Unlike thyrotropin, thyroid-stimulating antibodies do not activate phospholipase C in human thyroid slices

Abstract

The effects of thyroid-stimulating antibodies (TSAb) and of thyrotropin (TSH) were compared, on the generation of cyclic AMP and inositol phosphates (InsP), in human thyroid slices incubated in vitro, and on the Rapoport cyclic AMP bioassay. The TSAb positive sera were obtained from 19 patients with Graves' disease. In 14 experiments with the slices system, TSH significantly increased cyclic AMP accumulation (TSH, 0.03-10 mU/ml) as well as the cyclic AMP-independent inositol trisphosphate (InsP3) generation (TSH, 1-10 mU/ml). In the same 14 experiments, TSAb (0.10-28 mg/ml) enhanced cyclic AMP intracellular levels as expected while they did not induce any InsP accumulation. Even when TSAb increased cyclic AMP levels to the same or higher values as those obtained with TSH concentrations allowing InsP3 generation. TSAb were still unable to activate the phosphatidylinositol-Ca2+ cascade. The patterns of the response curves of TSAb and TSH on cyclic AMP accumulation were different, suggesting that different mechanisms may be involved. In addition, unlike TSH, TSAb were not able to stimulate H2O2 generation, which in human tissue mainly depends on the activation of the phosphatidylinositol-Ca2+ cascade. Immunoglobulins from six additional Graves' patients lacking measurable cyclic AMP-stimulating activity in both slices and cells systems did not activate phospholipase C either. In conclusion, our results show that TSAb do not share all the metabolic actions of TSH on human thyroid tissue. The data provide support for the concept that the pathogenesis of Graves' disease can be fully accounted for by the ability of TSAb to stimulate adenylate cyclase. This work also confirms that TSH activates the cyclic AMP and the phosphatidylinositol cascade by independent pathways in the human thyroid.

Full text

Unlike
Thyrotropin,
Thyroid-stimulating
Antibodies
do
not
Activate
Phospholipase
C
in
Human
Thyroid
Slices
E.
Laurent,*
J.
Van
Sande,*
M.
Ludgate,*
B.
Corvilain,*
P.
Rocmans,*
J.
E.
Dumont,*
and
J.
Mockel
*Institut
de
Recherches
Interdisciplinaires
en
Biologie
Humaine
et
Nucleaire,
School
of
Medicine;
*Department
of
Thoracic
Surgery
and
§Department
of
Endocrinology,
Erasme
University
Hospital,
Free
University
of
Brussels,
Brussels,
Belgium
Abstract
The
effects
of
thyroid-stimulating
antibodies
(TSAb)
and
of
thyrotropin
(TSH)
were
compared,
on
the
generation
of
cyclic
AMP
and
inositol
phosphates
(InsP),
in
human
thyroid
slices
incubated
in
vitro,
and
on
the
Rapoport
cyclic
AMP
bioassay.
The
TSAb
positive
sera
were
obtained
from
19
patients
with
Graves'
disease.
In
14
experiments
with
the
slices
system,
TSH
significantly
increased
cyclic
AMP
accumulation
(TSH,
0.03-
10
mU/ml)
as
well
as
the
cyclic
AMP-independent
inositol
trisphosphate
(InsP3)
generation
(TSH,
1-10
mU/ml).
In
the
same
14
experiments,
TSAb
(0.10-28
mg/ml)
enhanced
cyclic
AMP
intracellular
levels
as
expected
while
they
did
not
induce
any
InsP
accumulation.
Even
when
TSAb
increased
cyclic
AMP
levels
to
the
same
or
higher
values
as
those
obtained
with
TSH
concentrations
allowing
InsP3
generation,
TSAb
were
still
unable
to
activate
the
phosphatidylinositol-Ca2+
cascade.
The
patterns
of
the
response
curves
of
TSAb
and
TSH
on
cyclic
AMP
accumulation
were
different,
suggesting
that
different
mechanisms
may
be
involved.
In
addition,
unlike
TSH,
TSAb
were
not
able
to
stimulate
H202
generation,
which
in
human
tissue
mainly
depends
on
the
activation
of
the
phosphatidylino-
sitol-Ca2+
cascade.
Immunoglobulins
from
six
additional
Graves'
patients
lacking
measurable
cyclic
AMP-stimulating
activity
in
both
slices
and
cells
systems
did
not
activate
phos-
pholipase
C
either.
In
conclusion,
our
results
show
that
TSAb
do
not
share
all
the
metabolic
actions
of
TSH
on
human
thyroid
tissue.
The
data
provide
support
for
the
concept
that
the
patho-
genesis
of
Graves'
disease
can
be
fully
accounted
for
by
the
ability
of
TSAb
to
stimulate
adenylate
cyclase.
This
work
also
confirms
that
TSH
activates
the
cyclic
AMP
and
the
phospha-
tidylinositol
cascade
by
independent
pathways
in
the
human
thyroid.
