Exp. Clin. Endocrinol. 100 (1992) 28-31
Experimental and Clinical Endocrinology © 1992 Johann Ambrosius Barth
TSH-Receptor and Adhesion Molecules in Autoimmune Thyroid Disease F. Schuppert, M. Reiser and A. von zur Mühlen
Key words: Autoimmune thyroid disease - TSH-receptor Major histocompatibility complex - Intercellular adhesion molecule 1 - Endothelial leukocyte adhesion molecule I
zusätzlicher T-Zell-aktivierender Antigene wie ICAM-1 und
Zusammenfassung: Mittels Northern-Blot-Analyse und einer
Summary: Using a human TSH receptor (TSH-R) eDNA probe we investigated TSH-R expression levels in 20 human thyroid
menschlichen TSH-Rezeptor-(TSH-R-)cDNA-Sonde untersuchten wir das Niveau der TSH-R-Expression in 20 menschlichen Schilddrüsenfragmenten: 14 Basedow-Erkrankungn, 2 Hashimoto-Erkrankungen, 3 endemischen Strumen und eine gesunde Schilddrüse. Die TSH-R-Expression war niedrig in Schilddrüsen mit hohem Expressionsniveau des Haupthistokompatibili-
ELAM- 1 eine Rolle in der Initiierung der autoimmunen Schilddrüsenerkrankung spielt.
fragments by Northern blot analysis: 14 Graves' disease, 2 Hashimoto's disease, 3 endemic goiter and I healthy thyroid gland. TSH-R expression was low in those thyroids where expression of the major histocompatibility complex class I (MHC I), class II (MHC II) and intercellular adhesion molecule
interzellulären Adhäsionsmoleküls 1 (ICAM- 1), wohingegen die Expression des endothelialen Leukozyten Adhäsionsmoleküls I (ELAM-1) in keiner erkennbaren Verbindung zur TSHR-Expression stand. In-situ-Hybridisierung zeigte, daß neben den Lymphozyten (MHC II) Schilddrüsenzellen (MHC 11, ICAM-1) und Endothelzellen (ICAM-1, ELAM-1) die Quelle für die Transkripte dieser T-ZelI-aktivierenden Antigene waren. Wir schließen daraus, daß neben MHC I und II, die Expression
1 (ICAM-l) was high, whereas expression of the endothelial leukocyte adhesion molecule 1 (ELAM-1) bore no relation to TSH-R expression. In situ hybridization showed that next to lymphocytes (MHC II), thyroid cells (MHC li, ICAM-l) and endothelial cells (ICAM-!, ELAM-1) were the source of transcripts of these T-cell activating antigens. We conclude that next to MHC I and II, the expression of additional T-cell activating antigens such as ICAM-1 and ELAM-1 play a role in the initiation of auto-immune thyroid disease.
Introduction
Patients and Methods
Lymphocyte infiltration of the thyroid gland is one of the hallmarks of autoimmune thyroid disease (ATD). Using Northern blot analysis and in situ hybridization we investigated transcript levels of the recently cloned TSH receptor (TSH-R) (Libert et al., 1989; Nagayama et al., 1989; Parmentier et al., 1989; Misrahi et al., 1990; Heldin and
Clinical parameters
Westermark, unpublished) in relation to expression of specific adhesion molecules mediating the migration of lymphocytes to target cells (major histocompatibility complex class I [MHC II), to antigen presenting cells (MHC II and intercellular adhesion molecule 1 ICAM1]) and to endothelial cells (ICAM-I and endothelial
thyroid
tätskomplex Klasse I (MHC I), Klasse II (MHC Il) und des
leukocyte adhesion molecule 1 [ELAM-1]).
After approval of the local ethics committee, thyroid tissue was obtained from a total of 20 patients (3 male, 17 female, mean
age 42, range 15 to 78 yrs). 19 underwent modified subtotal thyroidectomy: 14 Graves' disease, 2 Hashimoto's disease and 3 endemic goiters. One patient underwent surgery of the paraglands
because
of primary hyperparathyroidism
("healthy thyroid AB."). In order to facilitate surgical preparation of the thyroid gland, patients received 400 mg iodide intravenously (ENDOJODIN®, Bayer, FRG) once a day for four days before surgery. Prior to iodide application treatment consisted of varying concentrations of antithyroid drugs with or without thyroid hormones.
Downloaded by: Boston University. Copyrighted material.
