dicates that there is a decrease of available acetylcholine. (AcCho) receptors at the ..... Fambrough, D. M., Drachman, D. B. & Satyamurti, S. (1973). Science 182 ...
Proc. Nati. Acad. Sd. USA Vol. 75, No. 7, pp. 3422-3426, July 1978 Medical Sciences
Effect of myasthenic patients' immunoglobulin on acetylcholine receptor turnover: Selectivity of degradation process (autoimmunity/skeletal muscle/tissue culture/acetylcholine receptor synthesis)
DANIEL B. DRACHMAN, C. WILLIAM ANGUS, ROBERT N. ADAMS, AND ING KAO Neuromuscular Unit, Department of Neurology, Johns Hopkins University, School of Medicine, 1721 E. Madison Street, Baltimore, Maryland 21205
Communicated by John E. Dowling, April 21,1978
ABSTRACT Antibodies in the sera of patients with myasthenia gravis are believed to play an important role in the pathogenesis of the disorder. They have recently been shown to accelerate the degradation of acetylcholine receptors in cultured mammalian skeletal muscle and at intact neuromuscular junctions. To elucidate the mechanism of the antibodyaccelerated degradation process, we have prepared cultures in which one set of acetylcholine receptors was exposed to myasthenic inmunoglobulin while a second set of acetylcholine receptors, newly incorporated after exposure to the immunoglobulin, was not. The set of acetylcholine receptors with bound myasthenic immunoglobulin was degraded at 2 to 3 times the normal rate, while the second set of acetylcholine receptors without bound immunoglobulin was degraded at the control rate. This suggests that the binding of antibody from myasthenic patients alters the acetylcholine receptors in some way that causes them to be selected for preferential degradation by the
muscle cells. New synthesis and incorporation of the acetylcholine receptors into the surface membrane of cultured skeletal muscle was unaffected by exposure to myasthenic immunoglobulin. Myasthenia gravis is a neuromuscular disorder characterized by weakness and fatigability of muscles. Recent evidence indicates that there is a decrease of available acetylcholine (AcCho) receptors at the neuromuscular junctions of myasthenic patients (1-5), which accounts for the typical clinical and physiological manifestations of the disorder (6). The pathogenesis of this abnormality is believed to involve an autoimmune attack directed against AcCho receptors (for reviews see refs. 7 and 8). Sera of most patients with myasthenia gravis contain antibody against AcCho receptor (9-11), which is capable of reproducing the basic features of the disease on passive transfer to mice (12, 13). We and others have recently reported that immunoglobulin (Ig) from myasthenic patients produces a marked acceleration of the rate of degradation of AcCho receptors of mammalian skeletal muscle in tissue culture (14-16). In this investigation, we have examined the interactions between myasthenic Ig and AcCho receptors further in rat muscle cultures. Our experiments were designed to distinguish between two possible effects of myasthenic Ig on receptor degradation: (i) A selective effect on AcCho receptors, with accelerated degradation only of that population of receptors to which antibody is bound, and (ii) a nonselective effect on the muscle cell's AcCho receptor-degrading mechanisms, with accelerated degradation of all AcCho receptors, regardless of the presence of bound antibody. We have tested this proposition by producing cultures in which one population of AcCho receptors has been exposed directly to myasthenic Ig while a second population in the same cultures has not. The degradation rates of these two populations of receptors have been separately followed. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Using similar methods, we have measured synthesis and incorporation of new AcCho receptors into the surface membrane of cultured skeletal muscle after exposure to myasthenic 1g.
