Exp. Clin. Endocrino!, loo (1992) 75-79
Experimental and Clinical Endocrinology © 1992 Johann Ambrosius Barth
Measurement of Stimulating TSH Receptor Antibodies in Sera of Patients with Graves' Disease by a Recombinant TSH Receptor Bioassay Laboratories of Endocrinology, University Clinic of Internal Medicine Bergmannsheil (Head: H. Schatz), Bochuni/Germany
Key words: Recombinant TSH receptor - Bioassay - Antibodies - Graves' disease
einem deutlichen Abfall der cAMP-Werte. Nach thyreostatischer Therapie wiesen Patienten mit einem Rezidiv signifikant
Zusammenfassung: Während die Bestimmung von TSH-
höhere cAMP-Werte auf, während in der Remission die cAMPWerte sich normalisierten oder nur leicht erhöht waren. Wegen
Rezeptor-Antikörpern in kommerziellen Rezeptorassays in der Diagnostik des Morbus Basedow etabliert ist, bleibt eine Differenzierung dieser Antikörper in In-vitro-Zellkulturassays aufgrund ihres hohen Aufwandes bislang wenigen Speziallabors vorbehalten. Die molekulare Klonierung und Expression des humanen TSH-Rezeptors ermöglichte die Entwicklung eines Bioassays, der eine vereinfachte Bestimmung von stimulierenden Antikörpern erlaubt. Wir etablierten einen rekombinanten TSH-Rezeptorbioassay (CHO-TSHr-Zellinie) und bestimmten stimulierende TSH-Rezeptorantikörper in 128 Seren von Patienten mit Morbus Basedow. Gemessen wurden jeweils die Antikörper-stimulierten cAMP-Konzentrationen und diese Werte
der Heterogenität der TSH-Rezeptor-Antikörper fanden wir zwar in der Gruppe der unbehandelten Basedow-Patienten, nicht jedoch im Gesamtkollektiv eine signifikante Korrelation zwischen cAMP- und TRAK-Werten. Am deutlichsten wurde
mit denen eines Rezeptorassays (TRAK) verglichen. In der
tionsparametern und Antikörpertitern kann eine Differenzierung der TSH-Rezeptor-Antikörper zu einer diagnostischen Klärung beitragen.
Gruppe der unbehandelten Basedow-Patienten (N = 33) fanden sich durchschnittlich Sfach höhere cAMP-Spiegel als in einem
diese Diskrepanz bei Patienten nach Radiojodtherapie, die aufgrund funktionsblockierender Antikörper hohe TRAK-Werte, aber niedrige cAMP-Werte aufwiesen. Wir folgern aus diesen Ergebnissen: Der CHO-TSHr-Bioassay ermöglicht eine verein-
fachte, präzise Differenzierung von TSH-Rezeptor-Antikörpern. In der Diagnostik des Morbus Basedow ist eine Bestimmung der TSH-Rezeptorantikörper im TRAK-Assay ausreichend. Bei einer Diskrepanz zwischen klinischem Bild, Funk-
Normalkollektiv. Unter thyreostatischer Therapie kam es zu
Hyperthyroidism and diffuse goitre in Graves' disease anse through the action of immunoglobulins (IgG) that
TSH receptor (TSI) antibodies. The first bioassays were
bind to the TSH receptor and inhibit TSH binding (Volpe, 1986). Since the discovery of these IgG it has become a
release from thyroids of different animals (Dorrington et al., 1966; McKenzie, 1968) or release of thyroid hormo-
major concern to establish assays that measure these
nes from thyroid dices in culture (Mann et al., 1986). Later assays were used that measure cAMP synthesis in thyroid cells of different species in response to TSI stimulation (Kasagi et al., 1982 and 1987; Rapoport et al., 1984). These assays were both time-consuming and expensive and thus their use for clinical routine was limited. A further disadvantage was the use of thyroid cells from others species or alternatively from human thyroid cells isolated from specimen of nodular goiters which are very heterogenous (Derwahl et al., 1989 and
antibodies. Two different types of assays have been used:
on the one hand, the receptor assays that determine the ability of Graves' IgG to inhibit labeled TSH binding to thyroid membrane preparations (Rees Smith and Hall, 1981). In order to avoid the occurrence of nonspecific binding effects these
crude membranes have been
replaced in the last years by detergent-solubilized thyroid
membranes which are now commercially available for measurement of these antibodies in unextracted serum (Southgate et al., 1984). On the other hand, bioassays were established to measure the bioactivity of stimulating
based on their ability to determine either radioiodine
1990).