(J.
Clin.
Invest.
1991.
87:1634-1642.)
Key
words:
hu-
man
thyroid
-
thyroid-stimulating
antibodies
-
thyrotropin
ino-
sitol
triphosphate
-
cyclic
AMP
Introduction
Thyroid
hyperfunction
and
growth
in
Graves'
disease
is
ascribed
to
the
thyrotropin
(TSH)'-mimicking
effect
of
autoan-
Address
correspondence
and
reprint
requests
to
E.
Laurent,
IRIBHN,
School
of
Medicine,
Campus
Erasme,
Bit.
C,
Route
de
Lennik
808,
B-1070
Brussels,
Belgium.
Receivedfor
publication
26
July
1990
and
in
revisedform
30
No-
vember
1990.
1.
Abbreviations
used
in
this
paper:
ASP,
ammonium
sulfate
precipi-
tates;
InsP,
inositol
phosphates;
KRB,
Krebs-Ringer
bicarbonate
buffer;
TSAb,
thyroid-stimulating
antibodies;
TSH,
thyrotropin.
tibodies
directed
against
the
TSH
receptor
on
the
surface
of
thyroid
cells
(for
a
review
see
references
1
and
2).
In
human
thyroid,
we
have
previously
shown
that
TSH
could
activate
the
two
main
pathways
of
cell
regulation
(3):
the
cyclic
AMP
cas-
cade
in
which
activation
of
adenylate
cyclase
generates
cyclic
AMP,
and
the
phosphatidylinositol
cascade
in
which
the
recep-
tor-activated
phospholipase
C
releases
two
second
messengers
from
phosphatidylinositol
4,5-bisphosphate:
diacylglycerol
and
inositol
1,4,5-trisphosphate
(Ins(1,4,5)P3)-Ins(1,4,5)P3
it-
self
activating
Ca2'
release
from
intracellular
stores
(4).
We
have
shown
that
InsP3
accumulation
was
cAMP-independent
(3).
Several
methods
are
currently
used
for
the
detection
of
TSAb,
mainly
based
on
adenylate
cyclase
stimulation,
as
mea-
sured
in
human
thyroid
slices,
cells
or
membranes,
or
in
FRTL5
cells
(2).
Enhancement
of
cyclic
AMP
accumulation
could
certainly
account
for
the
known
effect
of
TSAb
on
the
human
thyroid.
Indeed,
in
dog
and
human
thyroid,
hormone
secretion
is
stimulated
by
agents
that
increase
cyclic
AMP
lev-
els
and
inhibited
by
those
that
increase
Ca2+
levels
or
activate
protein
kinase
C
(5-7).
Cyclic
AMP
mediates
the
TSH-induced
thyroid
cell
differentiation
and
proliferation
(8,
9).
However,
claims
have
appeared
for
the
existence
of
a
growth-stimulating
immunoglobulin
(thyroid
growth-stimulating
antibodies
or
TGSAb),
separate
from
TSAb,
that
might
cause
thyroid
growth
in
Graves'
disease
and
some
euthyroid
goiters
through
a
path-
way
distinct
from
adenylate
cyclase
(10-13).
These
results
were
in
contradiction
with
the
findings
of
Zakarija
et
al.
who
showed
a
strict
parallelism
between
growth
and
cyclic
AMP
accumula-
tion-stimulating
activities
in
Graves'
immunoglobulins
(14).
These
in
vitro
studies
on
human
thyroid
slices
were
initiated
to
determine
whether
Graves'
disease
immunoglobulins
repro-
duced
one
or
both
of
the
primary
metabolic
effects
of
TSH
(adenylate
cyclase
and
phospholipase
C
activation)
that
might
be
relevant
to
the
increased
function
of
the
gland
and
essential
for
the
development
of
thyrotoxicosis.
Methods
TSAb.
Sera
were
obtained
from
25
patients
with
hyperthyroidism
due
to
Graves'
disease
(20
women,
5
men;
age
17-58,
mean
53).
The
diag-
nosis
was
based
on
the
classical
clinical
findings
of
thyrotoxicosis
(with
or
without
goiter
or
ophthalmopathy)
associated
with
elevated
T4,
T3,
and
undetectable
TSH
levels,
and
a
diffuse
radioactive
iodine
uptake.
Blood
samples
were
obtained
at
the
time
when
diagnosis
was
made
in
16
patients
while
9
of
them
were
under
methimazole
therapy.
The
number
of
sera
without
measurable
TSAb
activity
in
both
the
Rapo-
port
and
the
slices'
assay
systems
was
not
different
in
the
group
of
treated
patients
(4
of
16,
25%)
than
in
the
untreated
group
(2
of
9,
22%).
Serum
was
separated
by
centrifugation
of
50
ml
clotted
blood.
Control
sera
of
healthy
subjects
were
also
used.
Crude
IgG
was
prepared
by
ammonium
sulfate
precipitation
(15).
