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The Journal of Clinical Endocrinology & Metabolism 86(10):4814 – 4817
Copyright © 2001 by The Endocrine Society
Suppression of Serum TSH by Graves’ Ig: Evidence for a
Functional Pituitary TSH Receptor
LEON J. S. BROKKEN, JOLANDA W. C. SCHEENHART, WILMAR M. WIERSINGA,
MARK F. PRUMMEL
AND
Department of Endocrinology and Metabolism, Academic Medical Center, University of Amsterdam, 1100 DD Amsterdam,
The Netherlands
Antithyroid treatment for Graves’ hyperthyroidism restores euthyroidism clinically within 1–2 months, but it is well known
that TSH levels can remain suppressed for many months despite
normal free T4 and T3 levels. This has been attributed to a delayed recovery of the pituitary-thyroid axis. However, we recently showed that the pituitary contains a TSH receptor
through which TSH secretion may be down-regulated via a paracrine feedback loop. In Graves’ disease, TSH receptor autoantibodies may also bind this pituitary receptor, thus causing continued TSH suppression. This hypothesis was tested in a rat
model. Rat thyroids were blocked by methimazole, and the animals were supplemented with L-T4. They were then injected
with purified human IgG from Graves’ disease patients at two
G
RAVES’ DISEASE IS an autoimmune thyroid disorder
characterized by circulating Ig directed against the
TSH receptor (TSH-R) (1, 2). The majority of these TSH-R
autoantibodies (TRAb) act as agonists by mimicking TSH
binding leading to Graves’ hyperthyroidism and goiter. Antithyroid drug treatment usually restores euthyroidism
within 4 – 6 wk in patients with hyperthyroidism (3). However, it may take much longer for TSH values to normalize.
Many treated Graves’ disease patients who are clinically
euthyroid and have normal T4 and T3 serum levels continue
to show decreased TSH levels (4, 5).
The explanation for this continued suppression of TSH is
unknown, but it is usually attributed to a delayed recovery
of the pituitary-thyroid axis (6). We offer an alternative explanation, involving a direct effect of TRAb on TSH secretion
by the pituitary. We have recently postulated that in addition
to a negative feedback control by T4 levels, TSH secretion is
influenced through a negative ultra-short-loop feedback
mechanism within the pituitary. We indeed demonstrated
that the TSH-R is expressed in the human anterior pituitary
on folliculo-stellate (FS) cells (7). When TSH is secreted by the
thyrotrophs, it can bind to this receptor on FS cells, which
then signal the thyrotrophs to decrease their TSH secretion.
That the FS cells are involved in this feedback control is
likely, because they are well known for their paracrine regulatory capabilities (8, 9). Apart from this physiological control, the TSH-R on FS cells may also bind circulating TRAb,
which, by mimicking TSH, subsequently can cause a decrease in TSH secretion independently of thyroid hormone
Abbreviations: FS, Folliculo-stellate; FT4I, free T4 index; TBII, thyroid
hormone binding inhibitory Ig; TRAb, TSH receptor autoantibodies;
TSH-R, TSH receptor; TT4, total T4.
different titers or with IgG from a healthy control (thyroid hormone binding inhibitory Ig, 591, 127, and < 5 U/liter). Despite
similar T4 and T3 levels, TSH levels were indeed lower in the
animals treated with high TSH receptor autoantibodies containing IgGs; the 48-h mean TSH concentration (mean ⴞ SEM; n ⴝ
8) was 11.6 ⴞ 1.3 ng/ml compared with 16.2 ⴞ 0.9 ng/ml in the
controls (P < 0.01). The intermediate strength TSH receptor
autoantibody-treated animals had levels in between the other
two groups (13.5 ⴞ 2.0 ng/ml). We conclude that TSH receptor
autoantibodies can directly suppress TSH levels independently
of circulating thyroid hormone levels, suggesting a functioning
pituitary TSH receptor. (J Clin Endocrinol Metab 86: 4814 – 4817,
2001)
levels. Such a mechanism may very well be responsible for
the low TSH levels observed in otherwise euthyroid Graves’
patients receiving antithyroid drug treatment. TRAb often
remain present in patients treated for Graves’ disease (10, 11)
and can be responsible for the long-term suppression of TSH.
