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Electrophysiological effects of myasthenic serum factors studied in mouse muscle.

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Electrophysiologd Effects
of Myasthenic Serum Factors
Studied in Mouse Muscle
A. J, Lerrick, BSc,' D. Wray, DPhil,' A. Vincent, MB, MSc,? and J. Newsom-Davis, MDI
Miniature end-plate potential (mepp) amplitudes were investigated in mouse diaphragm exposed in vitro to different
serum fractions from seven patients with myasthenia gravis who had elevated serum anti-acetylcholine receptor
antibody levels and from controls. The mepp amplitudes were significantly reduced by whole myasthenic sera, restored
by washing, and not reduced by heated (56°C) myasthenic sera, which would inactivate complement but not antireceptor antibody. Immunoglobulin G (1gG)-depletedmyasthenic sera also significantly reduced mepp amplitudes, while the
IgG fraction alone or with normal serum did not. The results indicate that in vitro reduction of mepp amplitudes in
mouse muscle by myasthenic sera is not dependent on the IgG fraction alone, and requires a heat-sensitive factor.
Lerrick AJ, Wray D, Vincent A, Newsom-Davis J: Electrophysiological effects of myasthenic serum factors
studied in mouse muscle. Ann Neurol 13:186-191, 1983
The disorder of neuromuscular transmission in myasthenia gravis (MG) that causes fatigable muscle weakness is due to a postsynaptic defect. It is characterized
physiologically by a reduction in the amplitudes of the
miniature end-plate potentials (mepps) and end-plate
potentials (epps) (71, which represent the potential
changes evoked by, respectively, the spontaneous release of a single quantum of acetylcholine (ACh) and
the nerve-stimulated release of many quanta. This reduction can be accounted for by a decrease in the number of functional acetylcholine receptors (AChRs) in
the postsynaptic membrane. Consistent with this decrease, the sensitivity of the myasthenic end-plate to
iontophoretically applied ACh is also decreased El)
and there is a reduction in the number of binding sites
for a-bungarotoxin (a-BuTx), which specifically labels
AChRs (9, 12).
Anti-AChR antibody, an immunoglobulin G (IgG)
antibody detectable in the serum of most patients with
MG (4, 151, is implicated in the loss of functional
AChRs. Myasthenic IgG appears to !ead to reduced
density of AChRs by complement-mediated lysis [S,
201 and by increasing the rate of AChR degradation by
cross-linking of receptors [b}. Whether it can also
cause a direct immunopharmacological block of the receptor is uncertain. M G sera reduced the sensitivity of
cultured human (31, rat [21, and chicken [lo} muscle
cells to ACh. Sensitivity could not be restored by washing and was not dependent on the presence of comple-
ment. It is not clear whether the reduced sensitivity can
be attributed in part to direct block of AChRs rather
than wholly to accelerated AChR degradation in these
culture preparations.
Shibuya et al {22} demonstrated a significant reduction in mepp amplitudes within 90 minutes of application of M G serum to rat muscle, which was reversible
by washing. Whether this effect was dependent on
complement was not established. On the other hand,
no reduction in mepp amplitudes was observed by Albuquerque et al El} in rat muscle exposed to M G IgG.
In the present study, we confirmed that M G serum can
reduce mepp amplitudes in the mouse diaphragm and
investigated the serum fractions that lead to this effect.
From the Departments of 'Pharmacology and ?Neurological Science, Royal Free Hospital School o f Medicine, Rowland Hill St,
London N W 3 2 PF, England.
Received Apr 13, 1982, and in revised form June 4. Accepted for
publication June 6, 1982.
Methods
Seven patients with MG were studied, of whom six were
female. All had elevated serum titers of AChR antibody as
measured by an immunoprecipitation assay using a-BuTxlabeled human AChR obtained from amputated calf muscle.
Four patients had a thymoma, and three patients had thymic
hyperplasia. Clinical details are given in Table 1. Sera from
seven healthy laboratory workers were used as controls.
Diaphragm muscle was removed from BKTO mice anesthetized with chloroform. Hemidiaphragms were then transferred to well-oxygenated (95% 02h%COz) Krebs solution.
Intracellular voltage recording was carried out as previously described [24] using microelectrodes filled with 3 M
potassium chloride with resistances in the range of 5 to 30
Address reprint requests to Dr Wray.
