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Original Studies |
Department of Endocrinology, Academic Medical Center, University of Amsterdam (M.F.P., L.J.S.B., O.B., W.M.W.), 1105 AZ Amsterdam, The Netherlands; and Unité de Recherches Hormones et Reproduction, INSERM, U-135, Hopital de Bicêtre (G.M., M.M.), Le Kremlin- Bicêtre, France
Address all correspondence and requests for reprints to: Mark F. Prummel, M.D., Ph.D., Department of Endocrinology, F5-171, Academic Medical Center, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. E-mail: m.f.prummel{at}amc.uva.nl
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Introduction |
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We hypothesized that, in addition, TSH secretion might be fine-regulated in a short loop feedback at the pituitary level through the TSH receptor (TSH-R). This hypothesis was developed on two grounds. Firstly, because we were intrigued by the well known clinical observation that many patients with Graves’ hyperthyroidism continue to show suppressed plasma TSH levels despite adequate antithyroid treatment, resulting in clinical euthyroidism and normal (or even low) plasma T4 and T3 levels (1, 2). In our experience this is less often seen in patients with other forms of hyperthyroidism. We wondered whether TSH-R-stimulating antibodies might suppress TSH secretion by direct action on the pituitary. If so, the pituitary should contain a TSH-R. Several reports have now demonstrated that the TSH-R is present at extrathyroidal sites. In the intestine, for example, it appears to be involved in a local paracrine network of hormonal regulation of T cell homeostasis in response to locally produced TSH (3).
Secondly, we hypothesized that it would be more efficient for the fine-tuning of TSH secretion (i.e. to keep TSH plasma levels within a certain range) if the pituitary was able to measure and regulate its own TSH production, in analogy to modern heating boilers, which regulate their heat production through built-in temperature sensors in addition to the room thermostat.
A pituitary TSH-R might be involved in TSH secretion at the pituitary level in an autocrine fashion via a TSH-R on the thyrotroph itself. Alternatively, it might be mediated in a paracrine fashion through another cell type. A likely candidate for this is the folliculo-stellate (FS) cell (4), which is known to produce various cytokines and other regulatory factors, mostly notably IL-6 (5, 6).
To test our hypothesis, we set out to study the presence and cellular localization of the TSH-R in the human anterior pituitary using several, independent methods aimed at finding both TSH-R messenger ribonucleic acid (mRNA) and protein.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We used a human pituitary 
DR2 complementary DNA (cDNA)
library (CLONTECH Laboratories, Inc., Palo Alto, CA)
obtained from a pool of 12 Caucasian males and 6 Caucasian females
(age, 7–65 yr) who died from trauma. The library contained 2 x
106 independent clones, with an average insert
size of 1.8 kb (range, 0.6–4.7 kb). The titer was determined to be
8 x 109 plaque-forming units (pfu)/mL. For
screening, the library was plate-amplified using Escherichia
coli K802 and plated out on 8 150-mm plates (
4000 pfu/plate).
The plaques were transferred to nylon filters using standard techniques
and were screened with digoxigenin (DIG)-labeled cDNA probes
corresponding to the intracellular (ICD) and extracellular (ECD)
domains of the TSH-R (see below). For sequencing the clone was purified
to monoclonality by repeated Southern hybridization (i.e.
membrane lifts of the plaques were probed with the DIG-labeled cDNA
probes). The phage was converted to a plasmid by in vivo
excision according to the manufacturer’s manual (CLONTECH Laboratories, Inc.). We used a nonradioactive dideoxy chain
termination sequencing method on an automated sequencer (ABI Prism 377
DNA sequencer, Perkin-Elmer Corp., Foster City, CA). The
plasmid insert was sequenced using the pDR2 sequencing primers covering
the insert/plasmid boundaries. Further sequence information was
obtained using forward primers based on the published TSH-R sequence
(7, 8, 9), with an approximately 300-bp spacing creating
abundant internal overlap between sequences.
RT-PCR
Pituitary tissue (from a 75-yr-old man) was obtained from The
Netherlands Brain Bank (coordinator: Dr. R. Ravid). The tissue was
obtained at autopsy and was snap-frozen in liquid nitrogen. Before use
the anterior pituitary was dissected out (50 mg) and homogenized.