Due to the molecular cloning and stable expression of
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
M. Derwahi, H. Schatz, B. Bolle, A. Pohl and K. Meyer
Exp. Clin. Endocrino!, loo (1992) 1/2
76
disease. The results obtained in this bioassay were compared to those of a commercially available receptor assay for TSH receptor antibodies (TRAK).
Materials and Methods Transfection of CHO cells CHO cells were grown in lOO mm culture dishes in Ham F 12 medium supplemented with 10 % fetal calf serum and antibiotics (penicillin 100 U/ml and streptomycin 100 sg/ml). Cells
(HiTrap, Pharmacia) as recommended by the manufacturer. Sera from 25 subjects with normal thyroid function and lack
of thyroid antibodies were used as control and treated as described above.
Thyroid function was determined in all patients by measurement of free thyroxin (FT 4), triiodothyronine (T 3) and TSH by commercial RIA kits. TSH receptor antibodies were measured in the TRAK assay (Henning Berlin, Germany).
Results The human TSH receptor cDNA was stably transfected into Chinese Hamster Ovary (CHO) cells. Individual
clones were further isolated by limiting dilution. All
A o,
were transfected with the full length TSH receptor cDNA inserted into the expression vector pECE-SV2-NEO (kindly provided by Dr. Rapoport, San Francisco) using a modified calcium phosphate method (Chen and Okayama, 1987). For selection of transfected cells 400 sg/ml Geneticin (G 418. Gibco) were added to the medium. When non-transfected cells had died, surviving cells were pooled and further sub-
70-
30
FO < 20-
cloned by limiting dilution.
o
s)
10-
Measurement of cAMP accumulation Cells maintained in H5 medium for 5 days were incubated with the indicated concentrations of bovine TSH and 1 mM 1methyl-3-isobutylxanthol for 2 h at 37 °C. Then after removal
of the medium cAMP was extracted with 950/ ethanol and intracellular concentrations were measured by a RIA kit (Amersham-Buchler). Results were corrected to the protein
o
lo
iào
TSH mU/m'
B 12C
content of the cells U)
I-
Radiolabeled TSH binding N
CHO-TSHr cells were grown in 24 well plates to confluence. Binding studies were performed in Hank's modified buffer without NaCl (Tramontano et al., 1986). To maintain
isotonicity 280mM sucrose was added. Highly purified [1251]bTSH was a generous gift of Dr. Herzog (Henning Ber-
lin, Germany). Cells were incubated for 2h at 37 °C with ['251]bTSH and the indicated amount of unlabeled TSH (Sigma). At the end of the incubation cells were rinsed three
times with the same buffer (4C) and solubilized with 1 N NaOH. Radioactivity was measured in a y-counter. Nonspecific [1251]bTSH was determined in presence of l06 M unlabeled TSH, and this value subtracted from total binding. The data are expressed as the percentage of total [1251]bTSH binding added to each well.
Patients
o
ido
o.bi TSH
1000
(mU/m9
Fig. 1 A TSH-dependent cAMP synthesis in CHO-TSHr cells (clone RMD 12). cAMP accumulation was determined by a cAMP RIA kit after stimulation with the indicated TSH
concentrations. Mean ± SEM of 3 independent
experi-
ments. B Specific ['251]bTSH binding to CHO-TSHr cells expressing
the recombinant human TSH receptor. Cells were grown in 24 well plates to confluence. Studies of ['25IJbTSH binding were performed in modified Hank's buffer as described in Material and Methods. The data are expressed as the percentage of total [1251]bTSH binding added to each well. Mean ± SEM of 4 independent experiments in duplicate
Sera from 128 patients with Graves' disease (33 patients before treatment, 44 under antithyroid drug treatment, 24 in remission, li with a relapse after antithyroid drug treatment and 16 after radioiodine treatment) were analyzed. Sera were purified by precipitation with polyethylenglycol or in later experiments by means of protein A affinity chromatography
clones expressed a functional recombinant TSH receptor which is coupled to the adenylate cyclase system. As shown in Figure 1 for the clone RMD 12 TSH specifically bound to the recombinant receptor and stimu-
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
the human TSH receptor it became possible to measure both TRA and TSI in a recombinant human TSH receptor assay (Nagayama et al., 1989; Libert et al., 1989). We report here on the use of a CHO-TSHr bioassay for determination of stimulating TSH receptor antibodies from sera of patients with treated and untreated Graves'
77
M. Derwahi et al., Recombinant TSH Receptor Bioassay
lated intracellular cAMP accumulation. As calculated by Scatchard analysis the binding affinity (Kd) was 2.7 ± 0.9 i09 M1. Untransfected CHO cells used as the
A
control showed no significant TSH binding and only basal cAMP levels whether stimulated or unstimulated by TSH (data not shown). In this CHO-TSHr bioassay intracellular cAMP accumulation after stimulation with Graves' IgG was
measured
e o (o
o
Sera from
128
80
70 50
TRAK %
5o
o E
oo-
r = 0.32
patients with
B
40
30
80
0,
20
I..