To test this hypothesis, we used a modified long-acting
thyroid stimulator bioassay in which we measured the
plasma TSH response to the administration of TRAb in rats
that were unable to mount a thyroid response to TRAb because of prior antithyroid drug treatment.
Materials and Methods
Animals
Adult female Wistar rats (Harlan Sprague Dawley, Inc., Zeist, The
Netherlands), weighing approximately 325 g, were housed in cages at
21 C under a 12-h light, 12-h dark cycle (lights on at 0700 h and off at
1900 h). The animals received food and water ad libitum. The experiments
described here were approved by the animal welfare committee.
Experimental design
To suppress thyroidal T4 production, 24 rats were treated with the
antithyroid drug methimazole (1-methylimidazole-2-thiol; Sigma, St.
Louis, MO) at a concentration of 0.05% (wt/vol) in the drinking water
in combination with l-T4 (Sigma) dissolved in 0.9% NaCl (wt/vol) at 0.3
mg/ml and administered daily via a gastric tube in a dose of 1 ml/100
g BW. These dosages were determined in pilot experiments and resulted
in slightly elevated basal TSH levels between 5–10 ng/ml. After 1 wk,
the animals received a control IgG preparation (n ⫽ 8), a TRAbcontaining IgG preparation of intermediate strength (n ⫽ 8), or a preparation with a high TRAb titer (n ⫽ 8) at 0900 h. Blood was collected in
heparinized tubes immediately before and 1, 2, 4, 8, 24, and 48 h after
the administration of 1 ml of the appropriate IgG preparation and was
centrifuged at 3000 ⫻ g for 10 min at 4 C. Plasma was stored at ⫺20 C
for later analysis. Administration of IgG and subsequent withdrawal of
blood were performed via the tail vein under mild fentanyl fluanisone/
4814
Brokken et al. • Graves’ IgG Directly Suppress Serum TSH
J Clin Endocrinol Metab, October 2001, 86(10):4814 – 4817 4815
midazolam anesthesia (0.25 ml/100 g BW). Hematocrit was determined
before and 8 h after the onset of the experiment.
IgG purification
TRAb-containing serum was obtained from 32 patients with Graves’
disease who had thyroid hormone binding inhibitory Ig (TBII) titers
greater than 100 U/liter. Control serum was obtained from a healthy
subject. The pooled TRAb-containing serum and the control serum were
filtered through a 0.22-␮m pore size, low protein binding filter (Millipore
Corp., Bedford, MA), and the IgGs were isolated by affinity chromatography as described by Harlow and Lane (12). In short, the samples
were passed over a 5-ml protein G-Sepharose column (HiTrap Protein
G, Pharmacia Biotech, Piscataway, NJ) that was equilibrated with 0.1%
BSA in run buffer (20 mm sodium phosphate buffer, pH 7.0). After
washing with run buffer, the IgGs were eluted with 0.1 m glycine/HCl,
pH 2.7, and 1-ml fractions were collected in tubes containing 44 ␮l 1 m
Tris, pH 9.0, to neutralize the acid-labile IgGs. The protein-containing
fractions were pooled and concentrated by ammonium sulfate precipitation (50%, wt/vol). The IgG preparations were dissolved in a minimal
volume of PBS (pH 7.4) and finally dialyzed for 16 h at 4 C against several
changes of PBS. Both preparations were diluted in PBS to 30 mg/ml
protein. The TBII titer in the control IgG was less than 5 U/liter. The
TRAb-containing IgG had a TBII titer of 591 U/liter. To include an
intermediate strength preparation, this high TRAb pool was partly diluted with control IgG, yielding a TBII titer of 127 U/liter. The purity of
the IgG preparation and the yield of the different IgG isotypes were
assessed by immune electrophoresis and ELISA.
