Endocrinol. Japon. 1979, 26 (2), 285-289
Inactivation by
of Human Thyroid Stimulator Human Thyroid Extract
YOSHIMASASHISHIBA, MASARUTAKAISHI AND TAEKO SHIMIZU Division of Endocrinology and Endocrine Research Laboratory, Toranomon Hospital and Okinaka Memorial Institute for Medical Research, Tokyo, 107
Synopsis To
determine
Graves' the
inactivation
slices
cause
associated
of
the
in
allow
the
with
normal
obtained
all
by
normal
strating
almost
the or
to
even
incubation
pretreatment IgG was320•}
of
the
with
31
or
140•}
of
levels
IgG
were
25
were
particulate
not
138•}
cause
26f
influenced When the
respectively, to
fraction or
with
to to
incubated
fraction. or
moles/mg,
that required
particulate 142•}
without
the
thyroid thyroidal
is
slices
was
cAMP
25f
human
demonstrated
thyroid
with antigen,
human
of120min
slices
thyroid
patients
thyroid in
thyroid
disease
activity
of
with
was
human
thyroidal
Graves'
of the
it
human
in
basal
IgG
period
by
content
that
inhibition
When
pretreatment
IgG human
generation
the
experiment,
cAMP
of
a putative cAMP
on
with content
stimulate pretreating
after
complete
activity with
by
tissue.
with
indicating
incubated cAMP
an
IgG
preliminary
tissue,
IgG
were
pretreatment,
the
without
respectively,
slices
reaction
studied
penetrate
IgG
stimulating
the
stimulation,
from100mg
moles/mg,
the
In
to
of
was
generation IgG
thyroid with
property
vitro
fraction. cAMP
the
is
incubated
particulate
at
whether
disease
similar demon-
cAMP
generation
stimulation.
The immunoglobulin G (IgG) of patients with Graves'disease has been shown to possess an ability to stimulate human thyroid gland as is manifested by intracellular colloid droplet formation (Shishiba et al.; Onaya et al., 1973), cAMP generation (Onaya et al., 1973; McKenzie and Zakarija, 1976), adenyl cyclase stimulation (Orgiazzi et al., 1976), and triiodothyronine (T3)-immunoreactivity release from human thyroid tissue incubated in vitro (Shishiba et al., 1976). If these activities are triggered by the reaction of the IgG with a putative human thyroid antigen, they will be inactivated by pretreatment with human thyroid extracts. In order to test the vaReceived October11, 1978. This study was supported financially #248198, the Ministry of Education, Culture.
by the Grant Science and
lidity
of
this
hypothetical
mechanism,
we
pretreated the IgG of Graves'disease with human thyroid extract and examined for the effect of the pretreatment on the cAMP generation-stimulation
Materials
activity
and
of the
IgG.
Methods
Preliminary experiment To determine the appropriate period and mode of incubation for detecting the increase in cAMP in human thyroid tissue in vitro, a preliminary experiment was conducted. All through the experiment, IgG was semipurified with ammonium sulfate precipitation as described by MstIZ et al.(1964). Normal human thyroid tissueObtained at the surgery of adenoma was sliced with a Stadie-Riggs microtome. After being quickly rinsed with Earl's balanced salt solution, blotted and weighed, slices each weighing approximately30mg were incubated in the following three different modes.
286
SHISHIBA
a)
Mode
A:
containing tions
one
A
slice
of
the
for15min
bling
of
at37•Ž
gas
CO2.(i)
of
same
as
tubes
of
glucose,
bovine
solutions
above.
Mode
way
as
c)
Mode
A
of
normal
of
IgG,(iii)
the
of
containing
A,
TSH.
paper
and
C
and
frozen
were
extracted the
the
source
of
for
order reached
the
incubation
plus
the
human
human
thyroid
Graves'disease
gland was
containing10mM
tris-HCl
centrifugation
treatment
in
al.(1969). used
as
was
harvested
60min. terms
of
stated
in
the
buffer,
wash
was
suspended and
weight
results.
of The
washed
and
repeated
centrifugation and
activity
IgG at37•Ž.
at105000G tested
using
for the
After
cAMP
method
the after
for120min
was
membrane the
fraction
crude
membrane
was
determined
method
described As
adenyl with
is
by
shown
cyclase
the
shown
that the and caused
IgG the
Or-
in
activity
results
illustrates in
which
of
IgG
the
results
the
effect
with
fraction
human
for
Fig.