1.)
10
4..
:;j
t
0)
70
o
50
o
o-
40
E
Control
before
under Remiss. Relapse Ri
Treatment
Fig. 2 cAMP accumulation in response to Graves' lgG. CHO-TSHr cells were incubated for 2 hours with PEG-precipitated IgG from 128 patients with Graves' disease (see Materials and Methods), 33 hyperthyroid patients before treatment,
44 euthyroid patients under antithyroid drug treatment, 24 patients in remission, Il hyperthyroid patients with a relapse after antithyroid drug treatment and 16 after radioiodine treat-
ment. 25 patients with normal thyroid function and lack of thyroid antibodies were used as control. Each point represents the mean of three determinations
Graves' disease were tested, 33 hyperthyroid patients before treatment, 44 euthyroid patients under antithyroid drug treatment, 24 patients in remission, 11 hyperthyroid patients with a relapse after antithyroid drug treatment and 16 after radioiodine treatment. 25 patients with normal thyroid function and lack of thyroid antibodies
were used as the control. Before treatment the mean value of cAMP was 26.0 ± 18.3 pmol/106 cells. Under antithyroid drug treatment for 5 to 16 months cAMP was decreased to 14.1 ± 10.6 pmol/106 cells. Patients in remission for at least 6 months showed low cAMP levels (mean: 10.6 ± 4.4 pmol/106), whereas in the 11 patients with a relapse after antithyroid drug treatment cAMP
concentrations were increased with a mean value of 22.8 ± 12.9 pmol/106 cells (Figure 2). After radioiodine treatment cAMP levels were within the normal range. Comparison of results from the CHO-TSHr bioassay and a receptor assay (TRAK) for TSH receptor antibodies gave only a weak correlation in all sera tested (Figure 3). While in untreated hyperthyroid patients there was a significant correlation between both parame-
o-
-
30
s
20
.
:. .
lo 0
10
20
30
s
s
s 40
50
50
70
80
90
100
TRAK %
Fig. 3 Comparison ot cAMP and TRAK values in sera from 128 patients with Graves' disease (A) and in the subgroup of sera from radioiodine treated patients (B). In all sera tested there was a weak correlation between both parameters. In most euthyroid patients after treatment with radioiodine increased TRAK, but normal cAMP values were found
Discussion
CHO-TSHr cells are a permanent cell line that express the recombinant human TSH receptor (Nagayama et al. 1989; Derwahl et al., 1991). These cells display a specific TSH binding and synthesize cAMP in response to TSH (Figure 1). Using this cell line we established a bioassay for determination of stimulating TSH receptor antibodies (TSI) in sera of patients with Graves' disease. The bioassay bases on intracellular cAMP accumulation in response to TSI stimulation. We investigated sera from 128 patients with Grave's disease in this assay. 33 of these patients were untreated, 44 under treatment with methimazole, 11 were diagnosed to have had a relapse after antithyroid drug treatment and 24 patients were in remission for at least 6 months. In addition, sera of 16 patients with Graves' disease who had undergone radioiodine therapy, were analyzed.
In the group of hyperthyroid patients before treatment mean cAMP values were icnreased by more than 5-fold over the value of controls (Figure 2). Under antithyroid
ters, no correlation could be found in patients after
drug treatment for periods varying between 5 to 16
radioiodine treatment.
months, cAMP concentrations were decreased. In remis-
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
U)
(Figure 2).