Hormone assays
TBII titers were measured by TRAK assay (Brahms Diagnostica, Berlin, Germany). TSH plasma levels were determined in a highly sensitive
chemiluminescent enzyme immunoassay (Immulite Third Generation
TSH kit, rat TSH application, Diagnostic Products, Los Angeles, CA).
Total T4 (TT4) and total T3 plasma levels were determined by in-house
RIAs (13) using rat null plasma as diluent. As an estimate of free T4
levels, the free T4 index (FT4I) was calculated as the product of T4 and
T3 resin uptake. The latter was determined with a T3 Uptake Kit (OrthoClinical Diagnostics, Amersham Pharmacia Biotech, Little Chalfont,
UK). Mean plasma levels of TSH, TT4, FT4I, and T3 levels over the 48-h
period were calculated as the area under the curve divided by 48 h. All
samples were measured within one run. Data are expressed as the
mean ⫾ sem.
Statistical analysis
The data were analyzed using SPSS, Inc. software (version 7.5.2, SPSS,
Inc., Chicago, IL). Time series were analyzed by ANOVA with repeated
measurements and two grouping factors (time and treatment). A t test
was used to compare the 48-h mean plasma levels. Differences between
groups were considered significant at P ⬍ 0.05.
Results
The pooled serum samples yielded IgG preparations that
were more than 99% pure with respect to total protein. The
recovery of IgG1, IgG2, and IgG3 was more than 99% and 85%
for IgG4.
At baseline, there were no differences in TSH, TT4, T3, and
FT4I among the three groups (Fig. 1). After injection of IgG,
thyroid function remained unaffected, as documented by
similar T3 levels in all three groups. TT4 levels as well as FT4I
decreased transiently in all groups (Fig. 1, B and C, D). There
were no statistically significant differences in TT4, FT4I, and
T3 values among the three groups.
After injection of IgG, plasma TSH levels transiently increased in all three groups (Fig. 1A). However, TSH levels in
rats treated with high TRAb-containing IgG were lower during the entire observation period than those in rats that
FIG. 1. Plasma levels of TSH, TT4, FT4I, and TT3 in rats during a 48-h
period after the administration of 30 mg purified human IgG. E and
䡺 Data after administration of TRAb containing IgG obtained from
patients with Graves’ disease at intermediate and high concentrations, respectively. F, Data obtained after administration of control
IgG. Curves of TRAb-treated animals are statistically compared with
control curves by ANOVA with repeated measurements and two
grouping factors (time and treatment). P ⫽ 0.009, difference between
high serum TRAb vs. control.
received control IgG (P ⬍ 0.01, by ANOVA). Rats treated
with intermediate strength TRAb showed TSH levels in between those in the other two groups.
The 48-h mean plasma hormone levels were calculated and
showed no differences among the groups with respect to T3,
TT4, or FT4I (Fig. 2). However, 48-h mean TSH plasma levels
were significantly reduced in the rats treated with the highest
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J Clin Endocrinol Metab, October 2001, 86(10):4814 – 4817
FIG. 2. Mean TSH, TT4, FT4I, and T3 plasma levels calculated over
the 48-h time period in rats treated with control IgG (TBII, ⬍5 U/liter), intermediate strength TRAb (TBII, 127 U/liter), and high TRAb
(TBII, 591 U/liter) containing IgG. **, P ⬍ 0.01 compared with the
control group.
concentration of TRAb (P ⬍ 0.01). Hematocrit did not change
during the experiment (data not shown).
Discussion
In this rat model we showed that TRAb are capable of
suppressing TSH levels through an extrathyroidal pathway.