MATERIALS AND METHODS Tissue Culture. Cultures of rat skeletal muscle were prepared by conventional methods (14, 17). The limb muscles of 17- to 19-day Sprague-Dawley rat fetuses were minced, then trypsinized for 20 min in Hanks' balanced salt solution without divalent cations, containing 0.1% trypsin (wt/vol). The cells were strained through bolting silk, centrifuged, resuspended in culture medium, and plated for 45 min in a Falcon 75-cm2 plastic flask for selective removal of the rapidly adhering fibroblasts. The nonadhering cells, predominantly myoblasts, were transferred to a second flask and cultured for 24 hr. The cultures were trypsinized (0.025%) and replated in gelatincoated, 35-mm Falcon plastic petri dishes. Cultures were grown at 370 under an atmosphere of 10% CO2 in "standard medium," consisting of Eagle's minimal essential medium with Earle's salts, supplemented with 10% horse serum (vol/vol), 1% chick embryo extract (vol/vol), 100 units of penicillin per ml, 100 units of streptomycin per ml, and 2.5 ,gg of Fungizone per ml. Large numbers of dishes were prepared simultaneously so that the cultures would be at the same stage of development and would have closely comparable numbers of AcCho receptors. AcCho Receptor Degradation. Degradation of AcCho receptors was measured by an indirect method (14, 18, 19) that depends on the specific labeling of the receptors with 125Ilabeled a-bungarotoxin (125I-a-BuTx) and the release into the culture medium of radioactive material derived from degraded 125I-a-BuTx-AcCho receptor complexes. Sets of identical 7day-old cultures were first saturated with 125I-a-BuTx for 30 min [0.2 ,ug (25 fmol) in 1 ml; specific activity, approximately 3 X 104-5 X 104 Ci/mol], and the unbound toxin was removed by washing four times with culture medium. At 2-hr intervals the medium and a single washing were removed for measurement of released radioactivity, and fresh medium was added to the culture dishes. At the end of the experimental period, the cultures were extracted with two washings of 1% Triton X-100 in phosphate-buffered saline, pH 7.2, to solubilize the remaining cell-bound complexes of AcCho receptor and 125I-a-BuTx, and the radioactivity in the extracts was measured. The rate of degradation was calculated as the percent of total radioactivity released into the medium per hr. Preparation of Immunoglobulin. In order to test the effect of myasthenic patients' Ig on AcCho receptor turnover, we prepared Ig fractions individually from the sera of myasthenic patients and from a control pool (derived from 10 to 30 normal Abbreviations: AcCho, acetylcholine; l25I-a-BuTx, l25I-labeled cabungarotoxin. 3422
Proc. Natl. Acad. Sci. USA 75 (1978)
Medical Sciences: Drachman et al. myasthenigrasvis). other thian persons or patients with diseases The Ig was first precipitated with ammonium sulfate at '% saturation, then redissolved in Ringer's solution at half the original serum volume. It was then dialyzed overnight at 40 against two changes of Ringer's solution (20), giving a final IgG concentration of approximately 10-15 mg/ml. In the experimental cultures, the standard medium was replaced with medium in which an equal volume of control or patient's Ig was substituted for horse serum. Preparation and Labeling of AcCho Receptors Exposed and Not Exposed to Ig. The sequence of steps outlined in Table 1 was designed to produce two sets of cultures: set A, in which the labeled AcCho receptors were directly exposed to the test Ig; and set B, in which the cultured muscle was exposed to the test Ig at the same time as set A, but only the AcCho receptors incorporated after Ig treatment were labeled with 125I-a-BuTx. Thus, the labeled AcCho receptors in set B were never directly exposed to test Ig, although their muscle cells were. In order to permit valid comparisons between experimental subgroups, control and myasthenic Igs were always tested simultaneously, using a single large batch of cultures prepared at the same time. Four or five culture dishes were used for each subgroup in each experiment. Nonspecific adhesion of Ig was minimized by first incubating the cultures with medium containing 10% control human Ig. In set A, the receptors were labeled by incubation with 