Accelerated degradation of acetylcholine receptor from cultured rat myotubes with myasthenia gravis sera and globulins.

Altered geometry of the neuromuscular junction and a decreased number of acetylcholine receptors appear responsible for the defect of neuromuscular transmission in myasthenia gravis. We have used cultured rat myotubes as a model to study in vitro the potential role of myasthenic globulins in the pathological process. Acetylcholine receptor content was assayed by the extent of 125I-labeled alpha-bungarotoxin binding, and acetylcholine receptor function was assayed by the sensitivity to acetylcholine iontophoresis. The half-life of the acetylcholine receptor was 18.5 hr in the presence or absence of control sera. Myasthenic sera and globulins produced a gradual reduction in acetylcholine receptors, as assessed by biochemical and electrophysiological techniques. The half-life in the presence of myasthenic sera was 6 hr. The accelerated turnover was unaffected by puromycin but was slowed by lowered temperature (18-20 degrees), interference with energy metabolism (2,4-dinitrophenol), and interference with cytoskeletal structures (colchicine and cytochalasin B). We found no electrophysiological evidence to suggest globulin blockade of acetylcholine access to the acetylcholine receptor. Our observations suggest that circulating globulins in myasthenia gravis may contribute to the functional defects of neuromuscular transmission by accelerating the rate of internationalization and degradation of surface membrane acetylcholine receptors.     

Action of anti-mouse IgE on mouse and rat mast cells.

The action of anti-mouse IgE on normal or sensitized mouse and rat peritoneal mast cells as well as on the reaginic activity of mouse and rat antisera was studied. Anti-mouse IgE induced histamine release from normal mouse peritoneal mast cells in vitro and this effect was greatly enhanced when mast cells were actively sensitized. The action of anti-mouse IgE on normal rat peritoneal mast cells was slight; it was enhanced after sensitization of these cells with mouse reaginic antibodies as well as after active sensitization. Moreover, anti-mouse IgE is able to neutralise the reaginic activity of mouse and rat antisera. The results suggest that there exist some functional and structural similarities between mouse and rat IgE.     

Sorting receptor Rer1 controls surface expression of muscle acetylcholine receptors by ER retention of unassembled alpha-subunits

The nicotinic acetylcholine receptor of skeletal muscle is composed of five subunits that are assembled in a stepwise manner. Quality control mechanisms ensure that only fully assembled receptors reach the cell surface. Here, we show that Rer1, a putative Golgi-ER retrieval receptor, is involved in the biogenesis of acetylcholine receptors. Rer1 is expressed in the early secretory pathway in the myoblast line C2C12 and in mouse skeletal muscle, and up-regulated during myogenesis. Upon down-regulation of Rer1 in C2C12 cells, unassembled acetylcholine receptor α-subunits escape from the ER and are transported to the plasma membrane and lysosomes, where they are degraded. As a result, the amount of fully assembled receptor at the cell surface is reduced. In vivo Rer1 knockdown and genetic inactivation of one Rer1 allele lead to significantly smaller neuromuscular junctions in mice. Our data show that Rer1 is a functionally important unique factor that controls surface expression of muscle acetylcholine receptors by localizing unassembled α-subunits to the early secretory pathway.     

Acetylcholine receptors and myasthenia gravis

Recent years have seen considerable progress in understanding the nature of the molecular events involved in neuromuscular transmission. The acetylcholine receptor (AChR) has been purified to homogeneity and acetylcholine-induced ion transport has been reconstituted by incorporation of pure AChR into artificial membranes. Immunization against purified AChR induces a condition, clinically and physiologically similar to the human disease myasthenia gravis, which is due to circulating anti-AChR antibodies. This model, experimental autoimmune myasthenia gravis, is proving useful for investigating the role of genetic factors in determining the immune response to AChRs and for testing various experimental approaches to specific treatment. Myasthenia gravis is an autoimmune disease in which there is loss of acetylcholine receptors at the neuromuscular junction. Anti-AChR antibodies can be detected in the majority of patients and they cause loss of AChR by a variety of mechanisms. Anti-AChR antibody is heterogeneous and not restricted in idiotype. The role of the thymus in MG is still uncertain, but recent experiments implicate the presence of a cell type in MG thymus which may be involved in autosensitization to AChR.     

Receptor aggregation induced by antilutropin receptor antibody and biological response in rat testis Leydig cells.

