Calcium Channel Autoantibodies in the Lambert-Eaton Myasthenic Syndrome Katherine Leys, MSc, Bethan Lang, PhD, Irene Johnston, BSc, and John Newsom-Davis, MD
We have tested 36 patients with the Lambert-Eaton myasthenic syndrome for serum antibodies to voltage-gated calcium channels by using an immunoprecipitation assay with [1251) w-conotoxin-labeled voltage-gated calcium channels extracted from a human neuroblastoma cell line, SKN-SH. Forty-four percent of these patients had significant levels of antibody (30-1,466 pM) compared with healthy control individuals (< 15 pM). The incidence of positive sera in patients without associated small cell lung carcinoma (61%) was greater than in those patients with small cell lung carcinoma (28%). Results correlated strongly with results obtained using voltage-gated calcium channels extracted from the small cell lung carcinoma line, MARS. Anti-voltage-gated calcium channel antibody titers did not correlate with disease severity across individuals, but longitudinal studies in 2 patients receiving immunosuppressive therapy showed a clear inverse relation between antibody titer and an electromyographic index of disease se\-erity. l ' h e incidence of positive sera among patients with other neurological disorders was not significant, but 8 of 12 patients with rheumatoid arthritis or systemic lupus erythematosus had raised titers (30-82 pM). We conclude that the antibodies detected in this assay are heterogeneous and that some of them are likely to be implicated in this disorder of neuromuscular transmission. The assay should prove useful as an additional diagnostic aid in patients with Lambert-Eaton myasthenic syndrome. Leys K, Lang B, Johnston I, Newsom-Davis J. Calcium channel autoantibodies in the Lambert-Eaton myasthenic syndrome. Ann Neurol 1991;29:307-314
The Lambert-Eaton myasthenic syndrome (LEMS) is a disorder of neuromuscular transmission, characterized by reduced release of acerylcholine from the motor nerve terminals 111. It is associated with small cell lung carcinoma (SCLC) in approximately 60% of patients, and autonomic symptoms are also often present [2}. The evidence that LEMS is an autoimmune disease has recently been reviewed [3}. This mechanism is suggested by the association of LEMS with other diseases its correlation with the of autoimmune etiology {2, 4f, major histocompatibility complex and with IgG heavy chain markers { 5 ] , and the clinical response to immunosuppressive therapy {GI. Direct evidence was provided by the results of passive transfer experiments in which the main characteristics of E M S were induced in mice after the injection of IgG purified from the plasma of patients with LEMS [7, 8). The electrophysiological changes in the mice are consistent with a reduction in the number of functional, voltage-gated calcium channels (VGCCs) at motor nerve terminals [9]. Freeze-fracture elecrromicrographs of muscle from treated mice show a reduction in the number of active zone particles, thought to represent VGCCs [lo], and a disorganization of their normal array {I 11. This depletion was previously observed
at the nerve terminals of E M S patients [lZ], thus suggesting the presence of E M S autoantibodies to determinants on VGCCs. SCLC cells appear to express VGCCs. Direct recording has demonstrated that these cells can generate calcium spikes {13], and cultured SCLC lines have shown calcium influx after K'-induced membrane depolarization { 141. Furthermore, IgG purified from plasma of patients with LEMS substantially inhibits K+stimulated Ca" flux into these and other cell lines [14-171. Direct treatment of the rumor in patients with SCLC and E M S , by excision or x-irradiation, often results in improvement or remission of the neurological syndrome {l8], suggesting that in these patients, autoantibody production may be triggered by tumor VGCC determinants. Recently, a novel pepride, w-conotoxin (wCgTx), has been purified from the fish-eating snail, Conus geoxruphzls, and shown to block neuronal VGCCs [19} and also calcium channels in SCLC lines [IS]. High-affinity binding sites for { ' 2 5 1 ] ~ C g have T ~ been demonstrated in brain synaptosomes [20f, cultured neuronal cells [21], and SCLC lines [227.In this study, we describe the presence of high-affinity ['251]wCgTx binding sites in the human neuroblastoma cell line, SKN-SH, and a
From the Neurosciences Group, Institute of Molecular Medicine, [Jniversity of Oxford, John Radcliffe Hospital, Oxford, UK.
Address correspondence to Dr Lang, Neurosciences Group, Institute of Molecular Medicine. Universicy of Oxford, John Radcliffe OX3 UK.
Received .run 27, 1990, and in revised form Sep 13. Accepted for publication Sep 18, 1990.
Copyright 0 1991 by the American Neuroiogicd Association
307
human non-LEMS SCLC line, MARS. T h e s e two cell lines provided a source of solubilized VGCCs for irnmunoprecipitation assays t o investigate t h e presence of anti-VGCC antibodies in LEMS serum. Results using this assay t o test E M S and control sera have been briefly reported previously 1231. Similar assays have recently been described by Sher and colleagues [22, 241 and Lennon and Lambert [25}.
Methods iMembrane Preparation SKN-SH and MAR5 cells were grown in flasks containing RPMI 1640 medium with L-glutamine, 10% fetal calf serum, 50 Uiml penicillin, and 50 pg/ml streptomycin. At confluence, the cells were harvested, washed once with phosphatebuffered saline (PBS), and stored at - 70°C until required. Batches of membranes were prepared by resuspending cell pellets from approximately 30 confluent flasks in ice-cold 25 mM Tris-HCI/S mM HEPES buffer, pH 7.4, containing 0.32 M sucrose, 1 p M pepstatin, 2 p M leupeptin, 0.1 mM phenylmethylsulphonylfluoride, and 20 &ml soybean trypsin inhibitor. The cells were homogenized with a Polytron F T I O 35 homogenizer (Kinematica G m b H Lttau, Switzerland), pelleted at 18,000 rpm for 20 minutes, and washed twice with the previously mentioned buffer. Membranes were finally resuspended at a protein concentration of 10 to 20 mg/ mi, determined by the method of Lowry and colleagues [26] with bovine serum albumin (BSA) as standard, and stored in aliquots at - 70°C. {I2
’ I )uconotoxin Binding
Approximately 25 pg membrane protein was incubated with increasing amounts of [1251]wCgTx(2,000 Ci/mmol, Amersham International, Amersham, UK) in 250 p1 of the previously mentioned buffer containing 0.5% BSA (incubation buffer). Nonspecific binding was determined by the presence of 0.5 pM unlabeled wCgTx (Peninsula Laboratories [Europe] Ltd, St Helens, Merseyside, U.K.). After incubating for 45 minutes at room temperature, the membranes were collected on Whatman GF/C filters (BDH, Warwickshire, U.K.) that had been presoaked in 0.3% polyethylenimine (PEI). The filters were washed three times with 3 ml cold 50 mM Tris-HC1 buffer, p H 7.4, containing 1.5 mM CaCI2 and 0.2% BSA, and counted in a Packard gamma counter (Canberra Packard, Pangbourne, Berks, UK). Maximum binding capacity (Bmm)and affinity of {12SI]wCgTxfor membranes were determined by nonlinear regression analysis. To test the effect of IgG from patients with LEMS (prepared as previously described [27})on the number of [1251)wCgTx binding sites, SKN-SH cells were grown for 2 days in medium containing 2 mgirnl pooled IgG (from either 6 patients with LEMS or 6 control subjects). The cells were harvested and membranes prepared as described. Membranes were incubated with a saturating concentration of [1251)wCgTx (125 pM) and the number of binding sites compared with cells grown in medium alone.
lmmzlnoprecipitution Assay Aliquots of cell membranes were incubated in incubation x buffer with a saturating concentration of [l L S I f ~ C g T(200
308 Annals of Neurology
Vol 29 No 3 March 1991
pM). After 1 hour at 4”C, the unbound toxin was removed by centrifugation. The membrane pellet was washed once, resuspended in incubation buffer containing 1% Triton X100 (BDH, Warwickshire, UK), and left for 2 hours on ice. Unsolubilized material was spun down (30 minutes, 13,000 rpm) and the supernatant used as “extract” in the assay. A volume of extract containing 5 fmol [1251}~Cg?’x-VGCC complexes (calculated by counting a sample of supernatant) was added to test or control serum in a total volume of 100 p1. Increasing amounts of each serum sample between 0.25 and 5.0 pI were tested, but each tube was made up to a total of 5 p1 with carrier human serum (2 pooled healthy controls). After 2 hours at 4”C, 50 p1 goat anti-human IgG antibody was added to each tube. One milliliter of PTX buffer (20 mM phosphate buffer p H 7.4, 0.1% Triton X-100) was added after the formation of a visible precipitate (20-30 minutes) and left for a further 30 minutes. The tubes were then centrifuged at 13,000 rpm and the pellets washed twice with PTX before counting. Results were expressed as picomoles of [1251}wCgTxbinding sites precipitated per liter of serum (pM) and were calculated by regression analysis from the linear part of the titration curve. Each sample was assayed on at least two separate occasions.