78
range. In patients which had a relapse after antithyroid drug treatment, cAMP was again increased with a mean cAMP value close to that of the untreated hyperthyroid patients. In euthyroid patients treated with radioiodine cAMP concentrations were within the normal range or only slightly increased. However, comparing the groups of untreated patients, treated patients, patients in remission and patients with a relapse there was a wide range of overlapping values. Thus, with respect to the individual patient with Graves' disease measurement of TSI in the recombinant CHO-TSHr bioassay does not allow to discriminate between different groups. These results are in
accordance with those of other investigators who also found that measurement of TSH receptor antibodies cannot predict the course of the disease (Madec et al., 1984; Schleusener et al., 1989). Other studies did not confirm these finding (Rapoport et al., 1984; Wilson et al., 1985). One of the major differences between the geographical regions, where the studies were performed, is the dietary iodine intake of the patients. It therefore seems likely that measurement of TSH receptor antibodies may be valuable in predicting the course of Graves' disease in
regions with sufficient iodine intake, while in iodine deficient areas other additional factors may be important (Azizi, 1985). TSI determined in the CHO-TSHr bioassay and TSH receptor antibodies in the TRAK assay were found to be very weakly correlated (Figure 3 A). A significant correlation was only observed in patients before treatment. In
some euthyroid patient treated with radioiodine high TRAK values, but low or normal cAMP levels were detected. This is likely to indicate the presence of TSH receptor antibodies with blocking activity. Similar results
have been reported by other investigators in testing patients with Graves' disease following radioiodine therapy (Orgiazzi et al., 1976; Hashim et al., 1986). Studies on the binding of these antibodies suggest that they bind to the same region of the TSH receptor as TSH and TSI. Thus, in patients treated with radioiodine high IRAK values should be interpreted with caution. The CHO-TSHr bioassay provides a valuable tool for the measurement of stimulating TSH receptor antibodies. In comparison to other bioassays this assay is less expensive and time-consuming because of the short doubling-
time and the simple handling of these cells. From the clinical point of view, however, in diagnosis of Graves' disease the commercially available TRAK assay provides sufficient information. The application of the CHO-TSHr bioassay is recommended only in cases where antibodies with blocking activity are presumed. Acknowledgements
References Azizi, F.: Environmental iodine intake affects the response
to methimazole in patients with diffuse toxic goitre. J. Clin. Endocrino!. Metab. 61(1985) 374-377 Chen, C.; Okayama, H.: High-efficienty transformation of
mammalian cells by plasmid DNA. Mol. Cell. Bio!. 7 (1987) 2745-2752 Derwahl, M.; Studer, H.; Huber, G.; Gerber, H.; Peter, H. J.: Intercellular propagation of individually programmed growth bursts in FRTL-5 Cells. Implications for interpreting growth factor actions. Endocrinology 127 (1990)
2104-2110 Derwahl, M.; Huber, G.; Gerber, H.; Studer, H.: Heterogeneity of thyroid growth in vivo has its counterpart in the intercellular differences between single thyrocytes in culture. Acta Endocrino!. (Copenh.) 120 (Suppl. 1; 1989) 53
Dorrington, K. J.; Munro, D. S.: The long acting thyroid stimulator. Clin. Pharmacol. Ther. 7 (1966) 788-795 Hashim, F. A.; Creagh, F. M.; El Hawrani, A.; Parkes, A. B.; Buckland, P. R.; Rees Smith, B.: Characterisation of TSH antagonist activity in the serum of patients with thyroid disease. Clin. Endocrino!. (Oxf.) 25 (1986) 275-28 1
Kasagi, K.; Konishi, J.; lida, A.; Ikekubo, K.; Mon, T.; Kuma, K.; Tonizuki, K.: A new in vitro assay for human thyroid stimulator using cultured thyroid cells: effect of sodium chloride on adenosine 3',S'-monophosphate increase. J. Clin. Endocrino!. Metab. 54 (1982) 108-114 Libert, F.; Lefort, A.; Gerard, C.; Parmentier, M.; Perret, J.; Ludgate, M.; Dumont, J. E.; Vassart, G.: Cloning, sequencing and expression of the human thyrotropin (TSH) receptor: evidence for binding of autoantibodies. Biochem.
Biophys.
Res.
Commun.
165
(1989)
1250-1255 Madec, A. M.; Laurent, M. C.; Lorcy, Y.; Le Guerrier, A. M.; Rostagnat-Stefanutti, A.; Orgiazzi, J.; Allannic, H.: Thyroid-stimulating antibodies: an aid to the strategy of treatment of Graves' disease? Clin. Endocrino!. (Oxf.) 21(1984) 247-253 Mann, K.; Schneider, N.; Hörmann, R.: Thyrotropic activity of acidic isoelectric variants of human chorionic gonadotropin from trophoblastic tumors. Endocrinology 118 (1986) 1558-1566 McKenzie, J. M.: Humoral factors in the pathogenesis of Graves' disease. Physiol. Rev. 43 (1968) 252-259 Nagayama, Y.; Kaufman, K. D.; Seto, P.; Rapoport, B.: Molecular cloning, sequence and functional expression of the cDNA for the human thyrotropin receptor. Biochem. Biophys. Res. Commun. 165 (1989) 1184-1190 Orgiazzi, J.; Williams, D. E.; Chopra, I. J.; Salomon, D.H.: Human thyroid adenyl cyclase-stimulating activity in immunoglobulin G of patients with Graves' disease. J. Clin. Endocrinol. Metab. 42 (1976) 341-347 Rapoport, B.; Greenspan, F. S.; Filetti, S.; Pepitone M.: Clinical experience with a human thyroid cell bioassay for thyroid stimulating immunoglobulin. J. Clin. Endocrino!. Metab. 58 (1984) 332-338
Rees Smith, B.; Hal!, R.: Measurement of thyrotropin receptor antibodies.