125I-a-BuTx and the excess toxin was removed by washing four times. The cultures were then incubated for 2 hr with medium containing 10% test Ig (in place of horse serum and embryo extract), after which the test Ig was removed by repeated rinsing with standard medium. Steps 4, 5, and 6 in Table 1 duplicated the conditions in culture set B. Finally, degradation of the labeled receptors was measured over a 6- to 10-hr period, as described below. In set B, the cultures were initially exposed to medium containing 10% myasthenic or control Ig for 2 hr. After repeated washing to remove excess Ig, the remaining AcCho receptors were blocked with nonradioactive a-BuTx (0.2 yg/ml for 30 min at 370). This step ensured that none of the AcCho receptors that had been treated with Ig would be labeled by subsequent exposure to 125I-a-BuTx. The cultures were returned to the incubator to allow time (6 hr) for synthesis and incorporation of new AcCho receptors. At the end of this period, the new receptors were labeled by incubation with 125I-a-BuTx and the unbound toxin was removed by washing four times. At 2-hr intervals the medium was removed for determination
3423
relea~ed radioactivity and fresh medium was added to the The initial 2-hr collection was discarded because of the possibility that it might contain the washout of nonspecifically trapped 125I-a-BuTx, and the degradation rates were determined from subsequent collections. At the end of the 8or 10-hr collection period, the cultures were extracted with 1% Triton X-100 as described above. Synthesis and Incorporation of AcCho Receptors. We have used a modification of the protocol outlined for set B (Table 1) to measure the synthesis and incorporation of AcCho receptors after exposure of the cultures to control or myasthenic Ig (18, 19). Groups of culture dishes were preincubated with control human Ig to minimize nonspecific adhesion of the myasthenic Ig and were then exposed to the test Ig for 2 hr as before. After removal of unbound Ig by repeated rinsing, all existing AcCho receptors were blocked by saturation with unlabeled a-BuTx (0.2 ,ug/ml for 30 min at 37°). The excess a-BuTx was removed by rinsing, and the cultures were incubated for 6 hr to allow synthesis and incorporation of new receptors into the surface membranes. The newly incorporated AcCho receptors were then saturated with 125I-a-BuTx. The cultures were washed four times to remove unbound 125I-a-BuTx. The labeled AcCho receptors were extracted with Triton X-100, and their radioactivity was measured in a counter. Statistical Methods. Degradation rates were determined for 194 cultures, in seven separate experiments. Because of the variation between batches of cultures prepared at different times, statistical comparisons were always made with experimental subgroups prepared from a single batch of cultures. The significance of differences between treatment subgroups was calculated by Student's two-sample t test. Comparisons between subgroups were also expressed as "acceleration ratios," i.e., mean degradation rate of subgroup treated with myasthenic Ig divided by mean degradation rate of corresponding subgroup treated with control pool Ig (see Fig. 2). The same statistical methods were used for analysis of the receptor synthesis and incorporation data. of
culture dishes.
y
RESULTS Selective degradation of AcCho receptors The degradation rates of set A AcCho receptors, directly exposed to myasthenic patients' Ig, were increased to approximately 2.5 times the control rates (Figs. 1A and 2A), in confirmation of our previous findings (14). The mean degradation rate for receptors treated with control Ig was 4.05% + 0.51 (SD)
Table 1. Experimental sequence for producing two labeled sets of AcCho receptors Set B Set A AcCho receptors labeled after exposure of cultures to Ig Labeled AcCho receptors directly exposed to Ig 1. Preincubation with medium containing 10% control Ig to minimize nonspecific absorption of test Ig. 2. Label existing AcCho receptors with 1251-a-BuTx. Wash. 3. Incubation (2 hr) with test Ig. Wash. 4. Nonradioactive a-BuTx, 30 min. Wash. 5. Incubate with standard medium 6 hr. 6. No procedure. 7. Collect medium at 2-hr intervals to determine degradation rate of labeled AcCho receptors.
