Myasthenia Gravis, Thymectomy and Serum Thymic Hormone Activity
JEREMIAH 1. TWOMEY, M.B. VERNA M. LEWIS, MSc. BERNARD M. PATTEN, M.D. I louston, Texus GIDEON GOLDSTEIN, M.D., Ph.D. Ruritun, New Iersey ROBERT A. GOOD, M.D., Ph.D. New York, New York
From the Veterans Administration Hospital and Baylor College of Medicine, Houston, Texas: the Ortho Pharmaceutical Corp., Raritan, New ler-
sey; and the Sloan-Kettering Institute for Cancer Research, New York, New York. This research was supported by CA 19538, CA 19287 and CA17404 awarded by the National Cancer Institute, Department of Health, Education and Welfare, and the Kelsey and Leary Foundation. Requests for reprints should be addressed to Dr. J. 1. Twomey, Immunohematology Research Laboratory, Veterans Administration Hospital, 2002 Holcombe Boulevard, Houston, Texas 77211.Manuscript
accepted November 2,1978.
Serum thymic hormone activity was measured in 36 patients with myasthenia gravis and in 10 control subjects from each age decade. In all 25 patients under 50 years of age results were within, or close to, the normal range. Activity at levels considered normal for juveniles was detected in 10 of the 11older patients whereas levels normally decline in older subjects. One week after thymectomy, 13 of 17 patients (76 per cent) had no demonstrable serum thymic hormone activity. However, 10 months or longer after thymectomy only five patients (30 per cent) lacked thymic hormone activity in the serum. There was a significant correlation between clinical improvement and sustained lowering of serum thymic hormone activity after thymectomy. There is strong evidence that the neuromuscular block of myasthenia gravis is related to a deficiency of acetylcholine receptors at the neuromuscular junction [l-3]. It has been suggested that antibody to the acetylcholine receptor, which is found in the serum of almost 90 per cent of the patients with myasthenia gravis [4], causes this lesion. Both transient myasthenia gravis in neonates [5,6], suggesting humoral transmission across the placenta, and the clinical improvement following plasmaphoresis [7,8] support the concept that antiacetylcholine receptor antibody causes the neuromuscular block of myasthenia gravis. Experimental animals manifest a reduction of end plate potential amplitude and decrements to repetitive stimulation after injections of immunoglobulin from patients with myasthenia gravis [9] or by active immunization with acetylcholine receptor in an appropriate adjuvant [lo]. Recent data suggest that receptor antibody binds to acetylcholine receptors at the neuromuscular junction [u] to form bridges between receptors [12] which may contribute to accelerated receptor destruction. Such a series of events would result in a deficit of receptors as is found in myasthenia gravis [13]. Substantial evidence also implicates the thymus in the pathophysiology of myasthenia gravis. About 70 per cent of the patients manifest thymic germinal centers, whereas another 10 per cent have thymomas [14]. The B lymphocyte content of the thymus is modestly increased [15] and autoantibody is produced against myoid cells of the thymus [16]. More than 70 per cent of the patients benefit from thymectomy over a period of years [14,17]. Experimentally, it is possible to induce a myasthenia-like effect by immunizing animals with thymic tissue in adjuvant [18,19] or by administering a thymic extract [20], the purified thymic hormone, thymopoietin, [21] or a synthetic fragment of thymopoietin [zz]. The relative contributions of acetylcholine receptor antibody and
April 1979 The American Journal of Medicine Volume 66
639
MYASTHENIA GRAVIS, THYMECTOMY AND THYMIC HORMONE ACTIVITY-TWOMEY
;
20
= F L z
15
f
10
percentage of indicator cells induced to express Thy 1.2 antigen using antiserum to Thy 1.2 antigen,raised by immunizingAKR
@B NORMAL VAlUES PER ACE DECADE 112 SO1 n MVASlHENlA GRAVIS * MVASTHENIA GRAVIS WITH THVMOMA
mice with C3H thymocytes [33], guinea pig complement (GIBCO, Long Island, New York) and a sensitive enzymatic cytotoxicity test [34]. In this test, injured cells were solubilized by 30 minutes of incubation with 2.5 mg of protease/ml [Type VI, Sigma Chemical Co., St. Louis, Missouri) and the remaining cells were counted manually. A~I incubation standard, containing 1 pg/ml thymopoietin, a thymic peptide hormone [21,35], was included in each experiment. Results on serum were expressed as having equivalent inductive activity to known amounts of thymopoietin per milliliter.