Methods
Enzymol.
74
(1981)
405-429
The expert assistance of Sabine Hoppe and Heike Sippe! is
Sch!eusener, H.; Schwander, J.; Fischer, Ch.; Hol!e, R.;
gratefully acknowledged. This work was supported in part by a
Badenhoop, K.; Hensen, J.; Finke, R.; Bogner, U.;
grant of the Deutsche Forschungsgemeinschaft, Bonn, Germany, to M.D. (De 407/2-1).
Schatz, H.; Pickardt, C. R.; Kotulla, P.: Ergebnisse der thyreostatischen Behandlung bei Patienten mit BasedowHyperthyreose: Erfahrungen aus einer prospektiven mu!-
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
sion cAMP levels were found to be normal or in the lower
Exp. Clin. Endocrino!, loo (1992) 1/2
M. Derwahl et al., Recombinant TSH Receptor Bioassay
ardt, C. R.; Pfannenstiel, P.; Weinheimer, B. ,Georg Thieme
Verlag Stuttgart, pp. 37-46 [171 Southgate, K.; Creagh, F. M.; Teece, M.; Kingswood, C.; Rees Smith, B.: A receptor assay for the measurement of TSH receptor antibodies in unextracted serum. Clin. Endocrinol. (Oxf.) 20 (1984) 539-548 [181 Tramontano, D.; Ingbar, S. H.: Properties and regulation of the thyrotropin receptor in the FRTL-5 rat thyroid cell line. Endocrinology 118 (1986) 1945-1951 [19] Volpe, R.: Pathogenesis of autoimrnune thyroid disease.
In: The Thyroid. Eds. Ingbar, S. H.; Braverman, L. E. Philadelphia: JB Lippincott 1986, pp. 747-767 [20] Wilson, R.; McKillop, J. H.; Pearson, D. W. M.; Cuthbert, G. F.; Thomson, J. A.: Relapse of Graves' disease after medical therapy: predictive value of thyroidal technetium-99m uptake and serum thyroid stimulating hormone receptor antibody levels. J. Nucl. Med. 26 (1985) 1024-1031
Author's address: Priv.-Doz. Dr. M. Derwahl, Medizinische Universitätsklinik, Bergmannsheil, Gilsingstr. 14, W - 4630 Bochum, Germany
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
tizentrischen Studie. In: Schilddrüse 1987. Eds. Pick-
79
Experimental and Clinical Endocrinology © 1992 Johann Ambrosius Barth
Measurement of Stimulating TSH Receptor Antibodies in Sera of Patients with Graves' Disease by a Recombinant TSH Receptor Bioassay Laboratories of Endocrinology, University Clinic of Internal Medicine Bergmannsheil (Head: H. Schatz), Bochuni/Germany
Key words: Recombinant TSH receptor - Bioassay - Antibodies - Graves' disease
einem deutlichen Abfall der cAMP-Werte. Nach thyreostatischer Therapie wiesen Patienten mit einem Rezidiv signifikant
Zusammenfassung: Während die Bestimmung von TSH-
höhere cAMP-Werte auf, während in der Remission die cAMPWerte sich normalisierten oder nur leicht erhöht waren. Wegen
Rezeptor-Antikörpern in kommerziellen Rezeptorassays in der Diagnostik des Morbus Basedow etabliert ist, bleibt eine Differenzierung dieser Antikörper in In-vitro-Zellkulturassays aufgrund ihres hohen Aufwandes bislang wenigen Speziallabors vorbehalten. Die molekulare Klonierung und Expression des humanen TSH-Rezeptors ermöglichte die Entwicklung eines Bioassays, der eine vereinfachte Bestimmung von stimulierenden Antikörpern erlaubt. Wir etablierten einen rekombinanten TSH-Rezeptorbioassay (CHO-TSHr-Zellinie) und bestimmten stimulierende TSH-Rezeptorantikörper in 128 Seren von Patienten mit Morbus Basedow. Gemessen wurden jeweils die Antikörper-stimulierten cAMP-Konzentrationen und diese Werte
der Heterogenität der TSH-Rezeptor-Antikörper fanden wir zwar in der Gruppe der unbehandelten Basedow-Patienten, nicht jedoch im Gesamtkollektiv eine signifikante Korrelation zwischen cAMP- und TRAK-Werten. Am deutlichsten wurde
mit denen eines Rezeptorassays (TRAK) verglichen. In der
tionsparametern und Antikörpertitern kann eine Differenzierung der TSH-Rezeptor-Antikörper zu einer diagnostischen Klärung beitragen.