After
precipitation
and
centrifu-
gation,
water
was
added
to
the
pellet
until
complete
dissolution
and
the
IgG
concentration
was
measured
by
its
extinction
coefficient
at
280
nM.
Samples
were
then
dialyzed
against
NaCl
free
Hanks'
medium
for
experiments
on
cells
and
against
Krebs-Ringer
bicarbonate
(KRB)
1634
Laurent,
Van
Sande,
Ludgate,
Corvilain,
Rocmans,
Dumont,
and
Mockel
J.
Clin.
Invest.
©
The
American
Society
for
Clinical
Investigation,
Inc.
0021-9738/91/05/1634/09
$2.00
Volume
87,
May
1991,
1634-1642

buffer
for
experiments
on
slices.
Preparation
of
highly
purified
IgG
on
protein
A
sepharose
columns
was
performed
for
two
samples
and
pro-
vided
no
advantage
in
comparison
with
the
ammonium
sulfate
precipi-
tation
technique.
TSAb
activities
in
the
IgG
fractions
were
checked
in
the
Rapoport
bioassay
(15).
Briefly,
monolayer
cultures
of
human
thyrocytes,
which
had
been
frozen
in
liquid
N2
after
primary
culture
and
then
thawed,
were
exposed
to
TSH
or
TSAb
in
hypotonic
medium
(NaCl
free
Hanks)
for
4
h
at
370C.
Each
assay
contained
TSH
stan-
dards
diluted
in
control
IgG
used
at
the
same
concentration
as
the
TSAb
samples.
Cyclic
AMP
released
into
the
medium
was
measured
by
RIA
(15).
Human
thyroid
tissue.
For
slices
experiments,
human
thyroid
tis-
sue
was
obtained
from
euthyroid
patients
undergoing
lobectomies
for
resection
of
solitary
"cold"
nodules.
Only
the
healthy
normal
looking
nonnodular
tissue
was
used
and
cut
into
thin
slices
of
50
mg
wet
wt
with
a
Stadie-Riggs
microtome
within
10-30
min
of
surgical
resection.
Slices
were
randomly
assigned
to
inositol
phosphates
and
cyclic
AMP
experiments.
For
cells
in
culture,
thyroid
tissue
came
from
surgically
treated
Graves'
patients.
Inositol
phosphates
measurements.
Slices
were
incubated
at
370C,
under
an
atmosphere
of
0JCO2
(15:5,
vol/vol)
in
2
ml
of
KRB,
supple-
mented
with
8
mM
glucose,
and
0.5
g/liter
of
bovine
serum
albumin.
In
the
first
4-h
preincubation,
20
ICi/ml
3H-inositol
(sp
act
16.5
Ci/
mmol)
was
added
to
this
medium.
Tissue
was
then
transferred
to
fresh
unlabeled
incubation
medium
to
which
lithium
was
added
after
15
min.
After
a
further
5
min,
slices
were
put
for
1
h
in
a
third
series
of
flasks
containing
the
tested
agents
(TSH,
TSAb,
control
IgG)
in
the
presence
of
10
mM
LiCl.
Incubation
was
stopped
by
rapid
immersion
of
the
slices
in
(3%)
ice-cold
perchloric
acid.
After
homogenization
and
centrifugation
(2,000-g/l
0
min),
the
pellet
was
washed
with
1
ml
of
1%
cold
perchloric
acid
and
recentrifuged.
The
combined
supernatants
were
neutralized
to
pH
7.8
by
the
addition
of
760
mM
KOH
in
the
presence
of
a
380-mM
Hepes
buffer.
3H-labeled
inositol
phosphates
from
the
supernatants
were
eluted
in
a
stepwise
fashion
through
an
anion
exchange
column
of
AG,-X8
resin
(formate
form,
100-200
mesh;
Biorad,
Watford,
UK)
(16).
Intracellular
InsP3
is
expressed
as
cpm/100
mg
wet
wt
tissue.
Incorporation
of
3H-inositol
into
the
total
phosphatidylinositides
pool
was
estimated
after
chloroform/methanol
extraction
of
lipids
from
the
pellet
(
17).
Cyclic
AMP
measurements.
Slices
were
incubated
following
the
same
protocol
as
for
inositol
phosphates
measurements
but
without
3H-inositol.
For
the
test
incubation
of
1
h,
medium
was
supplemented
with
100
gM
Ro
20-1724
as
a
cyclic
AMP-specific
phosphodiesterase
inhibitor.
The
slices
were
then
dropped
into
boiling
water
for
5
min,
homogenized,
centrifuged,
and
the
supernatant
lyophilized.
The
tissue
extracts
were
resuspended
in
water
and
cyclic
AMP
was
measured
in
a
binding
assay
using
protein
kinase
from
bovine
skeletal
muscle
(18).
Intracellular
cyclic
AMP
concentrations
were
expressed
as
pmol/100
mg
wet
wt
tissue.
