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

Inactivation of human thyroid stimulator by human thyroid extract.

Endocrinol. Japon. 1979, 26 (2), 285-289 Inactivation by of Human Thyroid Stimulator Human Thyroid Extract YOSHIMASASHISHIBA, MASARUTAKAISHI AND TA...
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