186 0364-5 134/83/020186-06$1.50 0 1982 by the American Neurological Association
Table 1 . Clinical Details on the Seven Patients
with Myasthenia Gravis
Patient No.,
Age (Yd,
and Sex
1. 39, F
2. 60, F
3. 50, M
4. 52, F
5. 21, F
6. 24, F
7. 32, F
Duration
of Disease
6 Yr
2 Yr
3 mo
22 yr
2.5 yr
1 Yr
4 mo
Grade
Thymic
Abnormalities
IV
IIB
IIA
IV
IIB
IIA
I IB
Hyperplasia
T h ymoma
Thymoma
Thymoma
Hyperplasia
Hyperplasia
Thymoma
man muscle extract labeled with "'I a-BuTx, and IgG was
precipitated with antihuman IgG (Seward Laboratories Ltd,
London, UK) as previously described [18]. Antibodies crossreacting with mouse AChR were determined by substituting
a crude extract of mouse muscle for human muscle in the
assay.
The presence of factors inhibiting a-BuTx binding to human AChR was assessed by incubating excess whole serum
or serum fractions with human AChR, followed by addition
of ''1 a-BuTx (5 nM). The binding of a-BuTx was determined using a DEAE filter disc assay 1211. The number of a BuTx binding sites inhibited was expressed as a percentage of
the number precipitated per liter of serum.
Comparisons were made using Student's t test (two-tailed);
standard error of the mean (SEM) are quoted.
means
*
megohms. Hemidiaphragms were exposed for 4 hours to
oxygenated sera or to IgG in Krebs solution, and intracellular
recordings were carried out in the same serum or solution.
The muscle was then perfused with oxygenated Krebs solution (30 minutes) and intracellular recordings again were carried out. In other experiments, intracellular recordings were
made in freshly dissected muscles under constant perfusion
with oxygenated Krebs solution. All the exposures to solutions, as well as electrical recordings, were carried out at
room temperature (2 1" to 26°C). Intracellular voltage recordings, simultaneously at low and high gain, were stored on
magnetic tape (Store 4DS, Racal Recorders Ltd, Southampton, UK). End-plates were localized by moving the microelectrode until mepps were recorded with fast rise times (c1
msec). Amplitudes of mepps were measured by computer
(Cambridge Electronic Design System, Cambridge, UK) after
digitizing at 5 kHz. At 4 to 9 end-plates, an average of 111
(SD = 66) mepps were measured per end-plate, the mean
amplitude per end-plate was calculated, and these values
were used to obtain the grand mean for the end-plates in each
hemidiap hragm.
Sera from controls and M G patients were stored at
- 20°C. Before use, sera were thawed and spun at 5,000 rpm
for 5 minutes to remove any particles. They were then dialyzed against Krebs solution of the following Composition
(mM): Na', 143.0; K f , 5.9; M g Z f , 1.20; Ca2+, 2.52; C1-,
127.7; HC03-, 25.0; HzP04-, 1.2; S o d 2 - , 1.2; andglucose,
11.1. The p H of all solutions used for electrical recordings
ranged from 7.1 to 7.4. Decomplementation was performed
by heating the sera for 30 minutes at 56°C. The sera were
then respun as described and were then dialyzed.
IgG from one patient was prepared by the RivanoY
ammonium sulfate method 111). Selective removal of IgG of
subclasses 1, 2, and 4 was performed in two patients by adsorption of sera with immobilized staphylococcal protein A
(protein A Sepharose, Pharmacia Fine Chemicals, Ltd.) using
an equal volume of the column material. The passthrough
fractions were designated IgG-depleted serum and were
dialyzed against Krebs solution. The IgG adsorbed to the
column was eluted with a citrate-phosphate buffer, p H 2.5,
and the eluted material was then dialyzed extensively against
Krebs solution before use.
Sera and IgG fractions were assayed for anti-AChR antibodies as follows. Serum (1 to 5 ~ 1was
) incubated with hu-
Results
Examples of m e p p s in myasthenic sera are shown in
Figure 1. Histograms of m e p p amplitudes showed t h e
usual symmetrical distribution for controls and for myasthenic sera (Fig 2). A total of 54,248 m e p p s were
measured at 488 end-plates in this study. The mean
resting membrane potential was 64.6 t 0.2 mV (SD
= 4.6 mV).
The m e a n mepp amplitude in unheated MG sera was
0.85 t 0.06 mV (n = 6 muscles; each muscle exposed
t o o n e serum) and was significantly reduced compared
t o that in control (unheated, nonmyasthenic) sera (1.13
0.07 m V ; n = 6 ;p < 0.02) (Table 2 ) . The reduction
in m e p p amplitude did not correlate with t h e antiAChR antibody titer, n o r did it correlate with the titer
against mouse AChR, which was low in certain patients
(see Table 2).
