Anti-Acetylcholine Receptor Antibody in Myasthenia Gravis Relationship to Disease Severity and Effect of Thymectomy

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1 J. CIiii. Biochem. Nutr., 3, , 1987 Anti-Acetylcholine Receptor Antibody in Myasthenia Gravis Relationship to Disease Severity and Effect of Thymectomy Fumiyo MATSUBARA-MORI,1,* Mitsuhiro OHTA,1 Kiyoe OHTA,1 Hiroshi NISHITANI,2 and Kyozo HAYASHI3 Clinical Research Center, and 2Department of Neurology, Utano National Hospital, Narutaki, Kyoto 616, Japan Department of Biology, Gifu Pharmaceutical Gifu 502, Japan University, (Received July 22, 1987) Summary Levels of anti-acetylcholine receptor (AChR) antibodies were investigated in sera of 272 patients with myasthenia gravis (MG) and 75 patients with other autoimmune diseases by the immunoprecipitation method (RIA) using rat denervated muscle AChR as an antigen. Anti- AChR antibodies were found in over 7O% of the MG patients but not in those with other autoimmune diseases except for only 2 cases with border-line titer. Most patients with ocular MG without thymoma had insignificant levels of antibody titers. In patients with generalized MG, both the mean values of antibody titer and percentages positive for the antibody were roughly correlated with clinical seventies. Antibody titers and clinical features were estimated for a long-term period on MG patients who underwent thymectomy (24 cases with thymoma and 48 cases without thymoma). After the operation, anti-achr antibody levels tended to decrease significantly, corresponding to the clinical improvement; moreover, patients with thymic hyperplasia showed better clinical improvement than those with thymoma. Key Words: anti-acetylcholine receptor antibody, myasthenia gravis, thymectomy, thymoma, autoimmune disease Myasthenia gravis (MG) is a disorder characterized by easy fatigability and muscular weakness caused by a neuromuscular junction defect. MG patients have elevated levels of serum antibodies to acetylcholine receptor (AChR), while sera from normal subjects or patients with other disorders do not have significant titers of anti- *To whom correspondence should be addressed. 265

2 266 F. MATSUBARA-MORI et al. AChR antibodies [1-4]. Moreover, anti-achr antibodies have been reported to bring about not only a defect in neuromuscular transmission, but also a loss of available AChRs on the postsynaptic membrane [5-7]. Thus, the etiological significance of anti-achr antibody in MG has been strongly suggested, but the etiology of initiation of the immune response to AChR remains unclear. Thymic hyperplasia and thymoma are frequently implicated in MG. At present, the surgical removal of the pathological thymus gland or thymoma is one of the established therapeutic methods for MG [8-10]. Thymectomy increases the chance of clinical remission or improvement, particularly if performed in the early stage of the disease [11, 12]. Although it has been suggested that the effects of thymectomy may be due to the suppression of cell-mediated and/or humoral immunity to auto-achr through the alteration of T-cell subsets, the reports have not come to an agreement in regard to the actual effects on T-cell subsets [13-15] or concentration of anti-achr antibody [3, 16-21]. In this paper, we tested a large number of Japanese MG sera for anti-achr antibodies by immunoprecipitation method. Furthermore, these antibody titers were analyzed in relation to the clinical features and to the significance of thymectomy in MG. MATERIALS AND METHODS Subjects. Sera were obtained from 272 MG patients (46 with thymoma and 226 without thymoma). The diagnosis of MG was confirmed by a positive response to edrophonium, a decrementing motor action potential in response to repetitive nerve stimulation, and the jitter phenomenon as seen in single-fiber electromyography [22]. MG patients were classified into ocular type, remission type, and generalized mild, moderate, or severe type according to the clinical severity at the time of sampling. Sera from 48 patients with thymic hyperplasia and 24 patients with thymoma (mainly lymphoepithelial thymomas), who had positive anti-achr antibody titers and underwent the thymectomy, were sequentially examined for antibody titers in pre- and postoperative sera. Seventy-five patients with autoimmune disorders other than MG were also examined; 2 with hyperthyroidism, 56 with systemic lupus erythematosus (SLE), 5 with progressive systemic sclerosis, 10 with Sj ogren's syndrome, and 2 with dermatomyositis. Control subjects for the assay consisted of 70 sera from healthy persons. Assay for anti-achr antibody. a-bungarotoxin was purified from crude venom of the Formosan krait (Bungarus multicinctus) (Sigma Chemicals Co., USA) by Sephadex G-50 and carboxymethyl-cellulose chromatography [23]. The homogeneity of a-bungarotoxin was determined by polyacrylamide gel electrophoresis and amino acid analysis. AChRs were prepared from rat denervated skeletal muscles by the method of Almon and Appel [24]. The material with binding capacity for [125I]a-bungarotoxin (4.5 x 10-9 M) was stored in aliquots at - 80 C until used. J. Clin. Biochem. Nutr.