Antibodies against the lutropin receptor have been obtained by the monoclonal antibody technique. Mice were immunized with luteal membrane from ovaries from pseudopregnant rats, containing high lutropin receptor concentration. Hybridoma cells were obtained by fusing mouse myeloma cells with spleen cells from the immunized animal. Five clones were produced that secreted monoclonal antibodies that specifically inhibited lutropin binding to its receptor in a competitive fashion. Antibodies from three clones were capable of blocking biological response to lutropin (e.g., testosterone production by isolated rat Leydig cells). Antibodies secreted by two other clones, however, were capable of acting as Leydig cell stimulators. Immunofluorescence studies demonstrated the presence of receptor capping which may be associated with receptor-mediated testosterone production. Antagonist antibodies could be transformed into agonist by the addition of a second crosslinking anti-mouse IgG. The discovery of agonist antibodies against the receptor molecule proves that the biological information of the lutropin-receptor complex resides in the receptor and not in the hormone.     

Suppression of type II collagen-induced arthritis by monoclonal antibodies.

Some mouse monoclonal antibodies raised against chicken type II collagen suppressed or delayed the onset of chicken type II collagen-induced arthritis in DBA/1 mice. This was correlated with the suppression of anti-mouse type II collagen antibody responses following immunization with chicken type II collagen. The epitopes recognized by the suppressive antibodies were found to be present on cyanogen bromide (CB)-digested collagen peptides CB-11 and CB-12. This was also confirmed by the finding that administration of the CB-11 or CB-12 peptide suppressed the induction of arthritis.     

Anti-acetylcholine receptor antibodies directed against the alpha-bungarotoxin binding site induce a unique form of experimental myasthenia.

Of seven rat monoclonal antibodies directed against nicotinic acetylcholine receptor, the three whose binding was blocked by alpha-bungarotoxin produced acute paralysis in chicken hatchlings, whereas the four others had no effect. In the affected animals, weakness and decremental electromyographic responses appeared within 1 hr after intravenous injection and both abnormalities improved after anticholinesterase administration. No histological changes were seen in the muscle of injected animals. These data suggest that antibodies binding in relationship to the cholinergic binding site, and presumably producing pharmacologic blockade of acetylcholine receptor, may play an important role in the pathogenesis of the defective neuromuscular transmission in myasthenia gravis.     

Modulation of acetylcholine receptor by antibody against the receptor

Antibody against acetylcholine receptor induces an increase in the rate of degradation of acetylcholine receptors on a mouse cell line (BC(3)H-1) and cultured rat skeletal muscle. The increased rate of degradation results in a lowered density of acetylcholine receptors on muscle membrane and a lowered sensitivity to iontophoretically applied acetylcholine. The modulation of acetylcholine receptor is energy, temperature, and time dependent and may be related to antigenic modulation found in other systems. Acetylcholine noise analysis demonstrates that antibody against acetylcholine receptor reduces the channel mean conductance and mean open time slightly.It is concluded that antibody binds to the acetylcholine receptor, impairs its function, and induces receptor degradation. This results in a lowered density of acetylcholine receptor and a lowered sensitivity to acetylcholine. Patients with myasthenia gravis have antibodies to their acetylcholine receptor in their serum. Antigenic modulation of receptor in the muscle of patients with myasthenia gravis could contribute to the observed decrease in amplitudes of miniature endplate potentials and in muscle acetylcholine sensitivity, and the symptoms of muscular weakness.     

Modulation of experimental myasthenia gravis by IVIg

Myasthenia Gravis (MG) is an autoimmune disease mediated by antibodies directed against the acetylcholine receptor (AChR). Treatment by IVIg is effective in acute forms of myasthenia gravis. In order to determine the in vivo effects of the various fractions of human immunoglobulins, we used an experimental model of myasthenia gravis in SCID mice. To this end, thymic cells from MG patients are transferred to these mice according to a well defined protocol. When establishing of the model, we noticed the appearance of anti-AChR antibodies and the loss of AChR expression at the muscle level. After treatment with IVIgG or IVIgM, the mice displayed a lower anti-AChR antibody titer compared to control mice (albumin treated) and the loss of the AChR number at the muscle was significantly reduced. These results obtained from one MG patient indicate that the human immunoglobulin preparations induce significant effects on pathogenic parameters in the SCID mouse model. Therefore this model is interesting to approach the mechanisms of action of human immunoglobulins and deserves further investigation.     