Polyadenylated RNA was isolated using the QuickPrep Micro mRNA
purification kit (Pharmacia Biotech, Uppsala, Sweden) and
reverse transcribed into single stranded cDNA using the First Strand
cDNA synthesis kit with random primers (Roche Molecular Biochemicals, Mannheim, Germany). PCR was performed as described
previously (10), using the PCR Core kit (Roche Molecular Biochemicals). In short, the reaction mixture was
subjected to 35 cycles of 1 min at 92 C, 2 min at 55 C, and 3 min at 72
C after an initial 2 min at 92 C in a Personal Cycler (Biometra,
Gottingen, Germany).
In situ hybridization
Three human anterior pituitaries (from 76-, 82-, and 83-yr-old men) were obtained after autopsy from The Netherlands Brain Bank, snap-frozen in liquid nitrogen, and stored at -70 C. Thyroid tissue from a 23-yr-old woman diagnosed with Graves’ disease and liver tissue from an individual with no known history of thyroid pathology were obtained at surgery, stored in liquid nitrogen, and included as positive and negative control tissues, respectively. Ten-micron frozen sections were mounted on silane-coated slides, air-dried on a hot plate, and kept at -70 C until use. Tissue pretreatment consisted of fixation in 4% paraformaldehyde in PBS (pH 7.4), carboxylation in 0.1% active diethyl pyrocarbonate in phosphate-buffered saline, and equilibration in 5 x SSC (standard saline citrate). Sections were prehybridized for 1 h at room temperature in hybridization mixture (5 x Denhardt’s solution, 5x SSC, 50% deionized formamide, 200 µg/mL salmon sperm DNA, and 250 µg/mL yeast transfer RNA), which was replaced by 200 µL hybridization mixture containing 200 ng/mL antisense or sense TSH-R RNA probe (see below). Hybridization took place in a humidified chamber at 55 C overnight. The sections were washed for 5 min in 5 x SSC, for 5 min in 2 x SSC, and for 1 h in 0.2 x SSC at 65 C and equilibrated in Tris-buffered saline containing 0.5% Triton X-100 (TBS/T; Sigma, St. Louis, MO), pH 7.4. After blocking for 30 min in 1% blocking reagent (Roche Molecular Biochemicals) in TBS/T, the sections were incubated with alkaline phosphatase-coupled anti-DIG Fab (Roche Molecular Biochemicals; 1:5000 in TBS/T with 0.1% blocking reagent). The sections were washed thoroughly in TBS/T, equilibrated in detection buffer (100 mmol/L Tris-HCl, 100 mmol/L NaCl, and 50 mmol/L MgCl2, pH 9.5) and reacted overnight with 0.38 mg/mL nitro blue tetrazolium and 0.18 mg/mL 5-bromo-4-chloro-3-indolyl-phosphate (Roche Molecular Biochemicals) in detection buffer. To inhibit endogenous alkaline phosphatase activity, 0.24 mg/mL levamisole was added. Aspecific precipitate was removed by washing in 100% methanol. Finally, the sections were coverslipped in Kaiser’s glycerin for light microscopic examination (Axioskop, Carl Zeiss, New York, NY). Sections incubated with prehybridization mix only were included as controls in the immunochemical detection of the DIG-labeled RNA probes.
Immunohistochemistry
Human pituitary and thyroid tissue were subjected to immunohistochemistry to demonstrate TSH-R at the protein level. We used two different mouse monoclonal antibodies, both directed against the extracellular domain of the human TSH-R. The first, T5–317 (gift from Dr. M. Milgrom) (11), was used on formalin-fixed paraffin-embedded tissue. The second, A10 (gift from Dr. J. P. Banga), directed against amino acids 21–35 (12), was used on frozen sections. TBS/T was used as solvent for the antibodies and as rinsing buffer between incubations. To block aspecific antibody binding, 0.5% nonfat powdered milk was added during the incubations with the primary antibodies. In the case of formalin-fixed paraffin-embedded tissue, deparaffinized sections were pretreated in a microwave oven in citrate buffer, pH 6.0, for 15 min to retrieve antigenicity. The sections were then incubated for 15 min with serum-free protein block (DAKO Corp., Santa Barbara, CA) and reacted with antibody T5–317 at a concentration of 2 µg/mL overnight at 4 C in a humidified chamber. The bound Igs were revealed with a biotinylated antimouse antibody and streptavidin-peroxidase (LSAB 2 immunostaining kit, DAKO Corp.) used according to the manufacturer’s instructions. Antibody binding was visualized by reacting the sections to 0.5 mg/mL 3-amino-9-ethylcarbazole (Sigma) and 3% H2O2 in 50 mmol/L sodium acetate buffer, pH 5.0. Sections were mildly counterstained with hematoxylin.