3 ANTI-AChR ANTIBODY IN MYASTHENIA GRAVIS 267 In the assay system, to a solution of 300,ul of AChR preparation, which was diluted with 200 pl of Buffer I (0.05 M Tris-HCI, ph 7.4, containing 0.1 M NaCI, 0.1% Triton X-100, trasylol (100 U/ml), 1 mm PMSF and 0.02% sodium azide), 50 or 100 p1 of myasthenic sera diluted with Buffer I (1:100, total 100 pl with normal pooled sera diluted with Buffer 1(1:100)) were added and incubated for 16 h at 4 C. After the addition of 50 pl of [125IJa-bungarotoxin (0.5 pmol), the mixture was further incubated for 4 h at 37 C, followed by incubation for 16 h at 4 C with an optimal amount of anti-human IgG rabbit serum (r-chain specific). After the resulting complexes were precipitated by centrifugation (3,500 rpm) for 10 min at 4 C, the pellets were washed twice with 1.0 ml of saline solution. Radioactivity in the pellets was measured in an auto-gamma counter (Packard, type 5255), and the titers of anti-achr antibodies in the sera were estimated from the slope of two radioactivity counts at 50 and 100 pl of samples. Background counts in the control (without AChR) were subtracted from the total counts. The titer of anti- AChR antibody was expressed in terms of pmol of [125IJa-bungarotoxin binding sites precipitated per milliliter of serum. Values above the mean of normal controls plus 2SD ( pmol/ml) were considered positive. All experiments were carried out in duplicate. RESULTS Titers of anti-achr antibodies in patients with autoimmune diseases other than MG are shown in Fig. 1. Most sera had no antibodies except for only 2 cases of SLE with border-line values ( pmol/ml). Anti-AChR antibody titers in relation to the clinical severity of MG patients with or without thymoma are shown in Fig. 2, and mean values and frequency of these antibodies are summarized in Table 1. Frequency of antibodies was markedly higher in patients with thymoma (88%) than without (66 o). Levels of anti- Fig. 1. Titers of anti-achr antibody in 75 patients with autoimmune diseases other than MG. The dotted line represents normal upper limit (0.8 pmol/ml). SLE, Systemic lupus erythematosus; PSS, progressive systemic sclerosis. Vol. 3, No. 3, 1987

4 268 F. MATSUBARA-MORI et al. Fig. 2. Titers of anti-achr antibody and clinical states in 272 patients with MG. The horizontal axis represents the concentration of anti-achr antibody on a log scale. The dotted line and short vertical bars represent normal upper limit (0.8 pmol/ml) and mean values, respectively. Table 1. Mean values of and percentages positive for anti-achr antibody in the sera of patients with MG. bodies in the ocular MG patients without thymoma (33 juvenile type, 32 adult type) were negative except for 7 patients (11) with low titers. In generalized MG, the mean values of antibody titer and percentages positive for the antibody were roughly correlated with clinical severity. Particularly, there was a significant difference in the mean and frequency between mild and moderate groups. Several cases in clinical remission still showed significant, though not high, titers. J. Gun. Biochem. Nutr.