I n contrast to unheated sera, heated MG sera, which
retained anti-AChR antibody activity (see Table 2), had
no significant effect on mepp amplitudes. The mean
value (1.17 S 0.08 m V ; n = 7) was similar to that in
control (heated, nonmyasthenic) sera and was significantly greater than that in unheated myasthenic sera (p
< 0.01).
The reduced mepp amplitudes obtained in unheated
MG sera were restored t o control levels by washing for
+
UNHEATED YIISTHENIC SERUM
HEATED YYASTWLNIC SERUM
1.5."
L
&&
LLLL
*
u
F i g I . Examples of miniature end-plate potentials: intracellular
recordings 122°C) at end-plates of moue diaphragm muscle.t in
myasthenic serum with (right) and without (left)preziom heating to 56"Cfor 30 mintita. Calibrations are marked on lhe
figure.
Lerrick et al: Effects of Myasthenic Serum
187
::~
CONTROL
(unheated)
60
CONTROL
n
r
60
- -
n 30
c
O
20
b
0
E
10
$
0
0
30 minutes in Krebs solution (see Table 2). Similar
washing of muscles exposed to control sera (heated and
unheated) and to heated MG sera did not significantly
change the mean mepp amplitudes (see Table 2).
Further experiments were undertaken to investigate
the possible role of IgG in the observed effects of MG
serum. Serum fractions were prepared using sera (from
Patients 1, 2, and 3 ) that had been effective in decreasing mepp amplitudes. Unheated sera from which IgG
subclasses 1 , 2 , and 4 had been removed by adsorption
with staphylococcal protein A retained their capacity to
reduce mepp amplitudes compared with unheated control sera treated in the same way, and this reduction
was again significant (p < 0.01) (Table 3). The antiAChR concentrations in the IgG-depleted sera are
shown in Table 3.
In other experiments, the IgG fraction was prepared
either by Rivanol extraction (Patient 1) or by elution
from protein A,in which case IgG3 would have been
absent (Patients 1 and 3). The IgG preparations were
applied in Krebs solution at concentrations similar to
those of the original sera. No significant reduction in
mepp amplitudes was found compared with results in
control Krebs solution (see Table 3), although these
preparations contained considerable anti-AChR. In addition, no reduction in mepp amplitude was observed
on exposure to MG IgG (Rivanol extraction) (Patient
1) together with an equal volume of unheated control
serum, a value (1.08 ? 0.12 mV, 7 end-plates) that did
not differ from controls.
To see whether the inhibitory effect of IgG-depleted
(heated)
10
20
amplilude (mV)
n
r
0
amplitude (mV)
MYASTHENIC
(unheated)
MYAST HE N IC
(heated)
60
L
40
0
10
amplitude (m V)
omplilude (mV)
Fig 2. Examples of histograms of miniuture end-pbte potential
lmeppi umplitudes recorded 12.3" t o 24°C) in control sera und in
myasthenic sera with or u~ithoutpretioux heating t o 56"Cfor .30
minute.r.
Table 2. Effect of Heated and Unheated Myasthenic Sera on Mouse Muscle mepp Amplitudes"
Anti-AChR Titers
in Heated Serum
(nrnoVL)
mepp Amplitudes (mV)
Patient No.
Unheated Serum
Krebs Wash
Heated Serum
Krebs Wash
Human AChR
~
1
2
0.72 2
0.68 r
0.75 r
0.97 2
1.01 ?
3
4
5
6
0.94
7
Mean
Mean
control
2
0.07 (7)
0.12 (6)
0.05 (8)
0.09 (7)
0.10 (6)
0.06(7)
...
0.85 t 0.06 [(,Ib
1.13
2
0.07 [6]
1.23 2
1.08 _t
1.13 -+
1.50 t
1.33 t
1.38 t
...
1.28 ?
0.11 (7)
0.11 (6)
0.08 ( 6 )
0.13 (6)
0.21 (6)
0.15 (6)
0.06 [6]
1.25 i 0.06 [6]
1.45 t
1.12 t
0.96 t
0.94 t
1.46 t
1.14 t
1.11 _t
1.17
*
0.16 (5)
0.12 (7)
0.07 (6)
0.05 (7)
0.07 (6)
0.06 (6)
0.09 (6)
0.08 171
1.18 -t 0.07 [61
1.74 t
1.18 t
1.18 t
1.19 t
1.30 ?