1. Preincubation with medium containing 10% control Ig to minimize nonspecific absorption of test 1g. 2. No procedure. 3. Incubation (2 hr) with test Ig. Wash. 4. Nonradioactive a-BuTx, 30 min, to block all remaining AcCho receptor sites. Wash. 5. Incubate with standard medium 6 hrs to permit appearance of newly synthesized AcCho receptors. 6. Label new AcCho receptors with 1251-a-BuTx, 30 min. Wash. 7. Collect medium at 2-hr intervals to determine degradation rate of labeled new AcCho receptors.
This experimental protocol provides sister sets of myotube cultures. In set A, the initially present AcCho receptors were labeled, then treated with Ig. In set B, only the new receptors, which were synthesized and incorporated after exposure to Ig, were labeled. Except for the sequence of labeling, the cultures were treated in strictly parallel fashion. For details see text.
Medical Sciences: Drachman et al.
3424
I
Proc. Nati. Acad. Sci. USA 75 (1978)
n-_ .v -
cx ._E C).9*a C
0.90.80.7 0.6-
).3-
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.C 0
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A
B
0.1
4
2
6
8
0.1 2
4
6
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Time, hr
FIG. 1. Typical experiment showing degradation of set A and set B AcCho receptors in cultures exposed to Ig from a myasthenic patient or control pool. The experimental sequence is outlined in Table 1 and the text. Each point represents the mean of five measurements. Standard deviations were too small to be drawn. (A) Degradation of set A AcCho receptors exposed to Ig after labeling with 125I-a-BuTx. Note the marked acceleration of degradation induced by the myasthenic patient's Ig (0) as compared to the control (@). (B) Degradation of set B AcCho receptors, incorporated into the muscle membrane after exposure of cultures to 1g. Degradation rates of these receptors were identical for cultures treated with myasthenic (0) and control (@) Ig. (Curves were actually superimposed; "control" curve was offset upwards by artist.)
hr while the mean rate for receptors treated with myasthenic Ig was 10.71% + 1.82 per hr. Statistical comparison (see Materials and Methods) showed this increase to be highly significant in each of seven separate experiments, using Ig from four different patients (P < 0.001). By contrast, the degradation rates of set B AcCho receptors, incorporated into muscle membrane after exposure of the muscle cultures to myasthenic patients' Ig, showed no differ-
per
3LAA ** fA 0
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FIG. 2. Effect of exposure to immunoglobulin on degradation rates. The degradation ratio is the mean degradation rate of five cultures exposed to myasthentic Ig divided by the mean rate of five
cultures treated with control Ig in the same experiment. Note that the set A receptors (A), directly exposed to Ig, were accelerated approximately 2.5-fold by myasthenic Ig. The set B receptors (B), incorporated into the muscle membrane after exposure of the cultures to myasthenic Ig, showed no acceleration of degradation rates. The four different symbols represent Ig from four different myasthenic patients. Data from 160 cultures are summarized in this figure.