Patients Serum samples from a total of 36 patients with LEMS (Table 1)were tested using SKN-SH human neuroblastoma cells as the source of solubilized VGCCs. The diagnosis of E M S was made on the basis of clinical features [2] and electrophysiological recordings using surface electrodes, available in all but I patient. The resting compound muscle action potential (CMAP) in abductor digiti minimi was reduced in amplitude (< 8.5 mV) in 34 of 35 patients after supramaximal stimulation of the ulnar nerve. The percentage increment in CMAP amplitude after 15 seconds maximum voluntary contraction of the muscle was abnormal in all 35 patients (5% increment = 100 x [maximum CMAP amplitude - initial CMAP amplitude)/initial CMAP amplitude; upper limit of normal is 25% [6]). Seventeen of the 18 patients with E M S and lung cancer had biopsy-proven SCLC. Thirteen of the 18 patients without detectable lung cancer had been followed for at least 5 years, and 3 further cases were nonsmokers, making an underlying SCLC very unlikely. Of the 2 patients in this group who died, 1 had a lymphoma at autopsy; no autopsy was done in the other patient in whom SCLC cannot be excluded in view of a smoking history and preterminal hyponatremia and pneumonia. The following 48 control individuals were tested: 10 healthy subjects; 10 patients with SCLC but no E M S ; 8 patients with myasthenia gravis (MG); 8 patients with other neurological disorders (OND), comprising 4 with GuillainBarre syndrome, 2 with chronic inflammatory demyelinating polyneuropathy, 1 with chronic neuropathy, and 1 with ncuromyotonia; 12 patients with non-organ-specific autoimmune disorders, comprising 6 with systemic lupus erythematoms (SLE) and 6 with rheumatoid arthritis (RA). In further assays using MAR5 SCLC cells as the source of antigen, a total of 25 patients with LEMS and 36 control subjects were tested, representative samples being selected from each of the preceding groups.
Table 1 . Cltnical Detazh of the Putzents wzth Lambert-Eaton Myartbenrc Syndrom Patient No. /Smoking Habit
LEMS Duration" Age (yr)
Sex
It
44
2 3 4 5 6
53 64
F M M
+ + + + +
60 62 58 58 34 55 71 52 56 59 66 38 64 62 66 50 42 47 28 29 61 66 40 28 43 22 48 60 47 54
7 t
a+ 9 +
10 + 11 t 12 + 13 + 14 15 + 16 +
+
17 t
18 + 19 0 20 0 21 0 22 0 23 + 24 0 25 + 26 0 27 o 28 + 29 0 30 0 11 0 32 +
33
+
60
34 A 35 0 36 0
15 58
M M
F F M M F M F M F F F M M M
M M M F F M F F M M M M M M M M M
(yr)
0.4 0.3 0.8 0.3 1.3 1.0 0.8 1.0
1.3 0.2 1.2 0.5 1.7
0.4 3.6 0.9 1.2 0.7 11.3 10.0 I .o
8.0 0.5 4.2 3.1 13.0 0.3 3.3 4.0 2.8 1.5 1.6 1.2 1.0 0.7 3.0
CMAP Cancer Dctcctcd
Initial (mV)
1.2 1.5 0.5 1.0
SCLC SCLC SCLC SCLC SCLC SCLC SCLC SCLC SCLC SCLC Lung' SCLC SCLC SCLC SCLC SCLC SCLC SCLC
0.7
1,757
7.6 6.4 1.0
13191 1,040 333 1,169 65 3,275 188 180 205 93 3 250 650 500 1,800 1,96' 518 3,2'5 400 3,400 285 166 3'6 146 448
1.3 I10.5 0.4 6.6 4.0 1.8 1.5
.o
1 1.2
None None
1.5 0.3
Nonc None Lymphoma Breast None None None None
0.7
3.4 0.4 2.8
0.4 3.5 2.0 4.2 6.1
None
None None None None None
1.9 . . .d 1.5 2.0
Titer (pM)
27
2.1 3.2
1.6
None
($7) 1,213 327 2,445 200 3,900 315 411
0.1
None
Incrementb
0
46 7
0
93 3 960
40 23 15 14 7 0 0 12 40
54 218
4 0 2 4 4: 254 -
1,466 1,275 58 1,447 2 7 30 36 8
37 31 208 1
22
"At the rime of first sample '% increase in compound muscle action potentiai (CMAP) amplitude after 15 seconds of maximum voluntary contraction 'Histology nor available. '"or derermined.
LEMS
=
Lamben-Eaton myasthenic syndrome; SCLC
=
small cell lung carcinoma.
Results {'"l)wConotoxin Binding Saturable populations of specific {'251)wCgTxbinding sites were found in membranes prepared from both SKN-SH and MAR5 cells (Fig la). The presence of BSA in the incubation and wash buffers and the presoaking of the filters in PEI (see Methods) was crucial in preventing high levels of nonspecific binding. When
these precautions were taken, the nonspecific binding at K , concentrations was reduced to approximately 20% of the total binding in SKN-SH membranes, although it was still high (4096) in MAR5 membranes (Fig l b , c). SKN-SH cells had an approximately fourfold greater number of binding sites than MAR5 cells (Bmax= 56 k 9 and 13 1 fmol/mg protein respectively, mean
*
Leys et al: Ca"
Channel Antibodies in LEMS
3W
Table 2. The Eflect of IgG from Patients toith Lamberthfyasthenic Syndrome and from Control Subjec1.i on {12il}uConotoxinBinding to Cell Membranes
Specific binding (fmoles/mg)
O'
Eataton
I
% Specific [lz51]w-Conotoxin Binding Sites (n = 3-5)
SKN-SH
EMS Control (OND) Control (Healthy)
MAR5
43 i 7" 120 i 25 115 2 17
40 2 5" 93 i 25 ND
Values are mean -t SEiM. Results are expressed as the percentage of the values obtained with cells grown in medium alone. IgG from patients with LEMS significantly reduced the number of ['251]w-conotoxin binding sites compared with medium alone.
ap < 0.001. 40
20
0
60
80
100
= human neuroblastoma cell. line; MAR5 = non-EMS small cell lung carcinoma line; LEMS = Lambert-Baton myasthenic syndrome; OND = other neurological disorder; ND = not determined.
SKhT-SH
[ 1 2 5 - I - ~ C g T ~pM l
A
SKN-SH 160
rfmoles/mg
binding
120
0
20
40
60
80
100
[125-I-wCgTx] pM B
MAR5 fmoles/mg binding
100 r
0
20
40
60
80
loo
1125-I-wCgTxj pM C
Fig I . (A)Saturation binding cz4rue.r of {'2'I}wconotoxin (125I-WCgTx) t o mmbranes prepared from S K N S H neuroblu.stmma and MARS small cell lzlng carcinoma (non-LEMS) cells sells (3) (0). Spectj5s binding was calculated by subtracting the nonspecific binding measured in the praense ofO.5 pM unlabeled w-CgTx from the total binding. SKN-SH membranej-bound 56 ? 9 fmoll mg protein (KD = 18 f 6 p M ) . MARS membranes bound 13 2 Ifmolimg protein (K, = 7 5 I pM) (means and SEMS of 3 deteminations). (B and C) Total (A)and nonspecific(0) binding vf{'2sI}wCgTx to SKN-SH and MARS membranes, respectively. LEMS = kmbert-Eaton myasthenic syndrome.