1.24
...
1.31 t
_f
7.0
6.0
7.4
26.3
14.1
8.3
15.6
0.09 [ 6 ] 12.7
~
0.20 (7)
0.14 (6)
0.14 (6)
0.09 (6)
0.18 (6)
0.11 (6)
1.31 t 0.08 [ 5 ]
<0.2
Mouse AChR
~~~~
3.2
0.1
2.7
* 2.6 171
5.2
1.8
1.1
3.2
2.5
2
0.6 [7]
<O. 1
"The mepp amplitudes for each patient are means ( 2 SEMI from the number of end-plates shown in parentheses (each patient's serum was
exposed to one muscle). The grand mean and SEM of these values (one value per muscle) are shown at the bottom of the table (number of
muscles in brackets). Control values were obtained by identical manipulations, with test sera replaced by appropriate control sera.
bSignificant difference: p < 0.02 versus unheated control sera, p < 0.01 versus control heated sera and myasthenic heated sera, and p < 0.002
versus Krebs wash values. Other mean values for mepp amplitudes (bottom of table) were not significantly different (p > 0.05) from each other.
mepp
=
miniature end-plate potential; AChR = acetylcholine receptor.
188 Annals of Neurology
Vol 13 No 2
February 1983
Table 3. EHect of Myasthenic IgG-depfeted Sera and Myasthenic IgG on Mouse Muscle mepp Amplitudes"
IgG-depleted Serum
Patient No.
1
2
3
Anti-AChR
Titerb
Anti-a-BuTx
Site (5%)'
mepp
0.1
0
<5
0.65 2 0.07 (14, 2) 17.7
0.41 _c 0.08 (6,1)
...
0.67 2 0.09 (6,1)
5 .O
0.60 0.06 ~ 4 1 ~
1.13
Mean
Mean control
IgG in Krebs Solution
<5
16
Amplitude (mV)
1.02
?
Anti-AChR
Titerb
Anti-a-BuTx
Site ($23)'
mepp
14
...
0.91 ? 0.05 (23, 3)
...
1.00 ? 0.08 ( 6 , 1)
0.94 2 0.03 c41
1.01 ? 0.06 [S]
18
0.08 [GI
Amplitude (mV)
T h e mean mepp amplitude (and SEM) were obtained for each patient's serum or IgG by averaging over the number of end-plates, shown as the
first figure in parentheses (second figure is number of muscles).The mean mepp amplitude for each muscle was also obtained (not shown), and the
grand mean (and SEM) of these values (one value per muscle) are given at the bottom of the table (number of muscles in brackets). Controls were
obtained using unheated control sera (IgG-depleted) or Krebs solution, as appropriate. The results in this table were obtained in a different batch
of BKTO mice from those in Table 2. which accounts for differences in the sets of control values.
bNanomoles of a-BuTx binding sites precipitated per liter.
'Inhibition of c*-BuTx binding as percentage of anti-AChR.
dSignificantdifference: p < 0.01 versus IgG-depleted control sera and myasthenic IgG in Krebs solution, p < 0.002 versus Krebs solution. Other
mean values for mepp amplitudes (bottom of table) were not significantly different (p > 0.25) from each other.
mepp = miniature end-plate potential; AChR = acetylcholine receptor; a-BuTx = a-bungarotoxin.
sera was due to the presence of an IgG3 antibody directed against the a-BuTx binding site that inhibited
ACh binding to the receptor, the fractions were tested
for their ability to inhibit a-BuTx binding to solubilized
AChR. The results are shown in Table 3, where it can
be seen that two of the IgG-depleted preparations contained very little antibody and produced negligible
inhibition of toxin binding, yet gave a significant reduction in mepp amplitude. For the third patient, IgGdepleted serum contained anti-AChR and inhibited aBuTx binding, but the reduction in mepp mplitude
was no greater for this serum than for the other two.
Discussion
We found that mepp amplitudes were decreased in
mouse muscle exposed to MG serum in vitro, as
Shibuya et al {22] reported in rat muscles. A presynaptic action of the MG serum, though not ruled out, is
very unlikely since there is no evidence that quantum
size (the number of ACh molecules per packet) is reduced in MG; indeed, ACh content 1171, choline
acetyltransferse activity fl6], and ACh release [17} are
all increased in myasthenic muscle. The decrease in
mepp amplitudes is almost certainly due to a postsynaptic action of serum components. MG serum could
reduce AChR density by increasing the rate of degradation of junctional and extrajunctional AChR, but in
view of the rapid restoration of the reduced mepp amplitudes by washing, such a mechanism cannot underlie
our observations. MG serum thus appears able to produce a reversible block of AChR in vitro in mouse
muscle. Shibuya et al [22] attributed this blocking effect to anti-AChR antibodies. Binding of antibody to
receptor does not by itself adequately account for our
findings, however. Anti-AChR is known to be of high
avidity [23], and the reversibility of the effect argues
against receptor blockade by antibody. Furthermore,
the effect was not significantly altered by depleting
serum of IgG with a staphylococcal protein A column.