Gruppe der unbehandelten Basedow-Patienten (N = 33) fanden sich durchschnittlich Sfach höhere cAMP-Spiegel als in einem
diese Diskrepanz bei Patienten nach Radiojodtherapie, die aufgrund funktionsblockierender Antikörper hohe TRAK-Werte, aber niedrige cAMP-Werte aufwiesen. Wir folgern aus diesen Ergebnissen: Der CHO-TSHr-Bioassay ermöglicht eine verein-
fachte, präzise Differenzierung von TSH-Rezeptor-Antikörpern. In der Diagnostik des Morbus Basedow ist eine Bestimmung der TSH-Rezeptorantikörper im TRAK-Assay ausreichend. Bei einer Diskrepanz zwischen klinischem Bild, Funk-
Normalkollektiv. Unter thyreostatischer Therapie kam es zu
Hyperthyroidism and diffuse goitre in Graves' disease anse through the action of immunoglobulins (IgG) that
TSH receptor (TSI) antibodies. The first bioassays were
bind to the TSH receptor and inhibit TSH binding (Volpe, 1986). Since the discovery of these IgG it has become a
release from thyroids of different animals (Dorrington et al., 1966; McKenzie, 1968) or release of thyroid hormo-
major concern to establish assays that measure these
nes from thyroid dices in culture (Mann et al., 1986). Later assays were used that measure cAMP synthesis in thyroid cells of different species in response to TSI stimulation (Kasagi et al., 1982 and 1987; Rapoport et al., 1984). These assays were both time-consuming and expensive and thus their use for clinical routine was limited. A further disadvantage was the use of thyroid cells from others species or alternatively from human thyroid cells isolated from specimen of nodular goiters which are very heterogenous (Derwahl et al., 1989 and
antibodies. Two different types of assays have been used:
on the one hand, the receptor assays that determine the ability of Graves' IgG to inhibit labeled TSH binding to thyroid membrane preparations (Rees Smith and Hall, 1981). In order to avoid the occurrence of nonspecific binding effects these
crude membranes have been
replaced in the last years by detergent-solubilized thyroid
membranes which are now commercially available for measurement of these antibodies in unextracted serum (Southgate et al., 1984). On the other hand, bioassays were established to measure the bioactivity of stimulating
based on their ability to determine either radioiodine
1990).
Due to the molecular cloning and stable expression of
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
M. Derwahi, H. Schatz, B. Bolle, A. Pohl and K. Meyer
Exp. Clin. Endocrino!, loo (1992) 1/2
76
disease. The results obtained in this bioassay were compared to those of a commercially available receptor assay for TSH receptor antibodies (TRAK).
Materials and Methods Transfection of CHO cells CHO cells were grown in lOO mm culture dishes in Ham F 12 medium supplemented with 10 % fetal calf serum and antibiotics (penicillin 100 U/ml and streptomycin 100 sg/ml). Cells
(HiTrap, Pharmacia) as recommended by the manufacturer. Sera from 25 subjects with normal thyroid function and lack
of thyroid antibodies were used as control and treated as described above.
Thyroid function was determined in all patients by measurement of free thyroxin (FT 4), triiodothyronine (T 3) and TSH by commercial RIA kits. TSH receptor antibodies were measured in the TRAK assay (Henning Berlin, Germany).
Results The human TSH receptor cDNA was stably transfected into Chinese Hamster Ovary (CHO) cells. Individual
clones were further isolated by limiting dilution. All
A o,
were transfected with the full length TSH receptor cDNA inserted into the expression vector pECE-SV2-NEO (kindly provided by Dr. Rapoport, San Francisco) using a modified calcium phosphate method (Chen and Okayama, 1987). For selection of transfected cells 400 sg/ml Geneticin (G 418. Gibco) were added to the medium. When non-transfected cells had died, surviving cells were pooled and further sub-
70-
30
FO < 20-
cloned by limiting dilution.