H202
determinations.
Measurements
were
performed
according
to
the
method
of
Benard
and
Brault
(19)
based
on
the
conversion
of
the
nonfluorescent
substrate
homovanillic
acid
to
a
fluorescent
derivative
in
the
presence
of
H202
and
peroxidase.
Slices
were
preincubated
for
1
h
in
KRB
buffer
supplemented
with
8
mM
glucose
and
0.5
g/liter
BSA,
then
transferred
to
fresh
medium
containing
0.1
mg/ml
horse
radish
peroxidase
type
II,
440
,M
homovanillic
acid
and
the
agonist
tested.
The
fluorescence
of
the
medium
was
measured
90
min
later
(20).
Materials.
TSH
was
used
as
Thytropar
(4
IU/mg)
(Armour
Phar-
maceutical
Co.,
Phoenix,
AZ).
Myo-2
3H-inositol
came
from
New
En-
gland
Nuclear (DuPont-NEN,
Haren,
Belgium).
Ro
20-1724
was
a
gift
from
Hoffmann-La
Roche
(Nutley,
NY).
All
other
reagents
were
of
the
purest
grade
commercially
available.
Purified
bovine
TSH
(40
IU/mg)
was
a
gift
from
Dr.
J.
G.
Pierce
(UCLA,
Center
for
Health
Science,
Los
Angeles,
California)
and
purified
human
TSH
(11
IU/mg)
came
from
Porton
Products
(Salisbury,
Great
Britain).
Statistical
analysis.
For
the
comparison
of
agonist
and
control
groups
the
Student's
t
test
was
used.
Each
experimental
condition
was
conducted
with
triplicate
flasks
and
slices
for
InsP3
and
H202
measure-
ments
and
with
duplicates
for
cyclic
AMP
determinations.
P
<
0.05
was
chosen
as
the
level
of
significance.
Results
Effects
of
Graves'
immunoglobulins
on
cyclic
AMP
accumula-
tion.
Among
the
25
ammonium
sulfate
precipitates
(ASP)
tested,
6
were
negative
for
cyclic
AMP-stimulating
activity
in
both
the
Rapoport
and
the
slices
system,
while
19
of
them
were
active
in
one
or
both
of
the
assays.
In
the
Rapoport
bioassay
(15),
the
potency
of
the
TSAb
were
expressed
as
equivalents
of
mU
TSH
in
the
assay
and
ranged
from
0.001-10
mU
TSH
(Fig.
1).
All
ASP
were
then
used
for
cyclic
AMP
determinations
on
human
thyroid
slices,
in
which
their
activities
were
also
expressed
as
equivalents
of
mU
TSH
(range
0-3.3
mU
TSH).
As
expected
from
the
lower
sensitivity
of
the
slices
system,
three
sera
showed
cyclic
AMP
activities
in
the
first
assay
with-
out
any
activity
in
the
second
(No.
17-19).
One
ASP
(No.
14)
had
no
detectable
cyclic
AMP-generating
activity
in
the
Rapo-
port
bioassay,
but
it
was
positive
in
the
slices
system.
For
the
15
other
ASP,
there
was
a
good
correlation
between
the
activities
in
both
assays
(r
=
0.71,
n
=
15,
P
<
.01).
The
activity
of
TSAb
preparations
was
largely
independent
of
the
origin
of
the
hu-
man
thyroid
tissue
used
for
the
test.
Indeed,
7
ASP
used
at
constant
concentrations
had
the
same
relative
activity
versus
a
standard
TSH
concentration
action
curve
in
two
or
three
dif-
ferent
thyroid
tissues
(data
not
shown).
As
observed
earlier,
there
was
no
correlation
between
the
extent
of
cyclic
AMP
enhancement
and
clinical
parameters,
such
as
goiter
size,
sever-
ity
of
hyperthyroidism,
and/or
ophthalmopathy
(21).
Comparison
of
cyclic
AMP
and
InsP3
generations
induced
by
TSH
and
TSAb.
14
experiments
using
ASP
having
the
stron-
gest
TSAb
activities
(nb
1
to
14
on
Fig.
1)
were
performed
on
14
different
thyroid
tissues.
The
results
of
the
individual
experi-
ments
are
shown
in
Fig.
2.
In
all
the
experiments,
TSH
(0.03-
X
of
TSH
maximal
effect
(slices)
125I
100o.
5
0
6
2
3
8
7
9
0
0
14
75-
0
50-
25
1x
314
12
160
150
1707
I180n
C-)
0.001
0.01
0.1
1
10
(Log)
Equiv.
mU
TSH
(coils)
Figure
1.
Comparison
of
the
strength
of
cyclic
AMP-stimulating
ac-
tivities
for
Graves'
sera
No.
1-19
in
the
Rapoport
bioassay
(abscissa)
and
in
our
in
vitro
incubated
slices
system
(ordinate).
