial antibodies had anti-receptor antibodies of predominantly IgG class. Mice transfused with immunoglobulins from patients with PBC showed a reduction of skeletal muscle receptors comparable to that found in mice who had received immunoglobulins from myasthenia gravis (MG) patients. The antibody affinity was of the same order of magnitude in MG and PBC but antibodies with multiple affinities were more common in PBC. In PBC, the anti-receptor antibody associated idiotype repertoire was markedly different from that found in MG.

Spontaneous and pokeweed mitogen-induced production of specific autoantibodies were studied in cultures of peripheral blood mononuclear cells from patients with different clinical stages of myasthenia gravis. Receptor antibody-related idiotypes and anti-idiotypic antibodies were defined by binding to mouse monoclonal anti-idiotypic and anti-receptor antibodies, respectively. Patients with severe disease had a more complete spectrum of idiotypes in serum, and cells from such patients spontaneously produced more antibody species and higher concentration of both idiotypes and anti-idiotypes than patients with mild disease. The frequencies of antibody specificities in tissue culture supernatants more closely reflected disease activity than those in serum. Tissue culture for the study of different species of autoantibodies has proved to be a useful tool for monitoring the disease and the effects of treatment.

Antibodies to cholinergic receptor structures were found in 75% of 76 Finnish and 93% of 175 Swedish patients with myasthenia gravis. The amount of antibodies showed a positive correlation to the severity of the disease, and was reduced during immunosuppressive treatment, and by thymectomy. Thymoma patients had high values. The antibody was also found in the cerebrospinal fluid. Two healthy newborn babies of myasthenic mothers had antibodies during the first weeks of life, in spite of no clinical symptoms. The occurrence of IgM antibodies before IgM antibodies in two patients during the early stages of myasthenia gravis suggests that the antibody is not a primary cause of the disease.

Fifty-eight first-degree relatives to 40 patients with myasthenia gravis were investigated regarding presence of acetylcholine receptor antibodies, anti-idiotypic antibodies against the receptor antibodies and clinical and electrophysiological signs of disturbed neuromuscular function. No relative had clinical signs of muscle weakness. The prevalence of low concentrations of receptor antibodies was 54%, of anti-idiotypic antibodies 37% and of pathological and borderline single fibre EMG 45%. No sibling, only 2/11 children and 3/14 parents were normal in all three tests. A combination of receptor antibodies and anti-idiotypic antibodies was the most common finding and was especially frequent in children. In female siblings and children there was a positive correlation between the presence of HLA-antigen A1 and/or B8 and that of receptor antibodies and anti-idiotypic antibodies. Male siblings and children showed no such correlation but had a higher frequency of pathological single fibre EMG than females.

Low concentrations of acetylcholine receptor antibodies were found in 16 out of 17 patients with primary biliary cirrhosis. Seven patients were treated or had been treated with penicillamine. Ten untreated patients had antibody levels corresponding to those found in the treated group. Our data support the presence of receptor antibodies of both IgG and IgM class.

A 50-year-old woman with muscular fatigue and monoclonal gammopathy of unknown significance was shown to have high levels of antibodies against the acetylcholine receptor of the skeletal muscle endplate. The specific antibody activity was exclusively associated with the monoclonal component. There were no objective signs of myasthenia gravis. This is the first reported case with antibody activity against the acetylcholine receptor found in a monoclonal immunoglobulin fraction.

A monoclonal anti-idiotypic antibody against an anti-acetylcholine receptor antibody from a patient with myasthenia gravis was shown to bind the cholinergic ligand alpha-bungarotoxin. This binding could be inhibited by other cholinergic ligands, both antagonists and agonists. The anti-idiotype was also able to elicit the production of anti-receptor antibodies in mice. Thus, the anti-idiotype had functional properties similar to those of the original antigen, the acetylcholine receptor.