Production and characterization of monoclonal antibodies to soluble rat lung guanylate cyclase.

Four monoclonal antibodies to rat lung soluble guanylate cyclase [GTP pyrophosphate-lyase (cyclizing) EC 4.6.1.2] have been produced by fusing spleen cells from immunized BALB/c mice with SP-2/0 myeloma cells. The antibodies were detected by their ability to bind immobilized guanylate cyclase and by immunoprecipitation of purified enzyme in the presence of second (rabbit anti-mouse) antibody. After subcloning by limiting dilution, hybridomas were injected intraperitoneally into mice to produce ascitic fluid containing 2-5 mg of antibody per ml. The four antibodies obtained had titers of between 1:1580 and 1:3160 but were detectable at dilutions greater than 1:20,000. Soluble guanylate cyclase from several rat tissues were crossreactive with the four monoclonal antibodies, suggesting that the soluble enzyme from different rat tissues is antigenically similar. The antibodies also recognized soluble lung enzyme from rat, beef, and pig, while enzyme from rabbit was not crossreactive and mouse enzyme was recognized by only one of the antibodies. Particulate guanylate cyclase from a number of tissues had only minimal crossreactivity with the antibodies. Immunoprecipitated guanylate cyclase retained catalytic activity, could be activated with sodium nitroprusside, and was inhibited by cystamine. None of the antibodies were inhibitory under the conditions examined. These antibodies will be useful probes for the study of guanylate cyclase regulation and function under a variety of physiological conditions.     

Induction of a B-lymphocyte receptor for a T cell-replacing factor by the crosslinking of surface IgD.

The observation that anti-immunoglobulin antibodies and T cell-replacing factor (TRF) have a synergistic effect on the stimulation of B lymphocytes to differentiate into antibody-secreting cells suggested to us the possibility that the crosslinking of B-cell surface immunoglobulin by antigen or anti-immunoglobulin antibody might induce the expression of a B-cell receptor for TRF. In order to test this possibility we studied whether spleen cells from mice injected with 400-800 micrograms of anti-IgD antibody 1-3 days before sacrifice had an enhanced capacity to adsorb TRF activity from a partially purified culture supernatant of concanavalin A-stimulated mouse spleen cells. We found that spleen cells from euthymic or congenitally athymic mice injected 24-30 hr prior to sacrifice with either affinity-purified goat anti-mouse IgD antibody or a monoclonal allospecific anti-IgD antibody had greater than 100 times the TRF adsorptive capacity of spleen cells from control mice. In contrast, spleen cells from anti-IgD treated DBA/2Ha mice, which have been shown to have an immune defect associated with the lack of a TRF receptor, were unable to adsorb TRF activity from concanavalin A-stimulated helper supernatants. This suggests that the crosslinking of B-cell surface immunoglobulin may induce B lymphocytes to express a receptor or receptors for TRF and thus enhance B-cell responsiveness to this helper factor.     

Incidence of anti-mouse IgG in normal subjects and patients with autoimmune thyroid disease.

Serum antibodies to mouse IgG occasionally interfere with two-site immunometric assays in which mouse monoclonal antibodies are used. We examined the titers of antibodies to mouse IgG in serum samples from normal subjects and patients with autoimmune thyroid disease (AITD) using an enzyme-linked immunosorbent assay. Anti-mouse IgG antibodies were positive in 7/119 patients with Graves' disease (5.9%) and 3/60 patients with Hashimoto's thyroiditis (5.0%). One of the 15 patients with a thyroid neoplasm (6.7%) also had antibodies to mouse IgG, as did 5/60 healthy subjects (8.3%). These antibodies were either of the IgG or IgM class. There was no significant difference in the incidence of positive anti-mouse IgG antibody between normal subjects and patients with AITD. It is important to note this high incidence of antibodies to mouse IgG due to the potential of interference with immunometric assays employing mouse monoclonal antibodies.     