E 5 g = 2
5 ” 10
xl
30 40 AGE IN VEARS
50
60
JO !
Figure 1. Thy 1.2 antigen induction activity in normal serum and in serum from 36 patients with severe myasthenia gravis before thymectomy.
the thymus to myasthenia gravis remain to be clarified.
One hypothesis suggests that the primary immune event involves production of autoantibody in the thymus to acetylcholine receptors on myoid cells [23]. A second possibility is that thymopoietin causes the neuromuscular lesion and that the antiacetylcholine receptor antibody is an epiphenomenon [24]. A third possibility is that receptor antibody and thymopoietin act synergistically to cause myasthenia gravis. The present study was undertaken to further evaluate the role of the thymus in myasthenia gravis. MATERIALS AND METHODS Studies were carried out on 36 patients, including 22 females, with severe myasthenia gravis. The severity of the disease in all patients was Osserman grade II B or worse [25]; all patients had impaired grip and leg holding, and positive edrophonium chloride tests [26]. All patients tested demonstrated decrements of muscle action potential [27]. Fifteen of 31 patients had serum antibody that inhibited the binding of I251a-bungaratoxin to acetylcholine receptors [28]. At thymectomy, nine patients had lymphoid germinal centers [ll] and one patient had a clear cell lymphoepithelial thymoma. All patients received mestinon and most also received prednisone prior to thymectomy. Control studies were carried out on 10 patients from each age decade who were hospitalized for cardiac surgery. Serum was stored at -7O’C and was passed through PM30 membranes (Amicon Corp., Lexington, MA.) prior to assay to remove heteroantibody. The bioassay, which measures induction of the thymusderived antigen Thy 1.2 on null mouse lymphocytes, has been described elsewhere in detail [29,30]. Spleen cells from nude athymic mice were depleted of B lymphocytes and macrophages by passage through nylon columns [31]. Induction incubations were for 18 hours in a carbon dioxide enriched, humidified, 37°C incubator and included 0.5 X lo6indicator cells suspended in 0.1 ml of medium 199 plus 5 per cent bovine serum albumin, 0.1 ml serum filtrate and 125 pg/ml ubiquitin. Ubiquitin is a tissue peptide [32] which, at this concentration, inhibits nonspecific induction via /3adrenergic receptors and also increases the sensitivity of the assay to low concentrations of thymic hormone [29]. Activity was determined from the
640
ET AL.
April 1979
The American Journal of Medicine
RESULTS The normal range of Thy 1.2 antigen induction by serum from 10 control subjects in each age decade (f2 standard deviations) is depicted by the shaded area in Figure 1. Results on all 25 patients with myasthenia gravis who were under XI years of age were within or close to the normal range of results. Serum activity in the one patient who had a thymoma was only slightly higher than the normal values. In contrast, serum from 10 of 11 patients over 50 years of age had increased activity. However, these elevated values did not exceed normal levels for younger subjects. Results were not influenced by sex or by prednisone therapy. Seventeen patients with myasthenia gravis were tested shortly before and again one week after thymectomy, which included a wide resection of anterior mediastinal tissues. Postoperatively, 13 patients (76 per cent) had negligible serum inductive activity (Figure 21. A low level of activity persisted in the other four patients which may be ascribed to ectopic thymic tissue [36]. Serum from these 17 patients was tested again 10 to 31 months after thymectomy. Thirteen patients showed improvement during the postoperative period as evidenced by a one step or greater improvement in the Osserman scale and a reduced requirement for mestinon and prednisone. One patient died from complications of myasthenia gravis 10 months after surgery, and three other patients showed no improvement 12 to 17 months after thymectomy. Negligible serum T cell-inducing activity was recorded in only five of these 17 patients (Figure 3). However, all 13 patients who showed clinical improvement after thymectomy had an appreciable lowering of serum activity. In contrast, serum activity increased after surgery in three bf the four patients whose clinical status did not improve. The highest value was recorded shortly before death in the fatal case. These long-term postthymectomy values were significantly higher relative to preoperative values in patients who did not benefit from thymectomy than in patients who did benefit (p
JEREMIAH 1. TWOMEY, M.B. VERNA M. LEWIS, MSc. BERNARD M. PATTEN, M.D. I louston, Texus GIDEON GOLDSTEIN, M.D., Ph.D. Ruritun, New Iersey ROBERT A. GOOD, M.D., Ph.D. New York, New York
From the Veterans Administration Hospital and Baylor College of Medicine, Houston, Texas: the Ortho Pharmaceutical Corp., Raritan, New ler-
sey; and the Sloan-Kettering Institute for Cancer Research, New York, New York. This research was supported by CA 19538, CA 19287 and CA17404 awarded by the National Cancer Institute, Department of Health, Education and Welfare, and the Kelsey and Leary Foundation. Requests for reprints should be addressed to Dr. J. 1. Twomey, Immunohematology Research Laboratory, Veterans Administration Hospital, 2002 Holcombe Boulevard, Houston, Texas 77211.Manuscript
accepted November 2,1978.