Anti-acetylcholine receptor antibody associated idiotypes were defined by six murine monoclonal antibodies raised against purified receptor antibodies. Four of the monoclonal antibodies bound to idiotopes located within or close to the antigen binding site of the anti-receptor antibodies; the other two monoclonal antibodies were directed against framework determinants. These monoclonal antibodies recognized idiotopes present on immunoglobulins in 14-60% of patients presenting myasthenia gravis, indicating substantial idiotype sharing. These idiotopes were also found in patients with no detectable anti-receptor antibody activity in their serum. In all patients studied, the pattern of idiotypes fluctuated considerably during the course of the disease regardless of clinical symptoms. This suggests continuous modulation of the autoimmune process in myasthenia gravis.

The interaction of acetylcholine receptor antibodies with different kinds of human skeletal muscle receptor was investigated. The reaction of most receptor antibodies was strongest with receptor from a patient with myasthenia gravis and with receptor from denervated muscle. Results obtained with these receptors were well correlated. The binding of most receptor antibodies to receptor from functionally normal muscle was much weaker and also qualitatively different. In a few patients with moderate and severe clinical symptoms the existence of acetylcholine receptor antibodies was revealed only by their reaction with receptor from myasthenic muscle. One patient had a clone of receptor antibodies reacting exclusively with receptor from her own muscle.

The production of acetylcholine receptor antibody idiotypes and of antibodies directed against receptor antibody idiotopes were studied in cultures of blood mononuclear cells from 11 patients with myasthenia gravis and 11 healthy donors. The antibody specificities were defined by their reaction with monoclonal anti-receptor and anti-idiotypic antibodies. Cells from eight patients but no controls produced both kinds of antibodies. These antibodies were less frequently found in patients' sera. In no patient were antibody specificities present in serum not detected in cell cultures. It is concluded that the B-cell repertoire of specific autoantibodies is more completely disclosed by analysis of blood cell cultures than by serum antibody determinations. The results suggest an idiotype-anti-idiotype regulation of anti-acetylcholine receptor autoimmunity in myasthenia gravis.

Anti-idiotypic antibodies were prepared against purified acetylcholine receptor antibodies from two patients with myasthenia gravis. The purified idiotypes did not cross-react. Reaction with idiotypes from other patients were found in 8% and 37%, respectively, which suggests that shared idiotypic specificities occur. The anti-idiotypic IgG fractions had no receptor-like activity and did not bind cholinergic ligands. Receptor antibodies from two mothers and their newborn children with neonatal myasthenia gravis showed marked differences in the reactions with an anti-idiotypic antibody. This suggests that not passive transfer of maternal antibodies but a transient synthesis of a receptor antibody with a different specificity is an important factor in the pathogenesis of neonatal myasthenia gravis.

Acetylcholine receptor antibody was measured in serum and IgG fractions from patients with myasthenia gravis using different analyses for antibody interfering with the toxin binding site and for antibody directed against other sites. No correlation was found between the concentration of receptor antibody as measured with the two assays. The different antibody activities also showed different isoelectric spectra. Receptor antibody was purified about 1000 times from IgG by affinity chromatography on a partially purified human skeletal muscle receptor preparation. The purified antibody was homogeneous in SDS-gel electrophroesis and showed a polyclonal pattern in agarose gel electrophoresis and isoelectric focusing. Both kinds of light chains were demonstrated. The results imply that the acetylcholine receptor antibody found in myasthenia gravis is heterogeneous and of multiclonal origin.

Plasma exchange (PE) was performed in 6 patients with myasthenia gravis (MG). Acetylcholine receptor (AChR) antibodies and other MG-correlated antibodies were quantified each day before, during and after PE. The antibody concentrations decreased during PE with a consisting increase between two exchanges. Repeated PE induced a transient decline in the AChR antibody concentration. The clinical improvement was maximal during the week after PE and correlated in time with increasing concentrations of AChR antibodies.