ence from control rates (Figs. lB and 2B; P > 0.5 in each case). The degradation rates for the set A and set B populations of AcCho receptors were determined at the same time, in simultaneously prepared sets of cultures. A typical experiment is illustrated in Fig. 1. The mean degradation rate of set A AcCho receptors exposed directly to Ig from myasthenic patient 1 was 8.74% + 0.25 (SD) per hr, as compared with the mean control rate of 3.4% + 0.8 per hr. The set B receptors in cultures treated with Ig from myasthenic patient 1 had virtually the same degradation rate as those receptors in the control Ig-treated set B cultures (4.70% : 0.23 per hr and 4.72% ± 0.19 per hr, respectively). Control experiments Degradation Products: Column Chromatography. In order to determine whether the 125I-labeled material released from the Ig-treated cultures represented the product of degradation, column chromatography was carried out. Media from five experiments in which AcCho receptors were treated with myasthenic Ig, and had rapid degradation rates, were chromatographed on Bio-Gel P4 and Sephadex G-15 columns, with phosphate-buffered saline, pH 7.2/1% Triton X-100 as the eluant. Eighty-five to 98% of the applied radioactivity was recovered. Only 3-18% of the recovered radioactivity was in the void volume, while the remainder eluted in a single included peak that corresponded to the position of ['4C]tyrosine. Media from cultures treated with control pool Ig gave closely similar results. Our findings, which confirm those reported previously for chick embryo cultures without added human Ig (18), indicate that the great majority of released radioactivity is in a low molecular weight form (possibly iodotyrosine) and therefore not in such substances as a-BuTx or BuTx-ACh receptor complexes, which have higher molecular weights. The 1251release method for measuring AcCho receptor degradation has been extensively validated (14, 15, 18, 19, 21). The appearance of 125I in the medium has been shown to parallel the loss of receptor sites by the muscle under conditions in which new receptor synthesis is blocked. Moreover, 125I-a-BuTx itself does
Medical Sciences: Drachman et al. not alter the natural degradation of AcCho receptors by the muscle cells (18, 19). Pulse-chase experiments have confirmed the results of the 125I-release method for measuring AcCho receptor degradation (21). Time of Labeling of AcCho Receptors. The experimental protocol was designed so that the degradation rates in sets A and B were measured at the same time interval after exposure of the cultures to Ig (see Table 1). This necessitated a later time of labeling of set B receptors with 125I-a-BuTx and hence a shorter interval between labeling and collection of medium for measurement of degradation. As a direct test of the effect of time of labeling on degradation, we compared degradation rates of set A receptors labeled at step 1 with those labeled at step 6 (i.e., at the same time point used for labeling of set B receptors). In two separate experiments, the rates of those receptors labeled at the different time points were not significantly different (P > 0.2), thus excluding an effect of time of labeling in the present experiments. The mean degradation rates of AcCho receptors labeled at step 1 in two experiments were 3.49 0.12 (SD) and 3.85 i 0.15; rates of AcCho receptors labeled at step 6 were 3.55 0.15 and 3.97 ± 0.12, respectively. Responsiveness of Set B Receptors. Since our results showed that degradation of the set B receptors was unaffected by prior exposure of the cultures to myasthenic Ig, it was important to determine whether these receptors were unable to respond with accelerated degradation. To test this possibility, we prepared set B receptors as in Table 1 and then treated them with myasthenic Ig after labeling with 125I-a-BuTx. The mean acceleration ratios for set B receptors treated with Ig from patients 1 and 3 were closely similar to the corresponding ratios for set A receptors treated with the same Igs. The mean ratios for set A receptors treated with myasthenic Ig were 2.54 ± 0.18 (SD) and 2.74 + 0.05 in two experiments. The mean ratios for set B receptors treated with the same myasthenic Igs were 2.64 ± 0.18 and 2.82 + 0.05, respectively. Thus, the responsiveness of set B receptors to direct exposure to each myasthenic Ig was as great as that of set A receptors. Incubation of Myasthenic Ig with 125I--BuTx. 25I-a-BuTx was incubated with culture medium containing myasthenic Ig at 370 for 20 hr in the absence of muscle cells. Column chromatography showed that 99% of the radioactivity remained in the a-BuTx peak. This indicates that myasthenic Ig itself does not have the ability to degrade a-BuTx. Effect of Human Ig on Cultures. Additional control experiments were carried out to show that human Ig per se had no effect on AcCho receptor degradation. Degradation rates of cultures treated with control Ig did not differ from those of cultures incubated in standard medium containing horse serum and chick embryo extract (P > 0.2). The mean degradation rates of AcCho receptors treated with control human Ig were 3.45 ± 0.98 (SD) and 3.74 + 0.18 in two experiments. The corresponding degradation rates of AcCho receptors treated only with standard medium were 3.49 + 0.12 and 3.79 ± 0.30, respectively. Control Degradation Rate of Set B Receptors. An interesting incidental observation was that the normal degradation rates for the set B receptors were always 33-50% higher than those for set a receptors prepared at the same time in the same batch of cultures [mean control rates for set B = 5.95 + 0.76 (SD)]. This slightly higher degradation rate appears to be characteristic of newly incorporated AcCho receptors prepared as described above, regardless of whether the cultures were pretreated with myasthenic Ig, control Ig, or only standard medium without human Ig. In the present context, we make note of it only because the degradation rates of set B receptors in cultures treated with myasthenic Ig have appropriately been
Proc. Nati. Acad. Sci. USA 75 (1978)
3425
compared with those of set B receptors treated with control 1g.