Department of Clinical Endocrinology, Hannover Medical School, Hannover/Germany
F. Schuppert et al., TSH-Receptor and Adhesion Molecules in ATD
The following plasmids were used for probe preparation: hTSH-
R: human TSH-R cDNA in pUC 13 (kindly provided by Dr. N. E. Heldin, Heldin and Westermark, unpublished); MHC I: human HLA-B 8 cDNA in pUC9 (Pohla et al., kindly provided by Dr. E. H. Weiss); MHCII: human HLA-DR alpha (DR 1) cDNA in pcDV l-pL2 (Sekaly et al., 1986; ATCC); ICAM-1: human ICAM-1 in pCDM8 (Simmons et al., 1989; British
ELAM-]: human ELAM-1 in pCDM8 (Bevilacqua et al., 1989, British Biotechnology). As a calibration standard a 32P labelled oligo 28S rRNA probe was used Biotechnology);
(Gonzalez et al., 1985; Barbu and Dautry, 1989; MedProbe AS., Norway). 100 ng of cDNA was labelled using a random primed DNA labelling kit (Boehringer Mannheim, Mannhein, FRG Feinberg and Vogelstein, 1983; Feinberg and Vogelstein, 1984]) and alpha-[32P]-dCTP (Amersham, FRG). 1 sg of the oligonucleotide was labelled using T4 polynucleotide kinase (Bethesda Research Laboratories, USA) and gamma-[32P]-ATP (Amersham, FRG). Total RNA was isolated from thyroid fragments which had been frozen in liquid nitrogen immediately after surgical removal. Total RNA was extracted following the method as described by Chirgwin and co-workers (Chirgwin et al., 1979). Northern blot analysis was performed by separating 20 sg of denatured total RNA per lane on a formaldehyde 1.5 % agarose gel. Integrity of RNA was controlled after staining with
ethidium bromide under UY light before transfer. After an overnight transfer onto ZETAPROBE® nylon filters (Bio-Rad, FRG) and U.V cross-linking filters were prehybridized at 42 oc for 4 hours with 5 mL of hybridization fluid. After overnight hybridization at 42 °C filters were washed at increasing temperatures up to 60°C. Filters were exposed to Kodak X-OMAT
AR films (Kodak, USA) at 80°C using LIGHTNING PLUS CRONEX® XTRA LIFE intensifying screens (Du Pont, USA). Nylon filters were stripped and rescreened.
560 bp Xba-Pst I fragment from ELAM- 1. The probe was label-
led with 35S-UTP to a specific activity of approximately l0 cpmIsg using an in vitro transcription kit (Stratagene, FRG). Hybridization preceeded with 40 pL probe at 50°C overnight. As a control no labelling was seen when thyroid tissue sections were hybridized with 35S-labeled sense probe. Non-hybridized single-stranded RNA probe was then digested with RNase A (40 sg/mL) in ribonuclease buffer (0.5 mol!L NaCI, 10 mmol/L Tris pH 8.0, 1 mmol/L EDTA) at 37 °C for 30 min. Slides were washed at increasing temperatures in SSC of decreasing concentrations, finally twice at 60 oc in 0.1 X SSC for 15 min. The slide preparations were autoradio-
graphed by dipping into autoradiography emulsion (LM 1, Amersham, FRG) which was diluted 1: 1 with 0.6 mol!L ammonium acetate. Exposure preceeded at 4 °C in light tight boxes. Slides were developed with D19 developer (Kodak, USA) and counterstained with hematoxylin. Photographs were made on a Zeiss Axiomat photomicroscope (Zeiss, FRG).
Results Northern Blot Analysis Thyroids investigated by Northern blot analysis displayed a wide variation of TSH-R mRNA levels. A band of 4.4
to 4.6 kilobase (kb) of size was generated which is in accordance with previous studies using human thyroid tissue (Huber et al., 1991; Maenhaut et al., 1991). In some of the thyroids two faint bands above 7.5 kb of size could be visualized in addition. Expression of MHC I and
II was found in all thyroid glands including healthy thyroid tissue and closely correlated with each other (r = 0.886, p < 0.001). ICAM-1 expression was absent in some Graves' thyroids but overall positively correlated
to MHC I (r = 0.62, p < 0.05) and MHC II (r = 0.731,
Analysis of autoradio grap hic signals and statistical analysis Autoradiographic signals were quantified by laser scanning densitometry (Pharmacia LKB, FRG) and compared to signals
of oligo 28S rRNA in order to calibrate for potential RNA loading differences (e.g. TSH-R mRNA!28S rRNA ratio vs MHC II mRNA/28S rRNA ratio). Values of the healthy thyroid
p < 0.01) transcript levels. There was a tendency towards an inverse relation between MHC I, MHC II and ICAM- I
expression on one hand and TSH-R expression on the other. In contrast, ELAM-1 expression which was increased in some Graves' thyroids and one Hashimoto's thyroid showed no relation to expression of the MHC I, MHC II, TCAM-1 or TSH-R gene.