Intravenous administration of TRAb containing human IgG,
in contrast to normal control IgG, to methimazole-treated
rats induced a decrease in TSH levels without affecting T4 or
T3 levels. This extrathyroidal effect of TRAb is most likely
caused by the binding of these IgGs to the TSH-R in the
Brokken et al. • Graves’ IgG Directly Suppress Serum TSH
pituitary. In a recent study we have shown that the TSH-R
is expressed in the human anterior pituitary on the so-called
FS cells (7). Other researchers not only confirmed this finding
(14), but they also showed activation of adenylate cyclase by
TSH in a mouse FS cell line. These cells make up approximately 10% of the pituitary cell population and are known
for their regulatory effects on pituitary hormone secretion (8,
9). We hypothesized that these TSH-R-bearing FS cells play
a role in fine-tuning of TSH secretion by the thyrotrophs
through an ultra-short-loop negative feedback mechanism,
possibly mediated via cytokines. In Graves’ disease, this may
have a further consequence. The anterior pituitary resides
outside the blood-brain barrier and is thus accessible to circulating IgGs. Thus, TRAb may very well bind to the TSH-R
on FS cells, which may then send a paracrine signal to the
thyrotrophs to diminish their TSH secretion. The present rat
study strongly supports this postulate.
We do not think that the observed suppression of TSH
levels upon administration of TRAb-containing IgGs can
be explained otherwise. First, we included a TBII-negative,
control IgG preparation that was administered in the same
concentration as the two TBII-positive preparations, thus correcting for aspecific general effects of IgGs on the pituitarythyroidal axis. Next, we used two concentrations of TRAbcontaining IgGs and found an indication for a dose-response
effect. Thirdly, the thyroid hormone levels were similar in all
three groups over a 48-h period, and T4 and FT4I levels
actually decreased slightly in all groups. This not only shows
the effectiveness of the methimazole-induced block of thyroid hormone synthesis, but it also makes it highly unlikely
that TRAb suppressed TSH levels via stimulation of the
thyroid gland. In view of these considerations, we strongly
believe that the TRAb sera indeed suppressed TSH levels via
an extrathyroidal pathway.
We found that TSH levels increased rather sharply shortly
after the administration of IgGs in all groups. We suggest that
this was due to a stress response in the animals. Similar
increases in TSH levels were found upon skin incision in
patients undergoing cholecystectomy (14a). In addition, part
of the TSH increase may be explained by the naturally occurring morning surge in rats (15). We made our rats slightly
hypothyroid in view of the mildly elevated TSH levels. This
was done to ascertain that changes in TSH levels would
indeed be detectable by the TSH assay.
We suggest that these data can be extrapolated to the
human situation. When patients with Graves’ hyperthyroidism are rendered euthyroid, it is frequently seen that their
TSH levels remain suppressed for a long time despite normal
thyroid hormone levels (4, 5). It is also known that TRAb can
remain present for a variable period of months to even years,
and our data now support the hypothesis that TRAb may be
responsible for extrathyroidal TSH suppression. We believe
that this is a better explanation for continued TSH suppression than a delayed recovery of the pituitary-thyroid axis.
The hypothesis can also explain another poorly understood
phenomenon encountered in clinical practice. After 1 yr of
antithyroid drug treatment, approximately 50% of Graves’
patients relapse. This occurs in patients with large goiters, in
patients with high TBII titers (16, 17), and also in those who
continue to have suppressed TSH levels in the absence of
Brokken et al. • Graves’ IgG Directly Suppress Serum TSH
J Clin Endocrinol Metab, October 2001, 86(10):4814 – 4817 4817
detectable TBII titers (18). We suggest that these suppressed
TSH values result from biologically active TRAb below the
detection limit of routine TBII assays.
Acknowledgments
We thank Adrie Maas (Department of Experimental Internal Medicine, Academic Medical Center) for skillful technical assistance.
Received April 2, 2001. Accepted June 12, 2001.
Address all correspondence and requests for reprints to: Dr. Leon J. S.
Brokken, Department of Endocrinology, F5-171, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail:
[email protected].
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