Alternatively, frozen sections were fixed in 4% paraformaldehyde, blocked in 5% nonfat powdered milk, and incubated with A10 culture supernatant (1:10) for 1 h at room temperature, followed by overnight incubation at 4 C. Subsequently, the sections were incubated for 30 min with goat antimouse IgG conjugated to alkaline phosphatase (1:100; Dakopatts, Copenhagen, Denmark), reacted to nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate with levamisole, and finally washed in methanol.
Control incubations were carried out by replacing the primary antibodies with nonimmune mouse serum.
Combined in situ hybridization and immunohistochemistry
To determine the phenotype of the TSH-R mRNA-expressing cell type(s), some sections that were initially processed for in situ hybridization were subsequently subjected to immunohistochemistry with two cell type-specific antibodies: a monoclonal mouse anti-human leukocyte antigen (HLA)-DR [a determinant of major histocompatibility complex (MHC) class II; clone CR3/43, 1:200; Dakopatts] and a polyclonal rabbit anti-TSH (1:3200; Dakopatts), specifically staining dendritic cells and thyrotrophs, respectively. After blocking for 30 min in 5% nonfat powdered milk, the sections were incubated for 1 h with primary antibody and 0.5% nonfat powdered milk. Primary antibody binding was detected using the avidin-biotin peroxidase complex method (Vectastain, Vector Laboratories, Inc., Burlingame, CA) according to the manufacturer’s instructions. Antibody binding was visualized by reacting the sections to 3-amino-9-ethylcarbazole. Sections were mildly counterstained in methyl green (Vector Laboratories, Inc.).
Double immunohistochemistry
To characterize the TSH-R protein-expressing cell type, sections immunolabeled with A10 were in part double labeled with anti-HLA-DR and anti-TSH as described above for double labeling of sections subjected to in situ hybridization. In the case of anti-HLA-DR double labeling, sections were first treated for 2 h with 0.1 mol/L glycine/HCl, pH 2.2, to elute the reagents used for TSH-R labeling that might otherwise interfere with detection of the mouse monoclonal anti-HLA-DR antibody.
Synthesis of TSH-R probes and primers
We synthesized thyroidal cDNA that was used to produce 1) two
DIG-labeled TSH-R cDNA probes to screen a human pituitary library, 2)
sense and antisense DIG-labeled TSH-R complementary RNA probes for
in situ hybridization, and 3) a positive control in PCR
experiments. Thyroid tissue from a 43-yr-old man with Graves’
hyperthyroidism was obtained at surgery and snap-frozen in liquid
nitrogen. Approximately 200 mg tissue were homogenized, and
polyadenylated RNA was isolated and reverse transcribed as described
above. The TSH-R cDNA probes were then synthesized by PCR using the DIG
DNA labeling kit (Roche Molecular Biochemicals) with two
primer sets designed to amplify an ECD cDNA product (1.2 kb)
spanning exons 1–9 and an ICD cDNA product (1.4 kb) amplifying exon
10 of the TSH-R gene, as described previously (10). The
complementary RNA in situ hybridization probes were in
vitro transcribed from a PCR-derived cDNA template obtained with a
primer set designed to amplify a unique 152-bp sequence (bases
1048–1199 of the human TSH-R; forward primer,
5'-GCCTTGAATAGCCCCCTCCAC-3'; reverse primer,
5'-CCAAAACCAATGATCTCATC-3'). The T7 RNA polymerase promotor sequence
was added to either the forward or reverse primer, producing cDNA
templates for the in vitro transcription of DIG-labeled
antisense and sense RNA probes, respectively (DIG RNA labeling kit,
Roche Molecular Biochemicals).
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Results |
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lDR2 library screening and sequencing
We first used purified phage cDNA in a PCR and detected TSH-R
products of the expected sizes. We then screened approximately 32,000
pfu, and two plaques hybridizing with the ICD TSH-R probe were
identified. One of these was purified, and the DR2 phage was
converted into a pDR2 plasmid. The insert of this clone was estimated
to be approximately 3.0 kb long. The insert was fully sequenced,
starting with the pDR2 sequencing primers, and was completely identical
to the sequence of the thyroidal TSH-R published by Misrahi et
al. (7). Our sequence included the same polymorphism
at codon 601: TAT coding for Tyr, as opposed to CAT coding for His as
described by Libert et al. (9) and Nagayama
et al. (8).