Studies on monoclonal antibodies to TSH receptors--heterogeneity and pathophysiological significance of antibodies to TSH receptor

Recent studies revealed that anti-TSH receptor autoantibodies are involved in the pathogenesis of both Graves' disease and a part of hypothyroidism, but precise mechanism of action of these antibodies remained to be studied. In order to delineate the heterogeneity of these antibodies and their pathophysiological significance, we produced monoclonal antibodies to TSH receptor and studied their characteristics. Mouse monoclonal antibodies to TSH receptor were derived from spleen cells of mice immunized with partially purified human TSH receptor, which was prepared by TSH-coupled affinity chromatography of thyroid membrane solubilized with Triton X-100. By fusing spleen cells and mouse myeloma cells in the presence of polyethylene glycol and selecting with limiting dilution method, 5 hybridomas were obtained. Among 3 antibodies, which inhibited TSH binding to thyroid membrane (TSH displacing activity, TDA), 2 inhibited TSH stimulation of thyroid adenylate cyclase (AC) (human thyroid adenylate cyclase inhibitor activity, HTACI), and one showed no bioactivity. Among other 2 antibodies without TDA, 1 stimulated AC (human thyroid adenylate cyclase stimulator activity, HTACS) and the other inhibited TSH stimulation (HTACI). All activities of these antibodies were dependent on IgG concentration and disappeared by treatment of anti-mouse IgG antibodies. In addition, 4 human-human hybridomas were established by fusing human peripheral lymphocytes of patients with Graves' disease and nongoitrous hypothyroidism with human lymphoblastoid cell line. Among 2 antibodies with TDA, one antibody inhibited TSH stimulation of AC, inhibiting TSH binding competitively and another antibody stimulated AC, inhibiting TSH binding noncompetitively. Among the other 2 antibodies, which did not inhibit TSH binding but were shown to bind to TSH receptor by immunoprecipitation, one stimulated AC and the other inhibited TSH stimulation of AC. Among 2 antibodies with HTACI, one antibody with positive TDA inhibited stimulation of AC by stimulative antibodies with positive TDA, but the other without TDA inhibited stimulation of AC by both antibodies with or without positive TDA. These inhibitory antibodies did not inhibit stimulation of AC by Forskolin and Gpp(NH)p, which are known to affect other parts of receptor-AC system than receptor unit. These data suggest that anti-TSH receptor antibodies are heterogenous in the mode of binding to the receptor and in their bioactivities, and may be involved in the pathogenesis of both Graves' disease and a part of idiopathic hypothyroidism.     

Immunological relationship between acetylcholine receptor and thymus: a possible significance in myasthenia gravis.

A defined immunological cross-reaction was observed between acetylcholine receptor fraction from the electric eel, Electrophorus electricus, and two calf thymus fractions. The cross-reaction was demonstrated on the cellular level by means of the lymphocyte transformation technique, and on the humoral level, by means of the microcomplement fixation assay. In the human disease myasthenia gravis both acetylcholine receptor at the neuromuscular junction and the thymus are affected, probably by an autoimmune mechanism. The immunological cross-reaction between acetylcholine receptor and thymic components may explain the association between endplate and thymus disorders in myasthenia gravis.     

Specificities of antibodies to acetylcholine receptors in sera from myasthenia gravis patients measured by monoclonal antibodies.

The pattern of antibody specificities in sera from patients with myasthenia gravis (MG) was determined by the ability of monoclonal antibodies against defined determinants on the acetylcholine receptor molecule to inhibit binding of the serum antibodies to receptor from human muscle. We found that MG patients produce fundamentally the same pattern of specificities as that produced by animals immunized with receptor purified from fish electric organs or mammalian muscle. Most of the antibodies are directed at the "main immunogenic region' which is located on the extracellular surface of the alpha subunit and is distinct from the acetylcholine binding site. Regions on the beta and gamma subunits near the main immunogenic region are also significantly immunogenic. In one patient the proportions of antibodies to various regions are constant over time despite changes in total antibody amount and clinical state. Between patients there is no obvious correlation between antibody specificities and clinical state. These data suggest that the autoimmune response in MG is stimulated by human receptor rather than a crossreacting (e.g., viral) antigen and that in both MG and experimental autoimmune MG the pattern of specificities produced is determined by the inherently immunogenic structural features of the receptor molecule. They also suggest that the wide differences in clinical state sometimes observed between patients with the same total concentration of antireceptor antibody are due primarily to differences in endogenous factors which affect the safety factor for neuromuscular transmission rather than to the presence of especially pathogenic antireceptor specificities.     

Acetylcholinesterase of human erythrocytes and neuromuscular junctions: homologies revealed by monoclonal antibodies.