5 ANTI-AChR ANTIBODY IN MYASTHENIA GRAVIS 269 We analyzed the relation between the fluctuation in clinical state and anti- AChR antibody titers for a long-term period after thymectomy in 72 MG patients (48 without thymoma (Fig. 3A) and 24 with it (Fig. 3B)). A fairly good correlation was observed between the decrease in the antibody titer and the postoperative period in individuals. Namely, the longer the postoperative period, the lower the level of the antibody titer, especially in patients who had undergone the operation within the last 6 years. On the other hand, some of the patients who had received the operation over 6 years ago still had rather high antibody titers and did not show any clinical improvement. The monthly change in anti-achr antibody titer as a percentage of the preoperative value was studied in 19 MG patients without thymoma following thymectomy (Fig. 4). The titers of antibody decreased rapidly to 79% of the preoperative value within one month after the operation, and thereafter gradually to 58% within about 2 years. A temporary increase was seen around the third month, at which time unstable clinical features are often observed in many cases. The effects of thymectomy on the clinical state of patients (40 without thymoma, 21 with thymoma) as assessed annually are summarized in Table 2. Follow-up studies on these patients for more than one year after the operation were carried out for the evaluation and comparison between the pre- and postoperative clinical states. The frequency of remission and clinical improvement of MG that occurred after the operation was higher in non-thymoma cases (70%) than in thymoma cases (57%). Particularly, there was a markedly difference in the percentage of patients who went into remission between non-thymoma and thymoma groups. A B Fig. 3. Serial anti-achr antibody titers and postoperative periods in myasthenic patients without thymoma (A) and with thymoma (B). The titers in each individual person are linked by the solid line. Vol. 3, No. 3, 1987

6 270 F. MATSUBARA-M RI et a!. Fig. 4. Changes in anti-achr antibody titers in 19 myasthenic patients without thymoma after thymectomy. Each monthly value is estimated as a mean percentage of the preoperative titer. Table 2. Effects of thymectomy on clinical features of patients with MG. * Periods after thym ctomy. DISCUSSION In this study, we determined the levels of serum anti-achr antibodies using the immunoprecipitation method [2], by which we obtained a better correlation between the clinical features and the antibody titer compared with the concanavalin A-Sepharose method [25, 26] used in an investigation on neonatal MG [27, 28] and in a case involving a myasthenic crisis [29]. Anti-AChR antibody seems to be a diagnostic valuable indicator for MG, since insignificant titers were found in the most sera from patients with autoimmune disorders other than MG. Although it is more sensitive and specific to use the autologous antigen in the assay system rather than the cross-reacting antigen prepared from rat muscle, we prefer to use the latter material for routine assays or for the examination of a large number of patients because of the high concentration of AChR in rat muscle, its easy availability, its constant recovery in the extraction process, and its wellknown antigenic characteristics [30]. J. Clin. Biochem. Nutr.

7 ANTI-AChR ANTIBODY IN MYASTHENIA GRAVIS 271 When the myasthenic patients without thymoma were viewed as classified group, most of the patients with only ocular signs indicated insignificant or low antibody titers compared with those with generalized myasthenia, similar to those reported by others [31, 32]. This trend may be attributed to the heterogeneity not only in characteristics of anti-achr antibody but also in pathogenic mechanism of MG. In this respect Vincent and Newsom-Davis [33] reported that many of the ocular or generalized myasthenic sera with low antibody titers reacted better with human ocular or normal human muscle AChR than with denervated human muscle AChR. In regard to HLA antigen versus disease heterogeneity, the study on Hong Kong Chinese myasthenics [34] indicated that those with HLA Bw46 had a noticeably elevated prevalence of juvenile-onset ocular MG. In Japan, HLA and Gm have been defined as genetic markers in this disease, but there is no evidence which suggests the association of a specific HLA or Gm with myasthenia of ocular form or generalized form with negative titer of anti-achr antibody. The mean values of and percentages positive for antibody against AChR were roughly correlated with clinical severity in generalized forms with or without thymoma. A discrepancy was, however, observed between the level of antibody and clinical severity in some patients, e.g., several cases in remission yet having significant titers and active cases of generalized types lacking the antibody, which suggests that the pathogenesis of MG in such cases may relate to factors other than anti-achr antibody. Myasthenic patients with thymoma had a distinctly higher incidence of this antibody, and they usually showed worse prognosis and less significant remission by thymectomy than the non-thymoma patients. This may be due to that thymomabearing MG patients have different characteristics (distributions of age and sex) from those of non-thymoma groups [35, 36], the lack of the strong association with HLA, and/or an increased incidence of a particular Gm haplotype in Japanese cases [37]. Thymic hyperplasia and thymoma are frequently associated with MG [8], and thymectomy is one of the established therapies as substantiated by the clinical alleviation in patients having undergone the operation [8-10]. There have been conflicting reports of the effects of thymectomy on anti-achr antibody levels in patients without thymoma [3, 16-21]. Regarding our data, when the mean value of anti-achr antibody concentrations was estimated as a percentage of preoperative values at every month since thymectomy in 19 MG patients without thymoma, anti-achr antibody titer revealed a rapid decline within 1 month followed by fluctuation up to several postoperative months and stably gentle fall later correlated with the clinical state. These findings in Japanese MG are similar to the results by Lefvert et al. [3], Scadding et al. [16], and Vincent et al. [20]. Not only the examination of a large number of patients but also the long-term followup measurement at regular intervals seems to be necessary for the evaluation of the effect of thymectomy on MG. The decrease in antibody titers after the operation may be accounted for as Vol. 3, No. 3, 1987