Synthesis and incorporation of AcCho receptors Exposure of sets of cultures to myasthenic Ig did not alter the number of newly synthesized and incorporated AcCho receptors appearing during 6 hr of incubation, as measured by binding of 125I-a-BuTx (Table 2). The amount of 125I-a-BuTx (cpm) bound to cultures treated with IG from four different myasthenic patients did not differ significantly from the amount bound to control Ig-treated cultures (P > 0.1). This normal rate of synthesis and incorporation of AcCho receptors occurred at the same time that AcCho receptors with bound antibody were being rapidly degraded in parallel sets of cultures. DISCUSSION There is now abundant evidence that the decrease of junctional AcCho receptors in myasthenia gravis can be brought about by an antibody-mediated autoimmune process. Several studies have shown that Ig from myasthenic patients accelerates the degradation of AcCho receptors in tissue culture of skeletal muscle (14-16). Our recent finding that a similar process occurs at intact neuromuscular junctions (22) suggests that acceleration of AcCho receptor degradation potentially represents an important mechanism in the pathogenesis of human myasthenia gravis. The process of AcCho receptor degradation is believed to begin with endocytosis of the receptors from the muscle's surface membrane (18, 19). The ingested receptors undergo proteolysis within the muscle cells and the products of degradation are then released externally, into the surrounding medium. Most studies have followed the release of degradation products of 125I-a-BuTx-AcCho receptor complexes, as an indirect measure of the degradation of AcCho receptors (14-16, 18, 19, 22-24). The validity of this method has been thoroughly established in Table 2. Effect of Ig on AcCho receptor synthesis and incorporation
Myasthenic/control Patient
Bound 1251-a-BuTx, cpm ± SEM
MG 1 MG 2 Pool
5037 ± 505 4743 ± 274 5291 ± 205
MG3 MG4 Pool
4286 150 4173 131 4194 ± 292
Ig-treated cultures*
Experiment 1 0.95 0.90
Experiment 2 1.02 0.99
Synthesis and incorporation of new AcCho receptors in cultures treated with myasthenic Ig or control pool Ig. Results of two experiments are tabulated. Six cultures were used to test each Ig. Cultures were incubated with 0.1 ml of myasthenic or control Ig in 1 ml of medium for 2 hr at 37°. Remaining AcCho receptor sites were blocked with nonradioactive a-BuTx (0.2,ug). Cultures were incubated with standard medium for 6 hr and new AcCho receptors were then saturated with 1251-a-BuTx (0.2 ,g; 3.31 X 104 Ci/mol) at 370 for 30 min. After washing, the 1251-a-BuTx-AcCho receptor complexes were extracted with 1% Triton X-100 and radioactivity was measured. Note that new receptor synthesis and incorporation was not significantly different in sets of cultures treated with myasthenic Ig as compared with corresponding sets of cultures treated with control Ig (P >0.1). MG 1, 2, 3, and 4 are four different myasthenic patients. * Ratio of counts.