310 Annals of Neurology Vol 29 No 3 March 1991
i SEM of 3 determinations). The binding was of high affinity in both instances; in SKN-SH membranes, the KD value was 18 ? 6 pM and in MAR5 membranes, 7 5 1 pM (mean 2 SEM of 3 determinations). After 2 days in the presence of pooled IgG from 6 patients with LEMS, the number of [1'51]wCg'I'x binding sites was significantly reduced in both cell lines Cp < 0.001) compared with that found in cells grown in medium alone (Table 2). Control IgG pools prepared from sera from 6 healthy subjects or 6 patients with OND did not significantly affect the number of binding sites, compared with the number of binding sites in cells grown in medium alone. In these experiments, the cells were harvested and membranes prepared as described in Methods and IgG was not present in the final binding assay. Separate experiments, however, showed that the presence of IgG from patients with IEMS did not directly inhibit ['"I)wCgTx binding to membranes (data not shown).
Imrnunoprecipitution Assuys lJsing ['L51]wCgTx-labeledSKN-SH cells as the source of solubilized VGCCs, the antibody titers for the sera from 36 patients with E M S ranged from 5 to 1,466 pM, with a mean and SEM of 151 64 pM. Sixteen of these samples (44%) were positive for the presence of anti-VGCC antibodies, defined as a titer greater than 30 pM (3 SDs above the mean for the healthy control subjects). An example of VGCC precipitation obtained with the serum of a patient with LEMS positive for antibody compared with that of a healthy control is shown in Figure 2. The antibody titers for the 48 control samples had an overall mean and SEM of 15 ? 4 pM. All the healthy control subjects and those patients with OND had titers less than 20 pM. Two patients with SCLC
*
Table 3. Antz-Voltage-gated Calcium Channel Antibodzes rn Sera frnm Patzents with Lambert-Eaton Myasthenic Syndrome and from Control Sublects: S K N J H hTeuroblastoinaa\ a Source of Voltage-gated Culcium Channels
cprn precipitated
2500 r
ri
LEMS SCLC-EMS Non-SCLC-UhlS
36
Controla Healrhy
48
Positive for Antibody ( 2 30 pM) (9;)
151 -t 64 27 2 12 274 = 123 15 i 4 -G?4 14 i 5
18 18 LO 10
SCLC OND MG SXIRA
Fig 2. Precipitation of {1251i~conotoxin-labeledsoltage-gated ca/cium channels ({i2SI)oCgTx-VGCCs)extractedfronr SKNSH neuroblastoma cells bji the serum of a patient with LEMS (Patient 24, Table lj (OJ compared with the precipitation obtained with serum from a healthy control subject (0. The dotted lines show the amount of prec.ipitation obtained with the same 2 sera in the presence of 0.5 pV unlabeled uconotoxin. The specific aitivity of{'2'l}wCgTx in this experiment was 1,000 Ciimmol. LEMS = Lambert-Eaton mymthenii syndrome.
Anribody Titer (pM) (mean 2 S E X
Group
8
-l+j
8
1326
12
44 2 6
"p < 0.02, b~ < 0.01, x2 test vs. the healthy controls = Lamberr-Eaton myasrhenic syndrome; SCLC = small cell lung carcinoma; OND = other iieurological disorder; MG = myasrhenia gravis; SLE = sysrermc lupus erythernatosus; RA = rheumatoid mhritis.
LEMS
but not LEMS and 1 patient with M G had antibody titers slightly above the normal range as previously defined. Additionally, antibody was detected at raised titer in 8 of 12 patients with RA ( 5 patients) or SLE 13 patients), although in none was the titer higher than 90 pM (Fig 3). The mean values are summarized in Table 3. The patients with LEMS were divided into those with SCLC and those without detectable SCLC. The mean titers and SEMs for the 2 groups were 27 f- 12 and 274 2 123 pM, respectively. The proportion of patients positive for antibody was also different in the 2 groups (28% and 61%, respectively). By the x2 test, the incidence of anti-VGCC antibody-positive sera compared with the incidence in the healthy controls was significant in the group of patients with LEMS as
Fig 3. Serum antibodies in the Lambert-Eaton mymthenic syndrome (LEMS) and controls measured by inimunoprecipit/ntionof voltage-gated calcium channels (VGCCsJextractedfrom SKhT-SH neuroblastoma cells (see Methods), Antibod31 titers were coxridered positiue zf greater than -30pM (mean + 3 SDs for the healthy controlsubjects{HC}).SCLC = small celllung carcinoma;OND = other neuvohgical disorders: M G = myasthenia gravir; RA = rheumatoid arthriti.r; SLE = systemic lupus eythematos2c.c.
Anti-VGCC Antibody Titre (pM)
IA d
0
0
r-
0 0 0
2
0 I
1
I
I
l
l 1 -
I
I
I
I
1 4
I
a
1 1 1 1 -
Leys et al: Ca2+ Channel Antibodies in E M S
311
a whole ( p < 0.02) and in the group of patients with LEMS but without SCLC (J < 0.01), whereas the incidence in the group of patients with LEMS and SCLC was not significant. The incidence in the group of patients with SLE or RA (67%) was also significant ( p < 0.02), but the incidence in the other groups of control subjects was not. The results obtained with 25 sera from patients with E M S using antigen extracted from MAR5 cells were broadly similar to the results obtained with SKN-SH cells, although the antibody titers were, in general, slightly lower and the incidence of positive sera was less. There was a highly significant correlation, however, between the titers obtained for the two assays ( Y = 0.37, p < 0.001, n = 2 5). Similar correlations have been reported previously between two SCLC lines and between a SCLC line and the human neuroblastoma cell line, IMR-32 [25]. Of sera from 36 control subjects tested with SCLC antigen, 1 patient with MG, 2 patients with OND (those with neuromyoronia and chronic neuropathy j, 2 patients with SLE, and 2 patients with RA had antibody levels higher than 3 SDs above the mean for the healthy control subjects, using this antigen (29 pM). No clear correlation was evident between the titers for the group of patients with SLE or RA when tested with antigen from the two cell lines ( r = 0.56, n = 8). To investigate the pathological significance of the anti-VGCC antibodies in the patients with E M S , we compared the antibody titers obtained with SKN-SH antigen with the corresponding CMAP amplitudes and percentage increment in amplitude after 15 seconds of maximum voluntary contraction (see Table 1). There was no correlation between antibody levels and either of these two indices of disease severity ( r = 0.28 and 0.32, respectively; n = 35). We also undertook longitudinal studies using serum samples collected over several years from 2 patients with E M S receiving immunosuppressive drug therapy who had high initial levels of anti-VGCC antibody, comparing the titers with an electromyographic index of disease severity (CMAP amplitude). Neither of these patients had detectable SCLC. Patient 1, followed for 6 years, showed a pronounced clinical improvement and decrease in antibody titer during prednisolone therapy (Fig 4a). The antibody titers were inversely related to the CMAP amplitude. In Patient 2, followed for 11 years, antibody levels showed a similar inverse relation with CMAP amplitude (Fig 4b), although in this patient, the antibody titer remained greater than 500 phl even when the CMAP amplitude was in the normal range. Discussion The neurnnal calcium channel antagonist wCgTx bound specifically to membranes prepared from SKN-
312
Annals of Neurology
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No 3 March 1991
1: 2.5,
-
-
- 20
2
f
F i g 4. Change in serum anti-VGCC antibody titer (+, solid line) of 2 patients with LEMS, and the corresponding C M A P amplitudes (V,dotted line) in retrospective studies of 6 and 1I pears (Patients 24 and 23; rerpectitively;Table 1 ) . All ofthe serum jamples for each patient were assayed for antibody on the same occasion. The alteernateday ( a d , dose of prednisolone administered to the patients is illustrated diagrammatical4y beneath each graph. The patient in (a) was also treatedwith 120 mg 3,4-diaminopyridine hih and the patient in (b) with 100 mg azathioprine daily. throughout the period of study. V G C C = voltage-gated calcizm channel; LEMS = Lamhert-Eaton m-yasthenicsyndrome; C M A P = compound muscle potential.