Serum thymic hormone activity was measured in 36 patients with myasthenia gravis and in 10 control subjects from each age decade. In all 25 patients under 50 years of age results were within, or close to, the normal range. Activity at levels considered normal for juveniles was detected in 10 of the 11older patients whereas levels normally decline in older subjects. One week after thymectomy, 13 of 17 patients (76 per cent) had no demonstrable serum thymic hormone activity. However, 10 months or longer after thymectomy only five patients (30 per cent) lacked thymic hormone activity in the serum. There was a significant correlation between clinical improvement and sustained lowering of serum thymic hormone activity after thymectomy. There is strong evidence that the neuromuscular block of myasthenia gravis is related to a deficiency of acetylcholine receptors at the neuromuscular junction [l-3]. It has been suggested that antibody to the acetylcholine receptor, which is found in the serum of almost 90 per cent of the patients with myasthenia gravis [4], causes this lesion. Both transient myasthenia gravis in neonates [5,6], suggesting humoral transmission across the placenta, and the clinical improvement following plasmaphoresis [7,8] support the concept that antiacetylcholine receptor antibody causes the neuromuscular block of myasthenia gravis. Experimental animals manifest a reduction of end plate potential amplitude and decrements to repetitive stimulation after injections of immunoglobulin from patients with myasthenia gravis [9] or by active immunization with acetylcholine receptor in an appropriate adjuvant [lo]. Recent data suggest that receptor antibody binds to acetylcholine receptors at the neuromuscular junction [u] to form bridges between receptors [12] which may contribute to accelerated receptor destruction. Such a series of events would result in a deficit of receptors as is found in myasthenia gravis [13]. Substantial evidence also implicates the thymus in the pathophysiology of myasthenia gravis. About 70 per cent of the patients manifest thymic germinal centers, whereas another 10 per cent have thymomas [14]. The B lymphocyte content of the thymus is modestly increased [15] and autoantibody is produced against myoid cells of the thymus [16]. More than 70 per cent of the patients benefit from thymectomy over a period of years [14,17]. Experimentally, it is possible to induce a myasthenia-like effect by immunizing animals with thymic tissue in adjuvant [18,19] or by administering a thymic extract [20], the purified thymic hormone, thymopoietin, [21] or a synthetic fragment of thymopoietin [zz]. The relative contributions of acetylcholine receptor antibody and
April 1979 The American Journal of Medicine Volume 66
639
MYASTHENIA GRAVIS, THYMECTOMY AND THYMIC HORMONE ACTIVITY-TWOMEY
;
20
= F L z
15
f
10
percentage of indicator cells induced to express Thy 1.2 antigen using antiserum to Thy 1.2 antigen,raised by immunizingAKR
@B NORMAL VAlUES PER ACE DECADE 112 SO1 n MVASlHENlA GRAVIS * MVASTHENIA GRAVIS WITH THVMOMA
mice with C3H thymocytes [33], guinea pig complement (GIBCO, Long Island, New York) and a sensitive enzymatic cytotoxicity test [34]. In this test, injured cells were solubilized by 30 minutes of incubation with 2.5 mg of protease/ml [Type VI, Sigma Chemical Co., St. Louis, Missouri) and the remaining cells were counted manually. A~I incubation standard, containing 1 pg/ml thymopoietin, a thymic peptide hormone [21,35], was included in each experiment. Results on serum were expressed as having equivalent inductive activity to known amounts of thymopoietin per milliliter.