TSAb
were
used
for
maximal
concentrations
ranging
from
0.1
to
28
mg/ml
(as
expressed
in
Fig.
2).
Their
potencies
are
expressed
as
equivalents
of
mU
TSH
(Equiv.
mU
TSH)
and
ranged
from
0
to
10
for
cells.
For
slices,
some
TSAb
cyclic
AMP-stimulating
activities
(5,
8)
exceeded
highest
cyclic
AMP
stimulation
levels
obtained
with
TSH.
Correlation
coefficient,
r
=
0,71;
P
<
0.01.
Graves'
Sera
do
not
Activate
Phospholipase
C
in
Human
Thyroid
1635

1I
8/
^?
0
.3
.
313
'I
3.
io3MU/ams1
I
Ad-
(M.1
eOft0
wa.L
A-.A
.
o
O
*
i1
a2
10
.5
0
ONoan
Im
4.3
I
.1
a/B
0
.01
I1.33
1
3.
i10.11/mi
'6
4
'4
2
2
0
O
12
64
6
3
0
4.
0
.
6+
4
0
13
-V
I..w
-A
07
Igp
Figure
2.
Individual
results
of
the
14
experiments
using
TSAb
sera
No.
1-14.
The
number
of
the
experiment
corresponds
to
the
number
of
the
sera
expressed
in
Fig.
1.
(-
o
-)
Cyclic
AMP
concentrations-action
curves
in
response
to
TSH
or
TSAb.
(-v&
-)
InsP3
concentrations-action
curves
in
response
to
TSH
or
TSAb.
The
first
point
on
the
abscissae
for
the
TSAb
panel
refers
to
normal
sera
containing
nonspecific
immuno-
globulins
at
a
concentration
equivalent
to
the
highest
TSAb
concentration
used
in
the
experiment.
w.w.t.,
wet
weight
tissue.
Results
are
expressed
as
means±SEM
of
triplicates
for
InsP3
determinations
and
±SD
of
duplicates
for
cyclic
AMP
measurements.
SEM
and
SD
are
not
drawed
when
inferior
to
the
size
of
circles
and
triangle
symbols.
1636
Laurent,
Van
Sande,
Ludgate,
Corvilain,
Rocmans,
Dumont,
and
Mockel
qdA
(*Iu
f1t
e
wl
Q
o
-T
:1.
a
1.
0
A-A
*-6*
..
10
+
.
0
13
=
ltiOM
An)
6
4
2
2.
0
12
9
0
3
3.
0
4,0
2
4.
0
10
0
10
A
a
13
+
b
O
13
m/x
O
A
0
a
o
2
a
10
By
a
t

AMP
0o-0
3
b
2
1
&
0
4
3
2
7.
0
3
.
7.
0
(.102pm~s~100g
w~~t.
~
e10/tOMQ
w
"~.)
qdaW
(iomshq/100w
w..*
A-A
0-0o
4
4
3
3.1
2
0
0
i
I
0
ma
~.33
1
3.3
i
Om-umiUHQa;
.sJ01
0.
.
(
clot
(ld/lou
liSoo
3
2
I
0
12
0
..p
p
a
0
03
.1
.3
1
33
,
O
;
.1
a3
1
3.
1
MU/adH
TM
OA
S.
S
S
3
0
bf
(*laouloto
W
A-A
I
*
*
0
3
10
lo
no
*
0^OA
L
i
12m
sJ.~13b
Qof)
2
i
1
0
3
Figure
2.
(Continued)
Graves'
Sera
do
not
Activate
Phospholipase
C
in
Human
Thyroid
1637

-
aa(
w-
I
2
0
4
4
*
4
3
2
o
a
IN
Os)
S
S
.
a4
0
.05
.1
.3
1
3.
10
mU/Ml
0.1)
/
PUS
31
2
1
0
12odaJ
'U
1.2
41
A
.1
M
1
S
4
3
&~~~~1'.
4
f
2
.
.
.o
..
3.
10
MU/adUHM
OJg
0
Figure
2.
(Continued)
7Uo
03
0S
2.7
7
mg
I
1638
Laurent,
Van
Sande,
Ludgate,
Corvilain,
Rocmans,
Dumont,
and
Mockel
S
a
3
to.
0
i
of/ia
T
.
a6
4
2
0
I1.
3
3
0
No)
t
3
S
.
46'
2
12.
0
0
12
o/dTS
A
I
.1O
F,
ad*
Air
(*IV
Oftsumo
a
..
4
t
I
-7/

*~~~/
of/
*A-A
.6
'4
12
4
Figure
2.
(Continued)
10
mU/ml)
significantly
increased
cyclic
AMP
accumulation
and
InsP3
generation,
the
latter
at
higher
concentrations
(TSH,
1-10
mU/ml).
In
the
same
experiments,
TSAb
(0.10-28
mg/
ml)
also
enhanced
cyclic
AMP
levels
but
did
not
induce
InsP3
accumulation.