SH human neuroblastoma cells and from a human SCLC line, MARS. These binding sites were reduced in number when cells were cultured in IgG prepared from the serum of patients with E M S . A similar reduction has been reported in the neuroblastoma cell line, IMR-32 [24]. These studies provide further evidence that LEMS antibodies may downregulate VGCCs. Electrophysiological studies have demonstrated an inhibitory effect of IgG from patients with LEMS on VGCC function with no immediate pharma-
cological blockade [28-30). Also, morphological studies have shown that IgG from patients with E M S cross-links and reduces the density of active zone particles by accelerated internalization [ 31, 32). Using SKN-SH cells as a source of antigen, we have extracted {‘251}wCgTx-VGCCcomplexes for use in a radioimmunoassay to detect E M S antibodies. Fortyfour percent of tested sera from patients with LEMS contained antibodies that specifically precipitated these complexes. The percentage was higher among those patients without detectable lung cancer (61p%) than among those with lung cancer (28%)). Lennon and Lambert [25}, however, found a higher proportion of positives in the group of patients with LEMS and SCLC than in the group of patients without cancer. These results using radioimmunoassays contrast with the similarity in the range of inhibitory effects exerted by IgG from the 2 groups of patients on K’-stimulated calcium influx [14-17). We have found slightly raised VGCC precipitation (less than 40 pM) by a few sera from the groups of patients with MG or SCLC, but not by the sera from patients with OND. There was, however, a significant incidence of anti-VGCC antibody in patients with RA or SLE. Although none of the antibody titers in this group of patients w a high (all < 90 pM) the results imply caution in the use of this assay as a specific diagnostic test for E M S , unless the possibility of non-organ-specific autoimmune diseases such as RA and SLE can be excluded. Interestingly, Lennon and Lambert [ 2 5 ] also report raised anti-VGCC titers in a single patient with RA. It will be important to test whether the antibodies in these patients modulate VGCCs in intact cells, as do E M S anti-VGCC antibodies. If they do not, they may be “false positives” caused by their binding to other membrane proteins unrelated to calcium channel function that remain associated with VGCCs during the extraction procedure. Radioimmunoassays using antigen extracted from MAR5 SCLC cell membranes gave results similar to the results obtained with neuroblastoma cells. The highest titers again were in the group of patients with LEMS but without SCLC. The strong correlation between the results obtained for the sera from patients with LEMS using the two cell lines suggests antigenic similarity of the VGCCs extracted. The high incidence of antibody in sera from patients with RA or S E was confirmed with SCLC (MAR5) antigen, although no correlation was demonstrable between the titers obtained using SCLC and neuroblastoma VGCC. The 3 sera with raised titers among the other controls were from patients different from those positive in the SKN-SH assay. This lack of correlation suggests that the use of two sources of VGCC may help to eliminate false positives. Our results and those of other groups using similar
assays {22-25} indicate that anti-VGCC antibodies in a proportion of patients are not being detected. Are the antibodies detected by these assays responsible for the downregulation of VGCCs that leads to the defect in neuromuscular transmission? The poor correlation we have found across individuals, between antibody titers and the electromyographic index of disease severity, contrasts with the close correlation of this index with the functional effects of IgG on K+-stimulated calcium influx [17]. The results suggest that the antibody population detected by radioimmunoassay is heterologous, reminiscent of the finding in patients with MG in whom anti-acetylcholine receptor antibody titers correlate poorly with muscle weakness between individuals {33]. There is a much stronger correlation in longitudinal studies of individual patients with MG, particularly during plasma exchange and immunosuppressive therapy [34].In preliminary longitudinal studies, we have shown that there may be a similar inverse correlation between anti-VGCC antibody levels and disease severity in some patients with LEMS. There is, however, a further reason for questioning the role of the antibodies detected in the radioimmunoassay. I& from patients with LEMS interacts with channels that can be classified electrophysiologically and pharmacologically as L-type. It inhibits calcium flux through L-channels in a rodent neuroblastoma x glioma hybrid cell line [35] and in bovine adrenal chromaffin cells [36}, and also reduces rat pituitary hormonal release {37}. Although wCgTx is thought to inhibit both L and N channels on some neuronal cells [38}, it may not be active against L channels in neuroblastoma cell lines { Z l , 391. Furthermore, sensitivity to wCgTx has not been demonstrated at the mammalian neuromuscular junction E401. Thus, the conotoxin-labeled VGCCs extracted from neuronal cell lines may differ antigenically from those affected at the nerve terminal by E M S antibodies. The radioimmunoassay may detect some anti-VGCC antibodies that do not cross-react with VGCCs at the nerve terminal and would therefore not contribute to the pathophysiological changes of E M S at the neuromuscular junction, although they may play a part in the associated autonomic symptoms. References 1. Lambert EH, Elmqvist D. Quantal components of end-plate po-
tentials in the myasthenic syndrome. Ann NY Acad Sci 1971;183: 183-199 2. ONeill JH, Murray NMF, Newsum-Davis J. The Lambert-Eaton myasthenic syndrome: a review of 50 cases. Brain 1986;lll: 577-596 3. Vincent A, Lang B, Newsom-Davis J. Autoimmunity to the voltage-gated calcium channel underlies the Lambert-Eaton myasthenic syndrome, a paraneoplastic disorder. Trends Neurosci 1989;12~496-502 4. Lennon VA, Lambert EH, Whirtingham S, Fairbanks V. Auto-
Leys et al: Ca2’ Channel Antibodies in LEMS
313
5.
6. 7.
8.
immunity in the Lambert-Eaton myasthenic syndrome. Muscle Nerve 1982;5:S21-S25 Willcox N , Demaine AG, Newsom-Davis J, et al. Increased frequency of I& heavy chain marker Glm(2) and of HLAB8 in Lambert-Eaton myasthenic syndrome with and without associated lung carcinoma. Hum Immunol 1985;14:29-36 Newsom-Davis J, Murray NMF. Plasma exchange and immunosuppressive drug treatment in the Lambert-Eaton myasthenic syndrome. Neurology 1984;34:480-485 Lang B, Newsom-Davis J, Wray D, et al. Autoimmune aetiolugy fur myasthenic (Eaton-Lambcrt) syndrome. Lancet 1981;2: 224-226 Lang B, Newsom-Davis J, Prior C, Wray D. Antibodies to motor nerve terminals: an electrophysiological study of a human myasthenic syndrome transferred to mouse. J Physiul 1983;
3441335-345
9. Lang B, Newsom-Davis J, Peers C, et al. The effect of myasthe& syndrome antibody on presynaptic calcium channels in the mouse. J Physiol 1987;390:257-270 10. Pumplin DW, Reese TS, Llinas R. Are the presynaptic membrane particles the calcium channels? Proc Natl Acad Sci USA 1981;78:72 10-7213 11. Fukunaga H, Engel AG, Lang B, et al. Passive transfer of Lambert-Eaton myasthenic syndrome with IgG from man to mouse depletes the presynaptic membrane active zones. Proc Natl Acad Sci USA 1983;80:7636-7640 12. Fukunaga H , Engel AG, Osame M, Lambert EH. Paucity and disorganization of presynaptic membrane active zones in the Lambert-Eaton myasthenic syndrome. Muscle Nerve 1982; 51686-697 13. McCann FV, Pettengill OS, Cole JJ, et al. Calcium spike electrogenesis and other electrical activity in continuously cultured small cell carcinoma of the lung. Science 1981;212:1155-1157 14. Roberts A, Perera S, Lang B, et al. Paraneoplastic myasthenic syndrome IgG inhibits *'Ca'+ flux in a human small cell lung carcinoma line. Nature 1985;317:737-739 IS. D e Aizpurua HJ, Iambert EH, Griesmann GE, et al. Antagonism of voltage-gated calcium channels in small cell carcinomas of patients with and without Lambert-Eaton myasthenic syndrome by autoantibodies, w-conotoxin and adenosine. Cancer Res 1988;48:4719-4724 16. Lang B, Leys K, Vincent A, Newsom-Davis J. K+-stimulated Ca2+influx in cell lines derived from small cell lung cancer and neuronal turnours. In: Calcium channels: structure and function. Ann N Y Acad Sci 1989;560:294-296 17. Lang €3, Vincent A, Murray NMF, Newsom-Davis J. LambertEaton myasthenic syndrome: Immunoglobulin G inhibition of Caz+ flux in tumor cells correlates with disease severity. Ann Neurol 1989;25:265-27 1 18. Chalk CH, Murray NMF, Newsom-Davis J, et d.Response of the Lambert-Eaton myasthenic syndrome to treatment of associated small cell lung carcinoma. Neurology 1990;40:1552-1556 19. Kerr LM, Yoshikami D. A venom peptide with a novel presynaptic blocking action. Nature 1984;308:282-284 20. Cruz LJ, Olivera BM. Calcium channel antagonists: w-conotuxin defines a new high affinity site. J Biol Chem 1986;261: 6230-6233 21. Sher E, Pandiella A, Clemcnti F. wConotoxin binding and effects on calcium channel function in human neuroblastoma and rat pheochromocytonia cell lines. FEBS Lett 1988;235: 178-182