o
s)
10-
Measurement of cAMP accumulation Cells maintained in H5 medium for 5 days were incubated with the indicated concentrations of bovine TSH and 1 mM 1methyl-3-isobutylxanthol for 2 h at 37 °C. Then after removal
of the medium cAMP was extracted with 950/ ethanol and intracellular concentrations were measured by a RIA kit (Amersham-Buchler). Results were corrected to the protein
o
lo
iào
TSH mU/m'
B 12C
content of the cells U)
I-
Radiolabeled TSH binding N
CHO-TSHr cells were grown in 24 well plates to confluence. Binding studies were performed in Hank's modified buffer without NaCl (Tramontano et al., 1986). To maintain
isotonicity 280mM sucrose was added. Highly purified [1251]bTSH was a generous gift of Dr. Herzog (Henning Ber-
lin, Germany). Cells were incubated for 2h at 37 °C with ['251]bTSH and the indicated amount of unlabeled TSH (Sigma). At the end of the incubation cells were rinsed three
times with the same buffer (4C) and solubilized with 1 N NaOH. Radioactivity was measured in a y-counter. Nonspecific [1251]bTSH was determined in presence of l06 M unlabeled TSH, and this value subtracted from total binding. The data are expressed as the percentage of total [1251]bTSH binding added to each well.
Patients
o
ido
o.bi TSH
1000
(mU/m9
Fig. 1 A TSH-dependent cAMP synthesis in CHO-TSHr cells (clone RMD 12). cAMP accumulation was determined by a cAMP RIA kit after stimulation with the indicated TSH
concentrations. Mean ± SEM of 3 independent
experi-
ments. B Specific ['251]bTSH binding to CHO-TSHr cells expressing
the recombinant human TSH receptor. Cells were grown in 24 well plates to confluence. Studies of ['25IJbTSH binding were performed in modified Hank's buffer as described in Material and Methods. The data are expressed as the percentage of total [1251]bTSH binding added to each well. Mean ± SEM of 4 independent experiments in duplicate
Sera from 128 patients with Graves' disease (33 patients before treatment, 44 under antithyroid drug treatment, 24 in remission, li with a relapse after antithyroid drug treatment and 16 after radioiodine treatment) were analyzed. Sera were purified by precipitation with polyethylenglycol or in later experiments by means of protein A affinity chromatography
clones expressed a functional recombinant TSH receptor which is coupled to the adenylate cyclase system. As shown in Figure 1 for the clone RMD 12 TSH specifically bound to the recombinant receptor and stimu-
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
the human TSH receptor it became possible to measure both TRA and TSI in a recombinant human TSH receptor assay (Nagayama et al., 1989; Libert et al., 1989). We report here on the use of a CHO-TSHr bioassay for determination of stimulating TSH receptor antibodies from sera of patients with treated and untreated Graves'
77
M. Derwahi et al., Recombinant TSH Receptor Bioassay
lated intracellular cAMP accumulation. As calculated by Scatchard analysis the binding affinity (Kd) was 2.7 ± 0.9 i09 M1. Untransfected CHO cells used as the
A
control showed no significant TSH binding and only basal cAMP levels whether stimulated or unstimulated by TSH (data not shown). In this CHO-TSHr bioassay intracellular cAMP accumulation after stimulation with Graves' IgG was
measured
e o (o
o
Sera from
128
80
70 50
TRAK %
5o
o E
oo-
r = 0.32
patients with
B
40
30
80
0,
20
I..
1.)
10
4..
:;j
t
0)
70
o
50
o
o-
40
E
Control
before
under Remiss. Relapse Ri
Treatment
Fig. 2 cAMP accumulation in response to Graves' lgG. CHO-TSHr cells were incubated for 2 hours with PEG-precipitated IgG from 128 patients with Graves' disease (see Materials and Methods), 33 hyperthyroid patients before treatment,
44 euthyroid patients under antithyroid drug treatment, 24 patients in remission, Il hyperthyroid patients with a relapse after antithyroid drug treatment and 16 after radioiodine treat-
ment. 25 patients with normal thyroid function and lack of thyroid antibodies were used as control. Each point represents the mean of three determinations
Graves' disease were tested, 33 hyperthyroid patients before treatment, 44 euthyroid patients under antithyroid drug treatment, 24 patients in remission, 11 hyperthyroid patients with a relapse after antithyroid drug treatment and 16 after radioiodine treatment. 25 patients with normal thyroid function and lack of thyroid antibodies
were used as the control. Before treatment the mean value of cAMP was 26.0 ± 18.3 pmol/106 cells. Under antithyroid drug treatment for 5 to 16 months cAMP was decreased to 14.1 ± 10.6 pmol/106 cells. Patients in remission for at least 6 months showed low cAMP levels (mean: 10.6 ± 4.4 pmol/106), whereas in the 11 patients with a relapse after antithyroid drug treatment cAMP
concentrations were increased with a mean value of 22.8 ± 12.9 pmol/106 cells (Figure 2). After radioiodine treatment cAMP levels were within the normal range. Comparison of results from the CHO-TSHr bioassay and a receptor assay (TRAK) for TSH receptor antibodies gave only a weak correlation in all sera tested (Figure 3). While in untreated hyperthyroid patients there was a significant correlation between both parame-
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lo 0