Dilutions
of
Graves'
patients
IgG
did
not
reveal
any
InsP3
effect.
When
analysis
of
the
data
was
restricted
to
the
particular
subset
of
TSAb,
which
increased
cyclic
AMP
to
the
same
values
as
those
obtained
using
TSH
concentrations
allow-
ing
InsP3
generation
(experiments
1-10,
n
=
10),
no
TSAb
effect
on
the
latter
became
evident.
The
same
results
were
found
for
the
inositol
monophosphate
and
inositol
bisphos-
phate
fractions,
while
the
total
3H-inositol
content
of
the
phos-
pholipid
pellet
was
not
influenced
by
TSH
or
TSAb
(data
not
shown).
In
7
experiments
(2,
3,
4,
6,
11,
12,
13),
control
IgG
from
healthy
subjects
induced
a
slight
increase
in
InsP3
generation
of
the
same
magnitude
as
the
TSAb
in
the
same
experiments.
This
effect
was
thus
related
to
the
thyroid
sample
studied
and
not
to
any
differential
characteristics
of
the
normal
and
Graves'
IgG.
The
lack
of
effect
of
TSAb
on
inositol
phosphates
generation
was
not
related
to
a
negative
interaction
of
Graves'
IgG
with
receptor
activated
phospholipase
C,
since
TSH-induced
InsP3
responses
were
not
inhibited
by
Graves'
sera
(3
experiments
using
sera
9,
11,
14).
Fig.
3
shows
the
results
for
one
of
these
experiments
(serum
No.
1
1).
Cases
4,
5, 8,
9,
10,
and
12
in
Fig.
2
show
more
detailed
TSAb
and
TSH
concentration-response
curves
for
cyclic
AMP
and
InsP3
accumulations.
In
these
experiments,
the
TSH-in-
duced
cyclic
AMP
generation
curve
was
biphasic
(i.e.,
decreas-
ing
cyclic
AMP
for
the
highest
TSH
concentrations),
while
that
for
TSAb
showed
a
linear
concentration
dependence
or a
plateau
phase.
Effects
of
TSAb
in
comparison
with
TSH
on
H202
genera-
tion.
8
of
the
most
potent
TSAb
ASP
(No.
1-8)
were
tested
on
slices
in
order
to
determine
whether
they
could
induce
H202
production
(Table
I).
H202
values
for
TSAb-stimulated
slices
were
not
different
from
the
control
values,
except
for
TSAb
No.
6
which
inhibited
H202
generation.
TSH
10
mU/ml
in-
duced
a
12-fold
increase
of
H202
generation.
Comparison
of
cAMP
and
InsP3
generations
induced
by
thytropar,
bovine
or
human
purified
TSH
preparations.
Fig.
4
shows
that
the
biphasic
effect
of
TSH
on
cAMP
is
produced
by
the
commercial
and
purified
bovine
TSH
preparation,
as
well
as
by
purified
human
InaP3
(*103cpm/lOOmg
w-w-t.)
10
-.
5
o
TSH.
Figure
3.
Lack
of
effect
of
Graves'
IgG
(TSAb)
or
healthy
subjects'
IgG
(IgG)
on
TSH
3
and
IO
mU/ml-induced
InsP3
accumulations.
Serum
used
is
number
11
on
Fig.
1.
w.w.t.,
wet
T
g
i
g
weight
tissue.
Results
are
expressed
as
means±SEM.
*P
+TSAb
13g
12.3mg/mi
123mg/ml
<
0.05;
**P
<
0.0
1.
Graves'
Sera
do
not
Activate
Phospholipase
C
in
Human
Thyroid
1639
e-°
(1A2pmd"/
w
0O-O
T
cot
AM
(.IoV
/Om
W6W)
a
t
I
p
p4
p p p
i
1
L
&a
0
.
.1
.33
1
3.3
I0
MU/
O..u
4
d
2
+
13.
0
4
2
0
4
20
me/ad
1
A
&.
I0
aj
4
14.
0
0
.1
.33
1
3.3
10
MUKSA
W
OAW
0
kft
(*lo-3/loftg
WAQ
bft
(,olo-3WWIOOM
W.&Q
.
I
20
melw
W
20
me/ad
Tam
OAW

Table
I.
Effect
of
nonspecific
immunoglobulins
from
normal
subjects
(IgG),
TSAb
(1-8),
and
TSH
10
mU/ml
on
H202
generation
in
human
thyroid
slices
Agent
Concentratior
n
mg/ml
BSA
(3)
19
IgG
(3)
15
TSAb
1
(2)
13
2
(2)
4.6
3
(3)
10.9
4
(3)
20
5
(2)
19
6
(2)
12
7
(2)
20
8
(2)
25
TSH
(3)
10
mU/ml
Numbers
(TSAb1
to
TSAb8)
refer
to
the
Gra
1.