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22. Sher E, Pandiella A, Clementi F. Voltage-operated calcium channels in small cell lung carcinoma cell lines: pharmacological function and immunological properties. Cancer Res 1990;SO: 3892-3896 23. Leys K, Lang B, Vincent A, Newsom-Davis J. Calcium channel autobodies in Lambert-Eaton myasthenic syndrome. Lancet 1989;2:1107 24. Sher E, Gotti C, Canal N , et al. Specificity of calcium channel autoantibodies in Lambert-Eaton myasthenic syndrome. Lancet 1989;2:640-643 25. Lennon VA, Lambert EH. Autoantibodies bind solubilized calcium channel-o-conotoxin complexes from small cell lung carcinoma: a diagnostic aid for Lambert-Eaton myasthenic syndrome. Mayo Clin Proc 1989;64:1498-1504 26. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin-Phenol reagent. J Biol Chem 1951; 1931265-275 27. Horejsi J, Smetana R. The isolation of gamma-globulins from blood serum by Rivanol. Acta Med Scand 1956;155:65-70 28. Prior C, Lang B, Wray D, Newsom-Davis J. Action of LambertEaton myasthenic syndrome IgG at motor nerve terminals. Ann Neurol 1985;17:58?-592. 29. Lambert EH, Lennon VA. Selected IgG rapidly induces Lambert-Eaton myasthenic syndrome in mice: complement independence and EMG abnormalities. Muscle Nerve 1988;ll: 1133-1 145 30. Kim YI, Sanders DB, Johns TR, et al. Lambert-Eaton myasthenic syndrome: the lack of short-term effects of serum factors on neuromuscular transmission. J Neurol Sci 1988;87:1-13 3 1. Fukuoka T, Engel AG, Lang B, et al. Lamberc-Eaton myasthenic syndrome: early morphological effects of IgG on the presynaptic membrane active zones. Ann Neurol 1987;22:193-199 32. Nagel A, Engel AG, Lang B, et al. Lambert-Eaton myasthenic syndrome IgG depletes presynaptic membrane active zone particles by antigenic modulation. Ann Neurol 1988;24:552-558 33. Vincent A, Newsom-Davis J. Anti-acetylcholine receptor antibodies. J Neurol Xeurosurg Psychiatry 1980;43:590-600 34. Newsom-Davis J, Pinching AJ, Vincent A, Wilson SG. Function of circulating antibody to acetylcholine receptor in myasthenia gravis: investigation by plasma exchange. Neurology (NY) 1978;28:266-272 35. Peers C, Lang B, Newsom-Davis J, W Wray D. Selective action of myasthenic syndrome antibodies on calcium channels in a rodent neuroblastoma x glioma cell line. J Physiol 1990;421: 293-308 36. Kim YI, Neher E. IgG from patients with Lambert-Eaton syndrome blocks voltage-dependent calcium channels. Science 1988;239:405-408 37. Login IS, Kim YI, Judd AM, et al. Immunoglobulins of Lambert-Eaton myasthenic syndrome inhibit rat pituitary hormone release. Ann Ncurol 1987;22:610-614 38. McCleskey EW, Fox AP, Peldman DH, et al. w-Conotoxin: direct and persistent blockade of specific types of calcium channels in neurones but not muscle. Proc Natl Acad Sci USA 198?:84:4327-4331 39. Carbone E, Sher E, Clementi F. Ca currents in human neuroblastoma IMR32 cells: kinetics, permeahility and pharmacology. Pflugers Arch 1990;416:170-179 40. Yoshikami D, Bagabaldo 2, Olivera BM. The inhibitory effects of omega-conotoxins on Ca channels and synapses. Ann NY Acad Sci 1989;560:230-248
We have tested 36 patients with the Lambert-Eaton myasthenic syndrome for serum antibodies to voltage-gated calcium channels by using an immunoprecipitation assay with [1251) w-conotoxin-labeled voltage-gated calcium channels extracted from a human neuroblastoma cell line, SKN-SH. Forty-four percent of these patients had significant levels of antibody (30-1,466 pM) compared with healthy control individuals (< 15 pM). The incidence of positive sera in patients without associated small cell lung carcinoma (61%) was greater than in those patients with small cell lung carcinoma (28%). Results correlated strongly with results obtained using voltage-gated calcium channels extracted from the small cell lung carcinoma line, MARS. Anti-voltage-gated calcium channel antibody titers did not correlate with disease severity across individuals, but longitudinal studies in 2 patients receiving immunosuppressive therapy showed a clear inverse relation between antibody titer and an electromyographic index of disease se\-erity. l ' h e incidence of positive sera among patients with other neurological disorders was not significant, but 8 of 12 patients with rheumatoid arthritis or systemic lupus erythematosus had raised titers (30-82 pM). We conclude that the antibodies detected in this assay are heterogeneous and that some of them are likely to be implicated in this disorder of neuromuscular transmission. The assay should prove useful as an additional diagnostic aid in patients with Lambert-Eaton myasthenic syndrome. Leys K, Lang B, Johnston I, Newsom-Davis J. Calcium channel autoantibodies in the Lambert-Eaton myasthenic syndrome. Ann Neurol 1991;29:307-314
The Lambert-Eaton myasthenic syndrome (LEMS) is a disorder of neuromuscular transmission, characterized by reduced release of acerylcholine from the motor nerve terminals 111. It is associated with small cell lung carcinoma (SCLC) in approximately 60% of patients, and autonomic symptoms are also often present [2}. The evidence that LEMS is an autoimmune disease has recently been reviewed [3}. This mechanism is suggested by the association of LEMS with other diseases its correlation with the of autoimmune etiology {2, 4f, major histocompatibility complex and with IgG heavy chain markers { 5 ] , and the clinical response to immunosuppressive therapy {GI. Direct evidence was provided by the results of passive transfer experiments in which the main characteristics of E M S were induced in mice after the injection of IgG purified from the plasma of patients with LEMS [7, 8). The electrophysiological changes in the mice are consistent with a reduction in the number of functional, voltage-gated calcium channels (VGCCs) at motor nerve terminals [9]. Freeze-fracture elecrromicrographs of muscle from treated mice show a reduction in the number of active zone particles, thought to represent VGCCs [lo], and a disorganization of their normal array {I 11. This depletion was previously observed
at the nerve terminals of E M S patients [lZ], thus suggesting the presence of E M S autoantibodies to determinants on VGCCs. SCLC cells appear to express VGCCs. Direct recording has demonstrated that these cells can generate calcium spikes {13], and cultured SCLC lines have shown calcium influx after K'-induced membrane depolarization { 141. Furthermore, IgG purified from plasma of patients with LEMS substantially inhibits K+stimulated Ca" flux into these and other cell lines [14-171. Direct treatment of the rumor in patients with SCLC and E M S , by excision or x-irradiation, often results in improvement or remission of the neurological syndrome {l8], suggesting that in these patients, autoantibody production may be triggered by tumor VGCC determinants. Recently, a novel pepride, w-conotoxin (wCgTx), has been purified from the fish-eating snail, Conus geoxruphzls, and shown to block neuronal VGCCs [19} and also calcium channels in SCLC lines [IS]. High-affinity binding sites for { ' 2 5 1 ] ~ C g have T ~ been demonstrated in brain synaptosomes [20f, cultured neuronal cells [21], and SCLC lines [227.In this study, we describe the presence of high-affinity ['251]wCgTx binding sites in the human neuroblastoma cell line, SKN-SH, and a
From the Neurosciences Group, Institute of Molecular Medicine, [Jniversity of Oxford, John Radcliffe Hospital, Oxford, UK.
Address correspondence to Dr Lang, Neurosciences Group, Institute of Molecular Medicine. Universicy of Oxford, John Radcliffe OX3 UK.
Received .run 27, 1990, and in revised form Sep 13. Accepted for publication Sep 18, 1990.