8 272 F. MATSUBARA-MORI et al. follows : (i) the presence of AChR-like protein was demonstrated in thymus tissue, which cross-reacts with myasthenic IgG [38, 39]. Kao and Drachman [40] also reported the presence of AChR. protein in cultured rat and human thymuses. A decrease in antibody titer may thus occur by the removal of such an immunogenic stimulus; (ii) we have demonstrated that the anti-achr antibodies were detected at higher concentration in the thymic extract than in the serum [41]. The operation may results in the elimination of large amounts of antibodies in the abnormal thymus as suggested by the pathological findings of germinal centers and lymph follicles containing B-lymphocytes [42] ; and (iii) Scadding et al. [43] reported that anti- AChR antibodies can be synthesized by cultured thymic lymphocytes. The operation may suppress the antibody production through the removal of sensitized T-lymphocytes in the thymus and cause an annual decrease in the titer for a few years in parallel with the reduction in the number of long-lived T-lymphocytes which are necessary for cooperating with B-lymphocytes in the production of anti-achr antibody. Thymectomy may remove cells or some factors responsible for either suppressing or activating antibody production that may be important in the pathogenesis of MG [44]. Several patients who had undergone their operation 7 to 13 years prior to the study still retained a high antibody titer and did not show clinical improvement any greater than those whose gland had been removed 6 years or less prior. This could be related to the methodological difference in the surgical approach between the two groups. The method of the complete removal of surrounding fatty tissue in the anterior mediastinum and neck, which was used for the latter group, seems to be more reliable. Our data suggest that the nature of the humoral response in ocular myasthenia without thymoma or generalized myasthenia with thymoma is different from that in other forms of MG, in view of the levels of anti-achr antibody and/or the effect of thymectomy on both anti-achr antibody value and clinical state. Moreover, it was demonstrated that the significant reduction in anti-achr antibody after thymectomy plays an important role in the mechanism by which thymectomy benefits most cases of MG without thymoma. This work was supported in part by grants from the Research Committee of Intractable Neuroimmunological Disorders, the Ministry of Health and Welfare, and by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan. REFERENCES 1. Appel, S.H., Almon, R.R., and Levy, N. (1975) : Acetylcholine receptor antibodies in myasthenia gravis. N. Engl. J. Med., 293, Lindstrom, J.M., Seybold, M.E., Lennon, V.A., Whittingham, S., and Duane, D.D. (1976): Antibody to acetylcholine receptor antibody in myasthenia gravis, prevalence, clinical correlates and diagnostic value. Neurology, 26, Lefvert, A.K., Bergstrom, K., Matell, G., Osterman, P.O., and Pirskanen, R. (1978): Determination of acetylcholine receptor antibody in myasthenia gravis : clinical usefulness and J. Clin. Biochem. Nutr.

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11 ANTI-AChR ANTIBODY IN MYASTHENIA GRAVIS 275 in myasthenia gravis : evidence for altered cell populations. N. Engl. J. Med., 291, Scadding, G.K., Vincent, A., Newsom-Davis, J., and Henry, K. (1981): Acetylcholine receptor antibody synthesis by thymic lymphocytes : correlation with thymic histology. Neurology, 31, Elias, S.B., and Appel, S.H. (1979) : Current concepts of pathogenesis and treatment of myasthenia gravis. Med. Clin. North Am., 63, Vol. 3, No. 3, 1987