W.W.T,
wet
weight
tissue.
Results
are
expi
triplicates.
*
P <
0.05,
t
P
<
0.01;
n,
numbei
cates.
cyclic
AMP
(pmol.s/1
00mg
w.w.t.)
600-.
400
/
200;
0
0
1
lnsP3
(.1
o3
cpm/1
00
mg
w.w.t)
6
4-
2-
0
1
Figure
4.
Cyclic
AMP
and
InsP3
concentrat
thytropar
(-
o
-),
bovine
purified
TSH
(-
fied
TSH
(-
A
-).
w.w.t.,
wet
weight
tissue.
means±SD
of
duplicates
for
cyclic
AMP
and
InsP3
determinations.
H202
ng/100
mg
n
WWT
x
60
min
69+39
Effects
of
Graves'
immunoglobulins
without
TSAb
activities
on
InsP3
generation.
None
of
the
6
ASP
lacking
TSAb
activities
as
defined
in
Methods
was
able
to
increase
InsP3
accumulation
in
the
slices
(data
not
shown).
Discussion
%
-18
-P."
As
TSH
activates
both
the
cyclic
AMP
and
the
phosphatidyl-
60±28
inositol-Ca2'
cascades
in
human
thyrocytes
(3),
we
addressed
57±27
the
question
as
to
whether
TSAb,
the
antithyrotropin
receptor
75±24
antibodies
responsible
for
Graves'
disease,
also
activate
these
37±16
two
pathways.
As
expected,
the
thyroid
slices
system
is
less
42±10
sensitive
to
TSH
and
TSAb
than
that
using
thawed
cells
in
20±15*
hypotonic
medium:
some
sera
elicited
a
cyclic
AMP
accumula-
20±152
tion
in
the
latter
but
not
in
the
former
system.
However,
except
69±42
for
a
few
sera
(experiments
14,
17,
18,
19),
a
good
parallelism
72±37
was
achieved
between
cyclic
AMP
responses
to
TSAb
in
both
847±106t
systems
(thawed
cells
and
slices).
Thus,
the
more
physiological
system,
human
thyroid
slices
incubated
in
vitro
can
be
consid-
yves'
sera
presented
in
Fig.
ered
as
a
good
model
to
investigate
TSAb
activities
as
they
are
ressed
as
means±SD
of
usually
detected
and
defined
using
the
more
sensitive
Rapo-
r
of
experiments
in
tripli-
port
bioassay.
In
contrast
to
the
FRTL5
cell
line
and
dog
thyro-
cytes
in
primary
culture,
in
the
case
of
human
thyrocytes,
it
has
been
demonstrated
that
TSH
activates
the
PtdIns-Ca2+
cascade
in
the
same
range
of
concentrations
eliciting
other
physiologi-
cal
effects
(3).
These
facts raise
the
possibility
of
a
role
for
the
PtdIns-Ca2+
cascade
in
TSAb
action.
IgG
which
bind
to
thyroid
membranes
are
heterogenous
and
may
contain
activities
which
stimulate
or
inhibit
adenylate
cyclase
(22,
23).
Thus,
depend-
ing
on
the
titer
and/or
affinity
of
these
two
activities,
the
re-
sponse
in
the
TSAb
assays
could
clearly
vary
with
the
relative
concentrations
of
the
IgG
concerned
(such
variations
have
been
demonstrated
by
Zakarija
and
McKenzie
[22]).
There-
fore,
we
considered
it
important
to
assay
TSAb
samples
at
dif-
ferent
IgG
concentrations
since,
at
a
single
concentration,
some
may
appear
negative
while
positive
at
a
higher
or
lower
I
|
concentration.
None
of
the
concentrations
of
the
various
TSAb
10
mU
TSH
(Log)
tested
were
able
to
elicit
phospholipase
C
activation
as
mea-
sured
by
InsP3
generation.
This
lack
of
TSAb
effect
is
not
due
to
a
blocking
effect
of
the
sera,
as
the
TSH
action
on
the
InsP3
generation
itself
was
not
inhibited
in
the
presence
of
these
IgG.
These
results
show
that
at
serum
TSAb
concentrations
usually
measured
in
vivo,
no
activation
of
the
PtdIns-IP3
path-
way
is
observed.
Preliminary
data
obtained
by
B.
Corvilain
and
E.
Laurent
show
that
H202
generation
and
protein
iodination
in
human
thyroid
mainly
depend
on
the
activation
of
the
II
WPtdIns-InsP3-Ca2+
cascade
and
are
not
stimulated
by
agents
that
increase
cyclic
AMP
(24).
Our
findings
concerning
the
failure
of
TSAb
to
induce
any
H202
generation
are
thus
not
surprising.
This
suggests
that
activation
of
adenylate
cyclase
by
TSAb
also
fully
accounts
for
their
effects
on
human
thyroid
cells.