E 5 g = 2
5 ” 10
xl
30 40 AGE IN VEARS
50
60
JO !
Figure 1. Thy 1.2 antigen induction activity in normal serum and in serum from 36 patients with severe myasthenia gravis before thymectomy.
the thymus to myasthenia gravis remain to be clarified.
One hypothesis suggests that the primary immune event involves production of autoantibody in the thymus to acetylcholine receptors on myoid cells [23]. A second possibility is that thymopoietin causes the neuromuscular lesion and that the antiacetylcholine receptor antibody is an epiphenomenon [24]. A third possibility is that receptor antibody and thymopoietin act synergistically to cause myasthenia gravis. The present study was undertaken to further evaluate the role of the thymus in myasthenia gravis. MATERIALS AND METHODS Studies were carried out on 36 patients, including 22 females, with severe myasthenia gravis. The severity of the disease in all patients was Osserman grade II B or worse [25]; all patients had impaired grip and leg holding, and positive edrophonium chloride tests [26]. All patients tested demonstrated decrements of muscle action potential [27]. Fifteen of 31 patients had serum antibody that inhibited the binding of I251a-bungaratoxin to acetylcholine receptors [28]. At thymectomy, nine patients had lymphoid germinal centers [ll] and one patient had a clear cell lymphoepithelial thymoma. All patients received mestinon and most also received prednisone prior to thymectomy. Control studies were carried out on 10 patients from each age decade who were hospitalized for cardiac surgery. Serum was stored at -7O’C and was passed through PM30 membranes (Amicon Corp., Lexington, MA.) prior to assay to remove heteroantibody. The bioassay, which measures induction of the thymusderived antigen Thy 1.2 on null mouse lymphocytes, has been described elsewhere in detail [29,30]. Spleen cells from nude athymic mice were depleted of B lymphocytes and macrophages by passage through nylon columns [31]. Induction incubations were for 18 hours in a carbon dioxide enriched, humidified, 37°C incubator and included 0.5 X lo6indicator cells suspended in 0.1 ml of medium 199 plus 5 per cent bovine serum albumin, 0.1 ml serum filtrate and 125 pg/ml ubiquitin. Ubiquitin is a tissue peptide [32] which, at this concentration, inhibits nonspecific induction via /3adrenergic receptors and also increases the sensitivity of the assay to low concentrations of thymic hormone [29]. Activity was determined from the
640
ET AL.
April 1979
The American Journal of Medicine
RESULTS The normal range of Thy 1.2 antigen induction by serum from 10 control subjects in each age decade (f2 standard deviations) is depicted by the shaded area in Figure 1. Results on all 25 patients with myasthenia gravis who were under XI years of age were within or close to the normal range of results. Serum activity in the one patient who had a thymoma was only slightly higher than the normal values. In contrast, serum from 10 of 11 patients over 50 years of age had increased activity. However, these elevated values did not exceed normal levels for younger subjects. Results were not influenced by sex or by prednisone therapy. Seventeen patients with myasthenia gravis were tested shortly before and again one week after thymectomy, which included a wide resection of anterior mediastinal tissues. Postoperatively, 13 patients (76 per cent) had negligible serum inductive activity (Figure 21. A low level of activity persisted in the other four patients which may be ascribed to ectopic thymic tissue [36]. Serum from these 17 patients was tested again 10 to 31 months after thymectomy. Thirteen patients showed improvement during the postoperative period as evidenced by a one step or greater improvement in the Osserman scale and a reduced requirement for mestinon and prednisone. One patient died from complications of myasthenia gravis 10 months after surgery, and three other patients showed no improvement 12 to 17 months after thymectomy. Negligible serum T cell-inducing activity was recorded in only five of these 17 patients (Figure 3). However, all 13 patients who showed clinical improvement after thymectomy had an appreciable lowering of serum activity. In contrast, serum activity increased after surgery in three bf the four patients whose clinical status did not improve. The highest value was recorded shortly before death in the fatal case. These long-term postthymectomy values were significantly higher relative to preoperative values in patients who did not benefit from thymectomy than in patients who did benefit (p