Copyright 0 1991 by the American Neuroiogicd Association
307
human non-LEMS SCLC line, MARS. T h e s e two cell lines provided a source of solubilized VGCCs for irnmunoprecipitation assays t o investigate t h e presence of anti-VGCC antibodies in LEMS serum. Results using this assay t o test E M S and control sera have been briefly reported previously 1231. Similar assays have recently been described by Sher and colleagues [22, 241 and Lennon and Lambert [25}.
Methods iMembrane Preparation SKN-SH and MAR5 cells were grown in flasks containing RPMI 1640 medium with L-glutamine, 10% fetal calf serum, 50 Uiml penicillin, and 50 pg/ml streptomycin. At confluence, the cells were harvested, washed once with phosphatebuffered saline (PBS), and stored at - 70°C until required. Batches of membranes were prepared by resuspending cell pellets from approximately 30 confluent flasks in ice-cold 25 mM Tris-HCI/S mM HEPES buffer, pH 7.4, containing 0.32 M sucrose, 1 p M pepstatin, 2 p M leupeptin, 0.1 mM phenylmethylsulphonylfluoride, and 20 &ml soybean trypsin inhibitor. The cells were homogenized with a Polytron F T I O 35 homogenizer (Kinematica G m b H Lttau, Switzerland), pelleted at 18,000 rpm for 20 minutes, and washed twice with the previously mentioned buffer. Membranes were finally resuspended at a protein concentration of 10 to 20 mg/ mi, determined by the method of Lowry and colleagues [26] with bovine serum albumin (BSA) as standard, and stored in aliquots at - 70°C. {I2
’ I )uconotoxin Binding
Approximately 25 pg membrane protein was incubated with increasing amounts of [1251]wCgTx(2,000 Ci/mmol, Amersham International, Amersham, UK) in 250 p1 of the previously mentioned buffer containing 0.5% BSA (incubation buffer). Nonspecific binding was determined by the presence of 0.5 pM unlabeled wCgTx (Peninsula Laboratories [Europe] Ltd, St Helens, Merseyside, U.K.). After incubating for 45 minutes at room temperature, the membranes were collected on Whatman GF/C filters (BDH, Warwickshire, U.K.) that had been presoaked in 0.3% polyethylenimine (PEI). The filters were washed three times with 3 ml cold 50 mM Tris-HC1 buffer, p H 7.4, containing 1.5 mM CaCI2 and 0.2% BSA, and counted in a Packard gamma counter (Canberra Packard, Pangbourne, Berks, UK). Maximum binding capacity (Bmm)and affinity of {12SI]wCgTxfor membranes were determined by nonlinear regression analysis. To test the effect of IgG from patients with LEMS (prepared as previously described [27})on the number of [1251)wCgTx binding sites, SKN-SH cells were grown for 2 days in medium containing 2 mgirnl pooled IgG (from either 6 patients with LEMS or 6 control subjects). The cells were harvested and membranes prepared as described. Membranes were incubated with a saturating concentration of [1251)wCgTx (125 pM) and the number of binding sites compared with cells grown in medium alone.
lmmzlnoprecipitution Assay Aliquots of cell membranes were incubated in incubation x buffer with a saturating concentration of [l L S I f ~ C g T(200
308 Annals of Neurology
Vol 29 No 3 March 1991
pM). After 1 hour at 4”C, the unbound toxin was removed by centrifugation. The membrane pellet was washed once, resuspended in incubation buffer containing 1% Triton X100 (BDH, Warwickshire, UK), and left for 2 hours on ice. Unsolubilized material was spun down (30 minutes, 13,000 rpm) and the supernatant used as “extract” in the assay. A volume of extract containing 5 fmol [1251}~Cg?’x-VGCC complexes (calculated by counting a sample of supernatant) was added to test or control serum in a total volume of 100 p1. Increasing amounts of each serum sample between 0.25 and 5.0 pI were tested, but each tube was made up to a total of 5 p1 with carrier human serum (2 pooled healthy controls). After 2 hours at 4”C, 50 p1 goat anti-human IgG antibody was added to each tube. One milliliter of PTX buffer (20 mM phosphate buffer p H 7.4, 0.1% Triton X-100) was added after the formation of a visible precipitate (20-30 minutes) and left for a further 30 minutes. The tubes were then centrifuged at 13,000 rpm and the pellets washed twice with PTX before counting. Results were expressed as picomoles of [1251}wCgTxbinding sites precipitated per liter of serum (pM) and were calculated by regression analysis from the linear part of the titration curve. Each sample was assayed on at least two separate occasions.
Patients Serum samples from a total of 36 patients with LEMS (Table 1)were tested using SKN-SH human neuroblastoma cells as the source of solubilized VGCCs. The diagnosis of E M S was made on the basis of clinical features [2] and electrophysiological recordings using surface electrodes, available in all but I patient. The resting compound muscle action potential (CMAP) in abductor digiti minimi was reduced in amplitude (< 8.5 mV) in 34 of 35 patients after supramaximal stimulation of the ulnar nerve. The percentage increment in CMAP amplitude after 15 seconds maximum voluntary contraction of the muscle was abnormal in all 35 patients (5% increment = 100 x [maximum CMAP amplitude - initial CMAP amplitude)/initial CMAP amplitude; upper limit of normal is 25% [6]). Seventeen of the 18 patients with E M S and lung cancer had biopsy-proven SCLC. Thirteen of the 18 patients without detectable lung cancer had been followed for at least 5 years, and 3 further cases were nonsmokers, making an underlying SCLC very unlikely. Of the 2 patients in this group who died, 1 had a lymphoma at autopsy; no autopsy was done in the other patient in whom SCLC cannot be excluded in view of a smoking history and preterminal hyponatremia and pneumonia. The following 48 control individuals were tested: 10 healthy subjects; 10 patients with SCLC but no E M S ; 8 patients with myasthenia gravis (MG); 8 patients with other neurological disorders (OND), comprising 4 with GuillainBarre syndrome, 2 with chronic inflammatory demyelinating polyneuropathy, 1 with chronic neuropathy, and 1 with ncuromyotonia; 12 patients with non-organ-specific autoimmune disorders, comprising 6 with systemic lupus erythematoms (SLE) and 6 with rheumatoid arthritis (RA). In further assays using MAR5 SCLC cells as the source of antigen, a total of 25 patients with LEMS and 36 control subjects were tested, representative samples being selected from each of the preceding groups.
Table 1 . Cltnical Detazh of the Putzents wzth Lambert-Eaton Myartbenrc Syndrom Patient No. /Smoking Habit
LEMS Duration" Age (yr)
Sex
It
44
2 3 4 5 6
53 64
F M M
+ + + + +
60 62 58 58 34 55 71 52 56 59 66 38 64 62 66 50 42 47 28 29 61 66 40 28 43 22 48 60 47 54
7 t
a+ 9 +
10 + 11 t 12 + 13 + 14 15 + 16 +
+
17 t
18 + 19 0 20 0 21 0 22 0 23 + 24 0 25 + 26 0 27 o 28 + 29 0 30 0 11 0 32 +
33
+
60
34 A 35 0 36 0
15 58
M M
F F M M F M F M F F F M M M
M M M F F M F F M M M M M M M M M
(yr)
0.4 0.3 0.8 0.3 1.3 1.0 0.8 1.0
1.3 0.2 1.2 0.5 1.7
0.4 3.6 0.9 1.2 0.7 11.3 10.0 I .o
8.0 0.5 4.2 3.1 13.0 0.3 3.3 4.0 2.8 1.5 1.6 1.2 1.0 0.7 3.0
CMAP Cancer Dctcctcd
Initial (mV)
1.2 1.5 0.5 1.0
SCLC SCLC SCLC SCLC SCLC SCLC SCLC SCLC SCLC SCLC Lung' SCLC SCLC SCLC SCLC SCLC SCLC SCLC
0.7
1,757
7.6 6.4 1.0
13191 1,040 333 1,169 65 3,275 188 180 205 93 3 250 650 500 1,800 1,96' 518 3,2'5 400 3,400 285 166 3'6 146 448
1.3 I10.5 0.4 6.6 4.0 1.8 1.5
.o
1 1.2
None None
1.5 0.3
Nonc None Lymphoma Breast None None None None
0.7
3.4 0.4 2.8
0.4 3.5 2.0 4.2 6.1
None
None None None None None
1.9 . . .d 1.5 2.0
Titer (pM)
27
2.1 3.2
1.6
None
($7) 1,213 327 2,445 200 3,900 315 411
0.1
None
Incrementb
0
46 7
0
93 3 960
40 23 15 14 7 0 0 12 40
54 218
4 0 2 4 4: 254 -
1,466 1,275 58 1,447 2 7 30 36 8
37 31 208 1
22
"At the rime of first sample '% increase in compound muscle action potentiai (CMAP) amplitude after 15 seconds of maximum voluntary contraction 'Histology nor available. '"or derermined.