Such
a
conclusion
fits
in
well
with
the
fact
that
activation
of
the
cyclic
AMP
cascade
stimulates
dog
and
human
thyrocyte
secretion,
proliferation,
and
differentiation
expression
(8,
9)
(as
observed
in
Graves'
disease),
while
activation
of
other
cas-
10
mU
TSH
(Log)
cades,
the
phorbol
ester-protein
kinase
C
pathways,
or
the
ion-response
curves
to
growth
factor
protein-tyrosine
kinase
pathway,
leads
to
proli-
.
)
and
human
puri-
feration
and
loss
of
differentiated
expression
and
function
(9,
Results
are
expressed
as
25,
26).
±SEM
of
triplicates
for
As
previously
reported
(27),
the
effect
of
TSH
on
human
thyrocytes
cyclic
AMP
accumulation
is
often
biphasic,
with
a
1640
Laurent,
Van
Sande,
Ludgate,
Corvilain,
Rocmans,
Dumont,
and
Mockel
4
4
A
,---lj

clear
decrease
of
hormone
action
at
high
concentrations.
This
effect
is
not
due
to
the
presence
of
contaminants
in
the
bovine
TSH
preparation
used,
since
it
was
also
observed
when
using
purified
bovine
TSH
(40
IU/mg)
or
purified
human
TSH
(1
1
IU/mg).
Such
a
biphasic
effect
was
never
observed
with
TSAb.
These
facts
could
explain
that
at
high
TSAb
concentrations,
cyclic
AMP
accumulations
were
sometimes
higher
than
the
maximal
TSH
effects
(experiments
5
and
8
from
Fig.
2).
This
second
discrepancy
further
distinguishes
TSAb
and
TSH
ac-
tions.
The
decreased
effect
of
TSH
on
cyclic
AMP
production
was
generally
observed
for
hormone
concentrations
eliciting
the
InsP3
generation
effect.
It
is
therefore
possible
that
the
stim-
ulation
by
TSH
at
a
high
concentration
of
the
phosphatidyl
inositol
cascade
itself
could
inhibit
cyclic
AMP
generation.
Such
an
effect
could
result
from
direct
activation
of
the
inhibi-
tory
GTP
binding
protein
Ni
(as
for
norepinephrine
a,
effects),
from
the
inhibition
of
the
activating
GTP
binding
protein
N5
by
a
protein
kinase
C
induced
phosphorylation
or
from
Ca2`-
calmodulin
activation
of
a
cAMP
phosphodiesterase.
The
existence
of
IgG
stimulating
thyroid
growth
indepen-
dently
of
cyclic
AMP
in
the
serum
of
some
hyperthyroid
or
even
euthyroid
goitrous
patients
has
been
suggested
(10,
1
1,
28).
However,
these
results
were
sometimes
based
on
inade-
quate
methodologies
(29,
30)
and
the
experiments
were
never
obtained
with
human
thyroid
cells
or
tissue.
It
has
since
been
shown
that
cyclic
AMP
is
the
mediator
of
TSH
mitogenic
ac-
tion
in
the
FRTL5
cell
line
(31)
and
besides,
that
the
growth
promoting
action
of
Graves'
sera
parallel
their
action
on
cyclic
AMP
accumulation
in
these
cells
(14).
In
conclusion,
our
data
demonstrate
that
at
concentrations
similar
to
in
vivo
conditions,
Graves'
IgG
are
unable
to
activate
the
PtdIns-Ca2+
pathway
in
normal
human
thyroid
tissue.
Thus,
TSAb
do
not
share
all
the
metabolic
actions
of
TSH
on
normal
human
thyroid
tissue
incubated
in
vitro.
If
there
were
two
distinct
TSH
receptors,
one
controlling
phospholipase
C
and
the
other
adenylate
cyclase
activation
(such
as
is
the
case
for
many
other
hormones
and
neurotransmitters
[32-36]),
TSAb
would
only
activate
the
receptor
coupled
to
adenylate
cyclase.
If
there
is
only
one
TSH
receptor
controlling
two
inde-
pendent
pathways
(such
as
is
the
case
for
some
muscarinic
receptors
[37]),
TSAb
action
on
this
receptor
would
be
incom-
plete.
With
the
recent
cloning
of
a
human
TSH
receptor
the
tools
now
exist
to
distinguish
between
these
hypotheses
(38,
39).
Acknowledgments
We
thank
Mrs.
L.
Collyn,
Mr.
W.
Wasteels,
and
Mr.
C.
Massart
for
their
excellent
technical
help,
and
Mrs.
D.
Leemans
for
careful
prepara-
tion
of
the
manuscript.
Dr.
Laurent
is
a
research
fellow
of
the
Fonds
de
la
Recherche
Scientifique
(Belgium).
This
work
was
supported
by
grants
of
the
Fonds
de
la
Recherche
Scientifique
Medicale
(FRSM
3.4534.90).
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1642
Laurent,
Van
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Mockel