Acetylcholine receptor antibodies were studied in the serum of 21 myasthenic patients. In 18 cases antibodies directed against sites other than the toxin binding were present whereas in 10 cases only there was a measurable inhibition of the ligand binding site. These 10 sera were from the 6 patients in stage IIB, III and IV and from 4 of the 12 patients in stage IIA. Antibodies against both non-toxin and ligand binding sites were measured in IgG subclasses. Most of the antibodies of the first type belonged to either subclass 1 or 3. They were, however, never absent from subclasses 2 and 4. Antibodies of the second type were not found in subclasses 2 and 4 except in one case. In 3 cases they were present exclusively in subclass 3. In 3 patients there was no correlation between the subclass distribution of the antibodies for the different binding sites.

Antibodies against the acetylcholine receptor were found in a patient with primary biliary cirrhosis. The patient had no clinical or electrophysiological evidence of disturbed neuromuscular function. The antibodies were of both IgG and IgM isotype. Following passive transfer, these antibodies showed the same capacity to bind in vivo to mouse muscle receptors as immunoglobulins from patients with myasthenia gravis. The affinity of the antibodies was high and comparable to that found in myasthenia gravis patients.

Lymph IgG from patients with myasthenia gravis was shown to inhibit the binding of radio-labelled Naja naja siamensis alpha-neurotoxin to membrane fragments from the electric organ of Torpedo marmorata. This effect could be related to IgG subclass 3 and to the F(ab')2 fragment. These findings confirm the involvement of antibodies of postsynaptic structures in myasthenia gravis.

Forty patients with myasthenia gravis were HLA tissue typed and the amount of anti-acetylcholine receptor protein antibody determined. Sera from seven patients were tested for antibody titer by repeated determinations and the serum concentration was found to be stable. Patients with thymoma had higher titers than patients with normal thymus histology or hyperplasia. Individuals with thymoma lacking HLA-B8 were found to have a higher concentration of antibodies than HLA-B8 negative individuals with hyperplasia. No statistically significant differences were obtained when comparing the amount of antibody in HLA-B8 positive and negative individuals. Titers in patients subjected to thymectomy more than 8 years before sampling did not deviate from those in non-thymectomized myasthenics.

A specific immunoglobulin, the receptor antibody, can be found in most patients with myasthenia gravis. In order to study the kinetic properties of this antibody, serial determinations of receptor antibody, total IgG and IgG 3 were made during drainage of thoracic duct lymph in three patients. The values obtained were used in a mathematical model to calculate some kinetic parameters. Values for T 1/2 and fractional rates of synthesis and catabolism obtained for total IgG and IgG 3 by this method were shown to agree with those found with other techniques. Most of the receptor antibody activity was found in the IgG 3 fraction but the receptor antibody had a shorter T 1/2 and higher fractional rates of synthesis and catabolism than IgG 3. These kinetic characteristics are consistent with rapid variations in plasma concentration of the receptor antibody. The cause of this rapid turnover could be strong antigenic stimuli and rapid elimination by the antigen, the cholinergic receptor protein.

Aspects of acetylcholine receptor immunology and circulating receptor antibodies are reviewed with regard to both human and experimental myasthenia gravis. Receptor antibodies that have negligible cross-reactivity with skeletal muscle receptor can nonetheless cause destruction of the postsynaptic motor endplate area. Antibodies to mammalian skeletal muscle receptor can bind in-situ receptors. Transfer of myasthenic IgG increases neurophysiologic symptoms in rabbits showing slight signs of experimental myasthenia gravis, suggesting a block of in-situ receptors. Determination of receptor antibodies using RIA tests and partially purified human skeletal muscle receptor has been evaluated in the diagnosis of myasthenia gravis. Ninety percent of Swedish myasthenic patients in one study were found to have receptor antibodies. A rough correlation has been found between antibody titer and the severity of disease. Immunosuppressive treatment and thymectomy decrease the titer. In patients with thymoma, high antibody titers remain. When taken together, antibody-titer determination and electrophysiologic tests--particularly single-fiber electromyography--are very valuable diagnostic tools.