LEMS
=
Lamben-Eaton myasthenic syndrome; SCLC
=
small cell lung carcinoma.
Results {'"l)wConotoxin Binding Saturable populations of specific {'251)wCgTxbinding sites were found in membranes prepared from both SKN-SH and MAR5 cells (Fig la). The presence of BSA in the incubation and wash buffers and the presoaking of the filters in PEI (see Methods) was crucial in preventing high levels of nonspecific binding. When
these precautions were taken, the nonspecific binding at K , concentrations was reduced to approximately 20% of the total binding in SKN-SH membranes, although it was still high (4096) in MAR5 membranes (Fig l b , c). SKN-SH cells had an approximately fourfold greater number of binding sites than MAR5 cells (Bmax= 56 k 9 and 13 1 fmol/mg protein respectively, mean
*
Leys et al: Ca"
Channel Antibodies in LEMS
3W
Table 2. The Eflect of IgG from Patients toith Lamberthfyasthenic Syndrome and from Control Subjec1.i on {12il}uConotoxinBinding to Cell Membranes
Specific binding (fmoles/mg)
O'
Eataton
I
% Specific [lz51]w-Conotoxin Binding Sites (n = 3-5)
SKN-SH
EMS Control (OND) Control (Healthy)
MAR5
43 i 7" 120 i 25 115 2 17
40 2 5" 93 i 25 ND
Values are mean -t SEiM. Results are expressed as the percentage of the values obtained with cells grown in medium alone. IgG from patients with LEMS significantly reduced the number of ['251]w-conotoxin binding sites compared with medium alone.
ap < 0.001. 40
20
0
60
80
100
= human neuroblastoma cell. line; MAR5 = non-EMS small cell lung carcinoma line; LEMS = Lambert-Baton myasthenic syndrome; OND = other neurological disorder; ND = not determined.
SKhT-SH
[ 1 2 5 - I - ~ C g T ~pM l
A
SKN-SH 160
rfmoles/mg
binding
120
0
20
40
60
80
100
[125-I-wCgTx] pM B
MAR5 fmoles/mg binding
100 r
0
20
40
60
80
loo
1125-I-wCgTxj pM C
Fig I . (A)Saturation binding cz4rue.r of {'2'I}wconotoxin (125I-WCgTx) t o mmbranes prepared from S K N S H neuroblu.stmma and MARS small cell lzlng carcinoma (non-LEMS) cells sells (3) (0). Spectj5s binding was calculated by subtracting the nonspecific binding measured in the praense ofO.5 pM unlabeled w-CgTx from the total binding. SKN-SH membranej-bound 56 ? 9 fmoll mg protein (KD = 18 f 6 p M ) . MARS membranes bound 13 2 Ifmolimg protein (K, = 7 5 I pM) (means and SEMS of 3 deteminations). (B and C) Total (A)and nonspecific(0) binding vf{'2sI}wCgTx to SKN-SH and MARS membranes, respectively. LEMS = kmbert-Eaton myasthenic syndrome.
310 Annals of Neurology Vol 29 No 3 March 1991
i SEM of 3 determinations). The binding was of high affinity in both instances; in SKN-SH membranes, the KD value was 18 ? 6 pM and in MAR5 membranes, 7 5 1 pM (mean 2 SEM of 3 determinations). After 2 days in the presence of pooled IgG from 6 patients with LEMS, the number of [1'51]wCg'I'x binding sites was significantly reduced in both cell lines Cp < 0.001) compared with that found in cells grown in medium alone (Table 2). Control IgG pools prepared from sera from 6 healthy subjects or 6 patients with OND did not significantly affect the number of binding sites, compared with the number of binding sites in cells grown in medium alone. In these experiments, the cells were harvested and membranes prepared as described in Methods and IgG was not present in the final binding assay. Separate experiments, however, showed that the presence of IgG from patients with IEMS did not directly inhibit ['"I)wCgTx binding to membranes (data not shown).
Imrnunoprecipitution Assuys lJsing ['L51]wCgTx-labeledSKN-SH cells as the source of solubilized VGCCs, the antibody titers for the sera from 36 patients with E M S ranged from 5 to 1,466 pM, with a mean and SEM of 151 64 pM. Sixteen of these samples (44%) were positive for the presence of anti-VGCC antibodies, defined as a titer greater than 30 pM (3 SDs above the mean for the healthy control subjects). An example of VGCC precipitation obtained with the serum of a patient with LEMS positive for antibody compared with that of a healthy control is shown in Figure 2. The antibody titers for the 48 control samples had an overall mean and SEM of 15 ? 4 pM. All the healthy control subjects and those patients with OND had titers less than 20 pM. Two patients with SCLC
*
Table 3. Antz-Voltage-gated Calcium Channel Antibodzes rn Sera frnm Patzents with Lambert-Eaton Myasthenic Syndrome and from Control Sublects: S K N J H hTeuroblastoinaa\ a Source of Voltage-gated Culcium Channels
cprn precipitated
2500 r
ri
LEMS SCLC-EMS Non-SCLC-UhlS
36
Controla Healrhy
48
Positive for Antibody ( 2 30 pM) (9;)
151 -t 64 27 2 12 274 = 123 15 i 4 -G?4 14 i 5
18 18 LO 10
SCLC OND MG SXIRA
Fig 2. Precipitation of {1251i~conotoxin-labeledsoltage-gated ca/cium channels ({i2SI)oCgTx-VGCCs)extractedfronr SKNSH neuroblastoma cells bji the serum of a patient with LEMS (Patient 24, Table lj (OJ compared with the precipitation obtained with serum from a healthy control subject (0. The dotted lines show the amount of prec.ipitation obtained with the same 2 sera in the presence of 0.5 pV unlabeled uconotoxin. The specific aitivity of{'2'l}wCgTx in this experiment was 1,000 Ciimmol. LEMS = Lambert-Eaton mymthenii syndrome.
Anribody Titer (pM) (mean 2 S E X
Group
8
-l+j
8
1326
12
44 2 6
"p < 0.02, b~ < 0.01, x2 test vs. the healthy controls = Lamberr-Eaton myasrhenic syndrome; SCLC = small cell lung carcinoma; OND = other iieurological disorder; MG = myasrhenia gravis; SLE = sysrermc lupus erythernatosus; RA = rheumatoid mhritis.
LEMS
but not LEMS and 1 patient with M G had antibody titers slightly above the normal range as previously defined. Additionally, antibody was detected at raised titer in 8 of 12 patients with RA ( 5 patients) or SLE 13 patients), although in none was the titer higher than 90 pM (Fig 3). The mean values are summarized in Table 3. The patients with LEMS were divided into those with SCLC and those without detectable SCLC. The mean titers and SEMs for the 2 groups were 27 f- 12 and 274 2 123 pM, respectively. The proportion of patients positive for antibody was also different in the 2 groups (28% and 61%, respectively). By the x2 test, the incidence of anti-VGCC antibody-positive sera compared with the incidence in the healthy controls was significant in the group of patients with LEMS as
Fig 3. Serum antibodies in the Lambert-Eaton mymthenic syndrome (LEMS) and controls measured by inimunoprecipit/ntionof voltage-gated calcium channels (VGCCsJextractedfrom SKhT-SH neuroblastoma cells (see Methods), Antibod31 titers were coxridered positiue zf greater than -30pM (mean + 3 SDs for the healthy controlsubjects{HC}).SCLC = small celllung carcinoma;OND = other neuvohgical disorders: M G = myasthenia gravir; RA = rheumatoid arthriti.r; SLE = systemic lupus eythematos2c.c.