RT and PCR
To obtain further evidence for the presence of a TSH-R in the
pituitary, we used a human anterior pituitary gland obtained at
autopsy. Purified mRNA was used in a RT-PCR experiment with the same
TSH-R ICD and ECD primer sets as those used in our previous
experiments. ICD and ECD bands of the correct size (1.4 and 1.2 kb,
respectively) could be detected in this anterior pituitary tissue (Fig. 1
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Next, human pituitary sections were subjected to in
situ hybridization. The antisense TSH-R probe hybridized
specifically with cells scattered throughout the anterior pituitary. A
clear cytoplasmic staining pattern with a distinct negative nucleus was
observed (Fig. 2A). Hybridization with
the antisense probe was also detected in thyroid (Fig. 2B), but not in
liver (Fig. 2C) tissue, which were included as positive and negative
control tissues, respectively. Hybridization of pituitary sections with
the complementary sense probe did not result in any staining (Fig. 2D),
confirming the specificity of our TSH-R probe.
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Immunohistochemistry with two different monoclonal antibodies
against the TSH-R (clones T5–317 and A10) confirmed the presence of
TSH-R in the human anterior pituitary at the protein level. Both
antibodies specifically stained thyrocytes in control thyroid tissue
(Fig. 3, A and C). A10 was applied
to frozen sections and stained cells with a stellate-shaped morphology,
evenly distributed over the anterior pituitary (Fig. 3B). T5–317 was
used on formalin-fixed paraffin-embedded tissue. After microwave
treatment immune reactivity was observed in elongated cell types with
long processes that were evenly distributed over the anterior pituitary
(Fig. 3D).
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Subsequent double labeling of sections with anti-TSH revealed that
TSH-R mRNA expression did not colocalize with TSH immune reactivity
(Fig. 4A). However, when we double
labeled the hybridized sections with anti-HLA-DR serum, immune
reactivity was detected in dendritic-shaped cells with long slender
processes, and it appeared that TSH-R mRNA was expressed by a
subpopulation of HLA-DR-positive cells (Fig. 4B).
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Double labeling with A10 and anti-HLA-DR on paraformaldehyde-fixed
frozen pituitary sections demonstrated that TSH-R immune reactivity
also colocalized with a subset of cells positive for HLA-DR (Fig. 4D
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Again, no colocalization was observed when the sections were double
labeled with anti-TSH as the second primary antibody (Fig. 4C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, as shown in anterior pituitary cells cultured as
monolayers (20). Allaerts et al.
(14) showed that some of these MHC class II-expressing
dendritic cells coincide with FS cells, which constitute 5–10% of the
pituitary cells and were first identified in the anterior pituitary by
Rinehart and Farquhar (4). They are characterized as
agranular cells in the anterior pituitary, with a stellate-shaped
morphology and long cytoplasmic processes between the endocrine cells
(21). Roughly 10–20% of the FS cells express MHC class
II, and these have been shown to modulate anterior pituitary hormone
secretion (22, 23). Allaerts et al.
(24) showed in anterior pituitary cells cultured as
three-dimensional cell aggregates that FS cells are involved in
establishing a biphasic LH release at the pituitary level after GnRH
administration.
These results may be surprising because the TSH-R was thought to
be expressed only in the thyroid gland. However, it has now become
clear that the TSH-R is also functionally expressed in other
extrathyroidal tissues. For example, the TSH-R was also found to be
expressed on intestinal T cells, where it is involved in local
paracrine regulation of T cell homeostasis by sensing locally produced
TSH (3). Our findings are further supported by a recent
report by Ba
riaçik and Klein (25). These
researchers found that a proportion of murine dendritic cells express a
functional TSH-R on their surface. In our study we found the TSH-R
expressed by FS cells, which are known to be of dendritic cell lineage.
Both studies fit remarkably well with our hypothesis that a pituitary
TSH-R on FS cells may be involved in local fine-tuning of TSH
secretion. It should be made clear, however, that this hypothesis is
restricted to the fine-tuning only. The classical feedforward (TRH) and
feedback (T4) will prevail in hyper- and
hypothyroidism. In this model TSH is secreted by the thyrotrophs and
released in the extracellular space, where it binds to its receptor on
FS cells. Stimulation of this receptor might induce the release of
paracrine factors by the FS cells, which, in turn, modulate thyrotroph
TSH secretion. This possible feedback mechanism might not be limited to
TSH secretion, as it was recently shown that the human anterior
pituitary also contains GH receptors, which suggested that GH might
have autocrine and/or paracrine actions (26). Recently,
Asa et al. (27) confirmed this hypothesis in
transgenic mice devoid of the GH receptor, which showed a reduced GH
feedback inhibition on pituitary GH production.