Human erythrocyte acetylcholinesterase was used to immunize mice, and hybridomas were generated by fusion of mouse spleen cells with cells of the Sp 2/0 myeloma cell line. Five independently derived hybridoma clones produced antibodies that bound to purified erythrocyte acetylcholinesterase. All of these antibodies crossreacted with human and monkey neuromuscular junctions; immunocytochemical staining patterns corresponded to the distribution of junctional acetylcholinesterase. The monoclonal antibodies fell into at least four categories based on differences in crossreactivity with neuromuscular acetylcholinesterase of rabbit, dog, calf, and guinea pig, and competition tests indicated that the antibodies defined five different antigenic sites on the acetylcholinesterase molecule. It is concluded that there is a high level of homology between the acetylcholinesterases of erythrocytes and neuromuscular junctions.     

AUTOANTIBODIES AND ANTI-MOUSE ANTIBODIES IN THROMBOCYTOPENIC PATIENTS AS ASSESSed by DIFFERENT MAIPA assays

Two MAIPA (monoclonal antibody [MAb] immobilization of platelet antigen) assays were performed to determine (a) autoantibodies to platelet glycoproteins (GP) and (b) serum antibodies recognizing mouse MAbs used in the assay. In MAIPA I, control platelets were incubated simultaneously with human serum and a mouse MAb to a platelet glycoprotein (GP IIb–IIIa, Ib–IX, Ia–IIa, IV and p24). In MAIPA II, the control platelets were incubated first with the human serum and then, after washing, with the selected mouse MAb. A series of 25 patients with autoimmune thrombocytopenic purpura (ATP) associated or not with other autoimmune states were examined. Autoantibodies (both MAIPA I and MAIPA II positive) or anti-mouse Abs (MAIPA I positive and MAIPA II negative) were frequent in both groups of patients. Statistically significant differences existed in the incidence of anti-mouse Abs between patients (56.5%) and healthy donors (10%). This suggests that their production may be related to thrombocytopenias associated with autoimmune disease. We speculate that the presence of anti-mouse antibodies could reflect an abnormality in the immunological modulation of the idiotypic network.     

Plasma Exchange for Treatment of Myasthenia Gravis: Pathophysiologic Basis and Clinical Experience

Abstract: Myasthenia gravis is an autoimmune disease characterized by production of antibodies to acetylcholine receptors located at the motor end plate in skeletal muscles. The antibodies bind and subsequently induce degeneration of these receptors. Loss of acetylcholine receptors results in inadequate contraction of muscle fibers in response to acetylcholine released from nerve terminals and clinically apparent muscle weakness. Plasma exchange removes the circulating antibodies in myasthenic patients with short‐term clinical improvement. Plasma exchange may be indicated in patients with acute exacerbation of neuromuscular weakness with bulbar or respiratory compromise, preoperative optimization prior to thymectomy, and postoperative deterioration following thymectomy or other surgical procedures. Long‐term, intermittent plasma exchange for patients who do not adequately respond to standard treatment is another evolving indication.     

Immunization of naive BALB/c mice with human beta2-glycoprotein I breaks tolerance to the murine molecule

OBJECTIVE: Immunization of naive mice with beta2-glycoprotein I (beta2GPI) leads to the generation of pathogenic anticardiolipin antibodies associated with clinical manifestations of the antiphospholipid syndrome (APS). The aim of this study was to determine whether immunization of naive mice with human beta2GPI, which shares homology with mouse beta2GPI molecules, breaks tolerance to murine beta2GPI and leads to the generation of anti-mouse beta2GPI. METHODS: Twenty-four female BALB/c mice were immunized in the footpads with 10 microg of human beta2GPI. Twelve age- and sex-matched BALB/c mice were immunized in the same manner with Freund's complete adjuvant and served as controls. The reactivity of whole sera, polyclonal IgG, and affinity-purified anti-beta2GPI IgG antibodies against human, bovine, and mouse beta2GPI was evaluated by enzyme-linked immunosorbent assay. RESULTS: High titers of anti-human beta2GPI IgG antibodies were detected 1 month after immunization. Progressively increasing titers against murine and bovine beta2GPI were recorded 1-4 months after injection. CONCLUSION: Immunization of mice with human beta2GPI resulted in the generation of antibodies reacting with human, bovine, and murine beta2GPI. The loss of tolerance to mouse beta2GPI is attributable to the high interspecies homology of beta2GPI. These results may point to molecular mimicry as a possible cause of APS.