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s 40
50
50
70
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TRAK %
Fig. 3 Comparison ot cAMP and TRAK values in sera from 128 patients with Graves' disease (A) and in the subgroup of sera from radioiodine treated patients (B). In all sera tested there was a weak correlation between both parameters. In most euthyroid patients after treatment with radioiodine increased TRAK, but normal cAMP values were found
Discussion
CHO-TSHr cells are a permanent cell line that express the recombinant human TSH receptor (Nagayama et al. 1989; Derwahl et al., 1991). These cells display a specific TSH binding and synthesize cAMP in response to TSH (Figure 1). Using this cell line we established a bioassay for determination of stimulating TSH receptor antibodies (TSI) in sera of patients with Graves' disease. The bioassay bases on intracellular cAMP accumulation in response to TSI stimulation. We investigated sera from 128 patients with Grave's disease in this assay. 33 of these patients were untreated, 44 under treatment with methimazole, 11 were diagnosed to have had a relapse after antithyroid drug treatment and 24 patients were in remission for at least 6 months. In addition, sera of 16 patients with Graves' disease who had undergone radioiodine therapy, were analyzed.
In the group of hyperthyroid patients before treatment mean cAMP values were icnreased by more than 5-fold over the value of controls (Figure 2). Under antithyroid
ters, no correlation could be found in patients after
drug treatment for periods varying between 5 to 16
radioiodine treatment.
months, cAMP concentrations were decreased. In remis-
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U)
(Figure 2).
78
range. In patients which had a relapse after antithyroid drug treatment, cAMP was again increased with a mean cAMP value close to that of the untreated hyperthyroid patients. In euthyroid patients treated with radioiodine cAMP concentrations were within the normal range or only slightly increased. However, comparing the groups of untreated patients, treated patients, patients in remission and patients with a relapse there was a wide range of overlapping values. Thus, with respect to the individual patient with Graves' disease measurement of TSI in the recombinant CHO-TSHr bioassay does not allow to discriminate between different groups. These results are in
accordance with those of other investigators who also found that measurement of TSH receptor antibodies cannot predict the course of the disease (Madec et al., 1984; Schleusener et al., 1989). Other studies did not confirm these finding (Rapoport et al., 1984; Wilson et al., 1985). One of the major differences between the geographical regions, where the studies were performed, is the dietary iodine intake of the patients. It therefore seems likely that measurement of TSH receptor antibodies may be valuable in predicting the course of Graves' disease in
regions with sufficient iodine intake, while in iodine deficient areas other additional factors may be important (Azizi, 1985). TSI determined in the CHO-TSHr bioassay and TSH receptor antibodies in the TRAK assay were found to be very weakly correlated (Figure 3 A). A significant correlation was only observed in patients before treatment. In
some euthyroid patient treated with radioiodine high TRAK values, but low or normal cAMP levels were detected. This is likely to indicate the presence of TSH receptor antibodies with blocking activity. Similar results
have been reported by other investigators in testing patients with Graves' disease following radioiodine therapy (Orgiazzi et al., 1976; Hashim et al., 1986). Studies on the binding of these antibodies suggest that they bind to the same region of the TSH receptor as TSH and TSI. Thus, in patients treated with radioiodine high IRAK values should be interpreted with caution. The CHO-TSHr bioassay provides a valuable tool for the measurement of stimulating TSH receptor antibodies. In comparison to other bioassays this assay is less expensive and time-consuming because of the short doubling-
time and the simple handling of these cells. From the clinical point of view, however, in diagnosis of Graves' disease the commercially available TRAK assay provides sufficient information. The application of the CHO-TSHr bioassay is recommended only in cases where antibodies with blocking activity are presumed. Acknowledgements
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The expert assistance of Sabine Hoppe and Heike Sippe! is
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gratefully acknowledged. This work was supported in part by a
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grant of the Deutsche Forschungsgemeinschaft, Bonn, Germany, to M.D. (De 407/2-1).
Schatz, H.; Pickardt, C. R.; Kotulla, P.: Ergebnisse der thyreostatischen Behandlung bei Patienten mit BasedowHyperthyreose: Erfahrungen aus einer prospektiven mu!-
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Author's address: Priv.-Doz. Dr. M. Derwahl, Medizinische Universitätsklinik, Bergmannsheil, Gilsingstr. 14, W - 4630 Bochum, Germany
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