Anti-VGCC Antibody Titre (pM)
IA d
0
0
r-
0 0 0
2
0 I
1
I
I
l
l 1 -
I
I
I
I
1 4
I
a
1 1 1 1 -
Leys et al: Ca2+ Channel Antibodies in E M S
311
a whole ( p < 0.02) and in the group of patients with LEMS but without SCLC (J < 0.01), whereas the incidence in the group of patients with LEMS and SCLC was not significant. The incidence in the group of patients with SLE or RA (67%) was also significant ( p < 0.02), but the incidence in the other groups of control subjects was not. The results obtained with 25 sera from patients with E M S using antigen extracted from MAR5 cells were broadly similar to the results obtained with SKN-SH cells, although the antibody titers were, in general, slightly lower and the incidence of positive sera was less. There was a highly significant correlation, however, between the titers obtained for the two assays ( Y = 0.37, p < 0.001, n = 2 5). Similar correlations have been reported previously between two SCLC lines and between a SCLC line and the human neuroblastoma cell line, IMR-32 [25]. Of sera from 36 control subjects tested with SCLC antigen, 1 patient with MG, 2 patients with OND (those with neuromyoronia and chronic neuropathy j, 2 patients with SLE, and 2 patients with RA had antibody levels higher than 3 SDs above the mean for the healthy control subjects, using this antigen (29 pM). No clear correlation was evident between the titers for the group of patients with SLE or RA when tested with antigen from the two cell lines ( r = 0.56, n = 8). To investigate the pathological significance of the anti-VGCC antibodies in the patients with E M S , we compared the antibody titers obtained with SKN-SH antigen with the corresponding CMAP amplitudes and percentage increment in amplitude after 15 seconds of maximum voluntary contraction (see Table 1). There was no correlation between antibody levels and either of these two indices of disease severity ( r = 0.28 and 0.32, respectively; n = 35). We also undertook longitudinal studies using serum samples collected over several years from 2 patients with E M S receiving immunosuppressive drug therapy who had high initial levels of anti-VGCC antibody, comparing the titers with an electromyographic index of disease severity (CMAP amplitude). Neither of these patients had detectable SCLC. Patient 1, followed for 6 years, showed a pronounced clinical improvement and decrease in antibody titer during prednisolone therapy (Fig 4a). The antibody titers were inversely related to the CMAP amplitude. In Patient 2, followed for 11 years, antibody levels showed a similar inverse relation with CMAP amplitude (Fig 4b), although in this patient, the antibody titer remained greater than 500 phl even when the CMAP amplitude was in the normal range. Discussion The neurnnal calcium channel antagonist wCgTx bound specifically to membranes prepared from SKN-
312
Annals of Neurology
Vol 29
No 3 March 1991
1: 2.5,
-
-
- 20
2
f
F i g 4. Change in serum anti-VGCC antibody titer (+, solid line) of 2 patients with LEMS, and the corresponding C M A P amplitudes (V,dotted line) in retrospective studies of 6 and 1I pears (Patients 24 and 23; rerpectitively;Table 1 ) . All ofthe serum jamples for each patient were assayed for antibody on the same occasion. The alteernateday ( a d , dose of prednisolone administered to the patients is illustrated diagrammatical4y beneath each graph. The patient in (a) was also treatedwith 120 mg 3,4-diaminopyridine hih and the patient in (b) with 100 mg azathioprine daily. throughout the period of study. V G C C = voltage-gated calcizm channel; LEMS = Lamhert-Eaton m-yasthenicsyndrome; C M A P = compound muscle potential.
SH human neuroblastoma cells and from a human SCLC line, MARS. These binding sites were reduced in number when cells were cultured in IgG prepared from the serum of patients with E M S . A similar reduction has been reported in the neuroblastoma cell line, IMR-32 [24]. These studies provide further evidence that LEMS antibodies may downregulate VGCCs. Electrophysiological studies have demonstrated an inhibitory effect of IgG from patients with LEMS on VGCC function with no immediate pharma-
cological blockade [28-30). Also, morphological studies have shown that IgG from patients with E M S cross-links and reduces the density of active zone particles by accelerated internalization [ 31, 32). Using SKN-SH cells as a source of antigen, we have extracted {‘251}wCgTx-VGCCcomplexes for use in a radioimmunoassay to detect E M S antibodies. Fortyfour percent of tested sera from patients with LEMS contained antibodies that specifically precipitated these complexes. The percentage was higher among those patients without detectable lung cancer (61p%) than among those with lung cancer (28%)). Lennon and Lambert [25}, however, found a higher proportion of positives in the group of patients with LEMS and SCLC than in the group of patients without cancer. These results using radioimmunoassays contrast with the similarity in the range of inhibitory effects exerted by IgG from the 2 groups of patients on K’-stimulated calcium influx [14-17). We have found slightly raised VGCC precipitation (less than 40 pM) by a few sera from the groups of patients with MG or SCLC, but not by the sera from patients with OND. There was, however, a significant incidence of anti-VGCC antibody in patients with RA or SLE. Although none of the antibody titers in this group of patients w a high (all < 90 pM) the results imply caution in the use of this assay as a specific diagnostic test for E M S , unless the possibility of non-organ-specific autoimmune diseases such as RA and SLE can be excluded. Interestingly, Lennon and Lambert [ 2 5 ] also report raised anti-VGCC titers in a single patient with RA. It will be important to test whether the antibodies in these patients modulate VGCCs in intact cells, as do E M S anti-VGCC antibodies. If they do not, they may be “false positives” caused by their binding to other membrane proteins unrelated to calcium channel function that remain associated with VGCCs during the extraction procedure. Radioimmunoassays using antigen extracted from MAR5 SCLC cell membranes gave results similar to the results obtained with neuroblastoma cells. The highest titers again were in the group of patients with LEMS but without SCLC. The strong correlation between the results obtained for the sera from patients with LEMS using the two cell lines suggests antigenic similarity of the VGCCs extracted. The high incidence of antibody in sera from patients with RA or S E was confirmed with SCLC (MAR5) antigen, although no correlation was demonstrable between the titers obtained using SCLC and neuroblastoma VGCC. The 3 sera with raised titers among the other controls were from patients different from those positive in the SKN-SH assay. This lack of correlation suggests that the use of two sources of VGCC may help to eliminate false positives. Our results and those of other groups using similar
assays {22-25} indicate that anti-VGCC antibodies in a proportion of patients are not being detected. Are the antibodies detected by these assays responsible for the downregulation of VGCCs that leads to the defect in neuromuscular transmission? The poor correlation we have found across individuals, between antibody titers and the electromyographic index of disease severity, contrasts with the close correlation of this index with the functional effects of IgG on K+-stimulated calcium influx [17]. The results suggest that the antibody population detected by radioimmunoassay is heterologous, reminiscent of the finding in patients with MG in whom anti-acetylcholine receptor antibody titers correlate poorly with muscle weakness between individuals {33]. There is a much stronger correlation in longitudinal studies of individual patients with MG, particularly during plasma exchange and immunosuppressive therapy [34].In preliminary longitudinal studies, we have shown that there may be a similar inverse correlation between anti-VGCC antibody levels and disease severity in some patients with LEMS. There is, however, a further reason for questioning the role of the antibodies detected in the radioimmunoassay. I& from patients with LEMS interacts with channels that can be classified electrophysiologically and pharmacologically as L-type. It inhibits calcium flux through L-channels in a rodent neuroblastoma x glioma hybrid cell line [35] and in bovine adrenal chromaffin cells [36}, and also reduces rat pituitary hormonal release {37}. Although wCgTx is thought to inhibit both L and N channels on some neuronal cells [38}, it may not be active against L channels in neuroblastoma cell lines { Z l , 391. Furthermore, sensitivity to wCgTx has not been demonstrated at the mammalian neuromuscular junction E401. Thus, the conotoxin-labeled VGCCs extracted from neuronal cell lines may differ antigenically from those affected at the nerve terminal by E M S antibodies. The radioimmunoassay may detect some anti-VGCC antibodies that do not cross-react with VGCCs at the nerve terminal and would therefore not contribute to the pathophysiological changes of E M S at the neuromuscular junction, although they may play a part in the associated autonomic symptoms. References 1. Lambert EH, Elmqvist D. Quantal components of end-plate po-
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