3426
Medical Sciences: Drachman et al.
previous reports, as well as in the specific circumstances of the present investigation (see Results for details). As yet, the mechanism by which myasthenic Ig accelerates receptor degradation has not been elucidated. In this investigation two alternative hypotheses have been considered: First, Ig might interact directly with the AcCho receptors, presumably by binding to them. In this model, only the population of receptors with bound Ig would be rapidly degraded, while other receptors in the same cultures, but without bound Ig, would be degraded at the normal rate. Second, myasthenic Ig might act at the level of the muscle cell, accelerating its internal mechanisms involved in AcCho receptor degradation. This model would not result in a selective process; in a given culture treated with myasthenic Ig all AcCho receptors should be degraded at the same rapid rate. Our results clearly indicate that the accelerated degradation induced by myasthenic Ig is a selective process. Only the population of AcCho receptors directly exposed to myasthenic Ig (set A) was rapidly degraded; at the same time, a second population of AcCho receptors in the same cultures, newly incorporated into the muscle membrane after exposure to myasthenic Ig (set B), was degraded at the normal rate. The selectivity of this effect argues for a mechanism involving an interaction of myasthenic Ig with surface AcCho receptors, rather than an accelerating effect on the muscle cells' internal degradative mechanisms. It seems likely that binding of the myasthenic antibody to surface AcCho receptors produces some alteration that results in preferential degradation of the antibody-AcCho receptor complexes. Since binding of other ligands, such as a-BuTx, to AcCho receptors does not result in accelerated degradation (18), the antibody-receptor complex must have some special property that leads to its selective degradation by the muscle cells. Recent evidence suggests that the critical factor is the ability of antibodies to crosslink the receptors (25). The accelerated degradation of antibody-bound AcCho receptors may be analogous to the process of "modulation," by which antibodies to surface receptors (antigens) of lymphocytes produce aggregation and subsequent reduction of the receptors (26, 27). Such interactions between macromolecules and surface receptors may not be limited to the immune system; the action of certain hormones on appropriate receptors results in a similar reduction of surface receptors (28, 29). In addition to these studies of degradation, we have also measured the synthesis and incorporation of AcCho receptors appearing during a 6-hr period in cultures exposed to myasthenic Ig. It has previously been reported that cultured skeletal muscle contains a pool of precursor AcCho receptors sufficient for incorporation of new surface receptors for up to 3 hr (19, 30). When longer periods of incubation are used, as in the present experiment, the total number of new surface receptors measured by binding of '25I-a-BuTx should represent the incorporation of both preformed and newly synthesized receptors. The degradation of receptors synthesized during the 6-hr test period can be ignored for the present purposes, since we have shown it to be equal in cultures treated with myasthenic or controlIg. Our results (Table 2) indicate that the number of receptors appearing after exposure to myasthenic Ig was the same as that after controlIg. Thus, at the same time that degradation of Ig-treated receptors is proceeding at a rapid rate, synthesis and incorporation of new receptors remains unaffected. In conclusion, our findings have demonstrated that the accelerated degradation of AcCho receptors brought about by myasthenic Ig is a selective process, affecting only receptors directly exposed to the antibody. The surface interaction be-