roughly on
panel
had reachsubsequent
of
the
of
human
ex-
the
pre-
tissue
final
solution
as
pellet to
The
be
described
thyroid
thyroid
cAMP
slices
incubated
with
cAMP
content
of142•}25f
weight
tissue.
par-
generation
was
studied.
normal
IgG
The showed
moles/mg
When
IgG,
fraction
wet
of
weight
with
the
pretreated
particulate
wet
same
with
of
thyroid
from100mg
tissue,
thyroid
dose
human
prepared
thyroid
human
was
slices,
incubated
thyroidal
moles/mg
identical
with
with
cAMP
which
that
normal
pretreatment. IgG
The
did
was tested
was stimulaabove
was138•}26f
incubated
with
supernatant
for60min generation
the
of
human
was
the
slices
IgG
without
in
suspended
The
on
slices
almost
expressed
ultracentrifuged.
of
the
(d),
level
supernatant
thyroid was
C, slice
sucrose
pH7.4.
was
original
times.
for120min
of
at105000G
pellet
again
three
surgery
the
pellet
with10mg/ml
harvested
Mode
wet
of
the
at
ultracentrifuged
amount
incubated
after
tion
and
The
Mode
a
fraction,
in0.25M
for30min,
de-
chosen.
the
cyclase
to
ticulate
was em-
et
at
buffer
at1500G
in
crude With
Fig.2
according
particulate
homogenized
that,
al.(1976).
panel
was
cAMP
was
obtained
For
method
arbitrarilly
at4•Ž
correlated
periment
Tissue
Steiner
thyroid
was
cyclase,
and
et
filter
TSH.
obtain
equal-
stimulation.
experiment To
the
adenyl
adenyl
normal Main
is
homogenizer
Schwarz/Mann by
C
purpose.
confirm
prepared.
Mode
moist
and
Thytropar
human
or
present
C
(c), demonstrating ed adenyl cyclase
for
in
radio-immunoassay
by
Armour
(i)
enriched
ice.
(TCA)
by
1,
transferred
a
to
IgG
giazzi
of
slices
the
Mode
according
the
of120
as
glass
acid
of
further
on
B
experiments,
homogenized
plus10mg
incubated
motor-driven
described
required,
(i)
then
stimulating
Mode
same
one
solution
acetone-dry
a
supplied
method
When
was
Earl's
for
fraction,
same
blotted
measured
kits
the
slice
quickly
acetic
and
ploying
four
plus10mg/ml
incubation,
with
trichlor
(iv)
and
with
in6%
the
period
as
(i)
of
(a)).
subsequent
In
containing5mg/
same as
fresh
theophyllin
homogenized
to
same
After
a
solution
The
at37•Ž. B
(i)
same and
the
in
for
the
and
to
15min
as
Three
of
in
incubated
Earl's
BSA,(ii)
with10mM
scribed
at37•Ž.
at4•Ž
Graves'disease,
flask
albumin
TSH.
incubated
was
and
100ƒÊU/ml a
the
the
each
for120min
slice
ml
glucose
of
were but
solutions
One
of
of for
capable
(panel
suitable
solution
same
IgG,(iii) Graves'disease,
prepared
A,
four
ml
ly
serum the
human IgG of
IgG
thyroid
Japon,
bub-
salt
plus100ƒÊU/ml
Slices
Mode C:
min.(i)
detect
was
B: in
following
IgG
(i)
tube solu-
of95% O2and5% balanced
were
test
intermittent
Earl's
normal of
a
different
theophyllin,(ii)
replicate
b)
the
of
and10mM
plus10mg/ml as (i) plus10mg/ml the
in
four
consisting
ml
containing5mg/ml
(iv)
incubated
under
mixture
One
(BSA)
was following
Endocrinol. April1979
et al.
Thus,
with
human
not
influence
The a
slices
patient
shown (Shishiba
for
tent
thyroidal
et as
al.,
levels.
the
IgG
disease
positive
for
1978),
much
normal fraction
cAMP with
Graves'
be
of
particulate
incubated
with to
pretreatment
thyroid
from
which
was
LATS-protector
showed
as320•}31f
cAMP
con-
moles/mg
which
C.
significantly in
Results
the
(p