Apart from this physiological concept, a TSH-R at the pituitary level may have an important pathophysiological significance. Under normal circumstances there is an excellent negative correlation between plasma free T4 and TSH levels, and it is generally accepted to use TSH determinations to monitor thyroid status. There are, however, some notable exceptions.
During treatment of Graves’ hyperthyroidism it is frequently
observed in clinical practice that these patients continue to show
suppressed TSH levels while they are clinically euthyroid and have
normal (or even low) free T4 and
T3 levels (1, 2). To date, this has
been attributed to delayed recovery of the pituitary-thyroid axis from
the hyperthyroid state (28). However, our finding of a
pituitary TSH-R offers another, more plausible explanation, in that
TSH-R autoantibodies act as ligand for the pituitary receptor and cause
suppression of TSH secretion (just like TSH itself; Fig. 5). Because TSH-R autoantibodies can
remain present for a long time during adequate antithyroid treatment,
they might very well be the cause of the long-term TSH suppression seen
in these patients.
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Acknowledgments |
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Footnotes |
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Received April 8, 2000.
Revised July 26, 2000.
Accepted August 7, 2000.
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References |
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riaçik EM, Klein JR. 2000 The
thyrotropin (thyroid-stimulating hormone) receptor is expressed on
murine dendritic cells and on a subset of
CD45RBhigh lymph node T cells: functional role
for thyroid-stimulating hormone during immune activation. J
Immunol. 164:6158–6165.This article has been cited by other articles:
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  S. V. H. Grommen, S. Taniuchi, T. Janssen, L. Schoofs, S. Takahashi, S. Takeuchi, V. M. Darras, and B. De Groef Molecular Cloning, Tissue Distribution, and Ontogenic Thyroidal Expression of the Chicken Thyrotropin Receptor Endocrinology, August 1, 2006; 147(8): 3943 - 3951. [Abstract] [Full Text] [PDF]
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 A. Alkemade, E. C Friesema, G. G Kuiper, W. M Wiersinga, D. F Swaab, T. J Visser, and E. Fliers Novel neuroanatomical pathways for thyroid hormone action in the human anterior pituitary. Eur. J. Endocrinol., March 1, 2006; 154(3): 491 - 500. [Abstract] [Full Text] [PDF]
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 S. L. Okada, J. L. Ellsworth, D. M. Durnam, H. S. Haugen, J. L. Holloway, M. L. Kelley, K. E. Lewis, H. Ren, P. O. Sheppard, H. M. Storey, et al. A Glycoprotein Hormone Expressed in Corticotrophs Exhibits Unique Binding Properties on Thyroid-Stimulating Hormone Receptor Mol. Endocrinol., February 1, 2006; 20(2): 414 - 425. [Abstract] [Full Text] [PDF]
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W. Allaerts and H. Vankelecom History and perspectives of pituitary folliculo-stellate cell research Eur. J. Endocrinol., July 1, 2005; 153(1): 1 - 12. [Abstract] [Full Text] [PDF]
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 S. Kumar, M. J. Coenen, P. E. Scherer, and R. S. Bahn Evidence for Enhanced Adipogenesis in the Orbits of Patients with Graves' Ophthalmopathy J. Clin. Endocrinol. Metab., February 1, 2004; 89(2): 930 - 935. [Abstract] [Full Text] [PDF]
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L. J. S. Brokken, W. M. Wiersinga, and M. F. Prummel Thyrotropin Receptor Autoantibodies Are Associated with Continued Thyrotropin Suppression in Treated Euthyroid Graves' Disease Patients J. Clin. Endocrinol. Metab., September 1, 2003; 88(9): 4135 - 4138. [Abstract] [Full Text] [PDF]
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L. J. S. Brokken, J. W. C. Scheenhart, W. M. Wiersinga, and M. F. Prummel Suppression of Serum TSH by Graves' Ig: Evidence for a Functional Pituitary TSH Receptor J. Clin. Endocrinol. Metab., October 1, 2001; 86(10): 4814 - 4817. [Abstract] [Full Text] [PDF]
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