Proc. Natl. Acad. Sci. USA 75 (1978)
tween antibodies and membrane receptors may represent an important pathogenetic process in human myasthenia gravis, as well as in other autoimmune diseases. Further studies are needed to learn how the antibody-receptor complexes are recognized by the cells. We thank Mr. J. Michelson for his conscientious help and Drs. A. Pestronk, E. Stanley, and J. Griffin and Ms. C. Barlow for help in preparation of the manuscript. This work was supported in part by Grants 5 R01 HD 04817 and 5 P01 NS 10920 from the National Institutes of Health and by an academic career development award to I.K. from the National Institutes of Health. 1. Fambrough, D. M., Drachman, D. B. & Satyamurti, S. (1973) Science 182, 293-295. 2. Drachman, D. B., Kao, I., Pestronk, A. & Toyka, K. V. (1976) Ann. N.Y. Acad. Sci. 274,226-234. 3. Green, D. P. L., Miledi, R., de la Mora, M. P. & Vincent, A. (1975) Phil. Trans. R. Soc. London Ser. B 270,551-559. 4. Albuquerque, E. X., Rash, J. E., Mayer, R. F. & Satterfield, J. R. (1976) Exp. Neurol. 51, 536-3. 5. Engel, A. G., Lindstrom, J. M., Lambert, E. H. & Lennon, V. A. (1977) Neurology 27,307-315. 6. Satyamurti, S., Drachman, D. B. & Slone, F. (1975) Science 187, 955-957. 7. Drachman, D. B. (1978) N. Engl. J. Med. 298, 136-142 and
186-193. 8. Lennon, V. A. (1976) Immunol. Commun. 5,323-344. 9. Almon, R. R., Andrew, C. G. & Appel, S. H. (1974) Science 186, 55-57. 10. Bender, A. N., Engel, W. K., Ringel, S. P., Daniels, M. P. & Vogel, Z. (1975) Lancet i, 607-608. 11. Lindstrom, J. M., Seybold, M. E., Lennon, V. A., Whittingham, S. & Duane, D. D. (1976) Neurology 26, 1054-1059. 12. Toyka, K. V., Drachman, D. B., Pestronk, A. & Kao, I. (1975) Science 190, 397-499. 13. Toyka, K. V., Drachman, D. B., Griffin, D. E., Pestronk, A., Winkelstein, J. A., Fischbeck, K. H. & Kao, I. (1977) N. Engl. J. Med. 296, 125-131. 14. Kao, I. & Drachman, D. B. (1977) Science 196,527-529. 15. Appel, S. H., Anwyl, R., McAdams, M. W. & Elias, S. (1977) Proc. Natl. Acad. Sci. USA 74,2130-2134. 16. Bevan, S., Kullberg, R. W. & Heinemann, S. F. (1977) Nature
267,263-265. 17. Yaffe, D. (1973) in Tissue Culture, Methods and Applications, eds. Kruse, P. F. & Patterson, M. K., Jr. (Academic, New York), pp. 106-114. 18. Devreotes, P. N. & Fambrough, D. M. (1975) J. Cell Biol. 65, 335-358. 19. Devreotes, P. N. & Fambrough, D. M. (1976) Cold Spring Harbor Symp. Quant. Biol. 40,237-251. 20. Heide, K. & Schwick, H. G. (1978) in Handbook of Experimental Immunology, ed. Weir, D. M. (Blackwell Scientific Publications, Oxford), 3rd Ed., pp. 7.1-7.11. 21. Merlie, J. P., Changeux, J. P. & Gros, F. (1976) Nature 264, 74-76. 22. Stanley, E. F. & Drachman, D. B. (1978) Science, in press. 23. Patrick, J., McMillan, J., Wolfson, H. & O'Brien, J. C. (1977) J. Biol. Chem. 252,2143-2153. 24. Berg, D. K. & Hall, Z. W. (1975) J. Physiol. (London) 252, 771-789. 25. Drachman, D. B., Angus, C. W., Adams, R. N., Michelson, J. D. & Hoffman, G. J. (1978) N. Engl. J. Med., 298, 1116-1122. 26. Taylor, R. B., Duffus, W. P. H., Raff, M. C. & de Petris, S. (1971) Nature New Biol. 233,225-229. 27. Boyse, E. & Old, L. J. (1969) Ann. Rev. Genet. 3,269-290. 28. DeMeyts, P., Roth, J., Neville, D. M., Jr., Gavin, J. R. III & Lesnick, M. A. (1973) Biophys. Res. Commun. 55, 154-161. 29. Gavin, J. R. III, Roth, J., Neville, D. M., DeMeyts, P. & Buell, D. N. (1974) Proc. Natl. Acad. Sci. USA 71,84-88. 30. Devreotes, P. N. & Fambrough, D. M. (1976) Proc. Natl. Acad. Sci. USA 73,161-164.