In two patients this procedure removed virtually all
anti-AChR, including that against the a-BuTx binding
site. Moreover, no reduction in mepp amplitudes occurred in muscle exposed to the IgG fraction of MG
serum which, unfractionated, had exerted a significant
effect. This lack of effect of MG IgG alone confirms
the findings of Albuquerque et a1 [l] in rat muscle.
The observation that heated (56°C) MG sera had no
effect on mepp amplitudes, in contrast to the reduction
caused by unheated sera, further indicates that the
blocking effect depends at least in part on a heatsensitive component and not on IgG, which is not affected by such heat treatment. Complement, which
would be selectively inactivated by the heating procedure used in these experiments, can be fixed by antiAChR antibody, and it has been implicated in AChR
loss in both MG IS} and experimental autoimmune
MG 1141. Nevertheless, if complement is involved in
the reduction of mepp amplitudes observed in these
experiments, its action does not appear to depend on
the presence of anti-AChR, since two of the IgGdepleted fractions contained very little antibody. Lefvert et al [13] have suggested that most anti-AChR
directed against the a-BuTx site on the receptor is of
IgG3 subciass, and this antibody, if present, would not
be measured in the conventional immunoprecipitation
assay (where the a-BuTx site is occupied); it would be
Lerrick e t
al:
Effects of Myasthenic Serum
189
in the IgG-depleted fraction (from which IgG3 would
not have been removed). However, we found negligible activity against the a-BuTx site in the IgG-depleted
sera of these two patients.
Complement-mediated lysis of postsynaptic membrane secondary to antibody binding, a mechanism of
AChR loss in MG, would not readily explain the reversible nature of the reduction of mepp amplitude.
Moreover, the idea that anti-AChR and complement
might together be responsible for the electrophysiological effects we observed does not appear to be supported by the experiments in which exposure was
made to MG IgG with unheated control serum which
would have contained complement: no reduction in
mepp amplitude was observed. This raises the possibility that the putative heat-sensitive component may not
be present in non-MG serum. Indeed, in preliminary
experiments, heat-inactivated MG serum (Patient 1)
together with an equal volume of unheated control
serum did not significantly reduce mepp amplitudes,
suggesting that the heat-sensitive factor may be specific
for MG serum. Furthermore, it is possible that this
factor acts with IgG3, which also could be present in
very small amounts.
The fractional reduction in mepp amplitudes in
mouse muscle exposed to MG sera is less than that
found in myasthenic muscle [l, 7, 121. The acute nature of the experiments could account for this difference, but the lower affinity of human anti-AChR for
mouse AChR may play a part [23], and similar studies
using human rather than mouse muscle might show a
greater effect.
The mechanisms underlying the reversible blocking
effect we observed do not seem likely to contribute to
the reduced mepp amplitudes found in biopsied myasthenic muscle, which are not restored by washing. This
does not exclude the possibility that a degree of reversible block exists in vivo. In vitro studies of myasthenic
muscle are not undertaken in a medium equivalent to
the donor’s own serum, but in Krebs solution (or
equivalent), where any reversible effect would no longer be evident. Clinical observations may provide some
support for the presence of a reversible block in vivo.
Plasma exchange, which depletes plasma factors, typically leads to an increase in strength after a minimum
time lag of 48 hours [l8}, but electromyographic studies often show improvement within 24 hours 1191.
While these results suggest that much of the improvement could be due to synthesis of new AChR
free of antibody, it is possible that reversal of blockade
contributes to this early response, in view of the time
taken for IgG equilibration between the intravascular
and extravascular compartments. Furthermore, occasional patients have been reported in whom improvement has been clinically evident immediately fol-
190 Annals of Neurology
Vol 13 No 2
February 1983
lowing plasma exchange [ 5 ] . If that improvement is
attributable to remission of genuine myasthenic weakness, presumably it reflects a reversible mechanism of
the kind observed here.
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Lerrick e t al: Effects of Myasthenic Serum
191
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