tissue were arbitrarily assigned as unity value. Correlation analysis of expression levels was performed and p
Experimental and Clinical Endocrinology © 1992 Johann Ambrosius Barth
TSH-Receptor and Adhesion Molecules in Autoimmune Thyroid Disease F. Schuppert, M. Reiser and A. von zur Mühlen
Key words: Autoimmune thyroid disease - TSH-receptor Major histocompatibility complex - Intercellular adhesion molecule 1 - Endothelial leukocyte adhesion molecule I
zusätzlicher T-Zell-aktivierender Antigene wie ICAM-1 und
Zusammenfassung: Mittels Northern-Blot-Analyse und einer
Summary: Using a human TSH receptor (TSH-R) eDNA probe we investigated TSH-R expression levels in 20 human thyroid
menschlichen TSH-Rezeptor-(TSH-R-)cDNA-Sonde untersuchten wir das Niveau der TSH-R-Expression in 20 menschlichen Schilddrüsenfragmenten: 14 Basedow-Erkrankungn, 2 Hashimoto-Erkrankungen, 3 endemischen Strumen und eine gesunde Schilddrüse. Die TSH-R-Expression war niedrig in Schilddrüsen mit hohem Expressionsniveau des Haupthistokompatibili-
ELAM- 1 eine Rolle in der Initiierung der autoimmunen Schilddrüsenerkrankung spielt.
fragments by Northern blot analysis: 14 Graves' disease, 2 Hashimoto's disease, 3 endemic goiter and I healthy thyroid gland. TSH-R expression was low in those thyroids where expression of the major histocompatibility complex class I (MHC I), class II (MHC II) and intercellular adhesion molecule
interzellulären Adhäsionsmoleküls 1 (ICAM- 1), wohingegen die Expression des endothelialen Leukozyten Adhäsionsmoleküls I (ELAM-1) in keiner erkennbaren Verbindung zur TSHR-Expression stand. In-situ-Hybridisierung zeigte, daß neben den Lymphozyten (MHC II) Schilddrüsenzellen (MHC 11, ICAM-1) und Endothelzellen (ICAM-1, ELAM-1) die Quelle für die Transkripte dieser T-ZelI-aktivierenden Antigene waren. Wir schließen daraus, daß neben MHC I und II, die Expression
1 (ICAM-l) was high, whereas expression of the endothelial leukocyte adhesion molecule 1 (ELAM-1) bore no relation to TSH-R expression. In situ hybridization showed that next to lymphocytes (MHC II), thyroid cells (MHC li, ICAM-l) and endothelial cells (ICAM-!, ELAM-1) were the source of transcripts of these T-cell activating antigens. We conclude that next to MHC I and II, the expression of additional T-cell activating antigens such as ICAM-1 and ELAM-1 play a role in the initiation of auto-immune thyroid disease.
Introduction
Patients and Methods
Lymphocyte infiltration of the thyroid gland is one of the hallmarks of autoimmune thyroid disease (ATD). Using Northern blot analysis and in situ hybridization we investigated transcript levels of the recently cloned TSH receptor (TSH-R) (Libert et al., 1989; Nagayama et al., 1989; Parmentier et al., 1989; Misrahi et al., 1990; Heldin and
Clinical parameters
Westermark, unpublished) in relation to expression of specific adhesion molecules mediating the migration of lymphocytes to target cells (major histocompatibility complex class I [MHC II), to antigen presenting cells (MHC II and intercellular adhesion molecule 1 ICAM1]) and to endothelial cells (ICAM-I and endothelial
thyroid
tätskomplex Klasse I (MHC I), Klasse II (MHC Il) und des
leukocyte adhesion molecule 1 [ELAM-1]).
After approval of the local ethics committee, thyroid tissue was obtained from a total of 20 patients (3 male, 17 female, mean
age 42, range 15 to 78 yrs). 19 underwent modified subtotal thyroidectomy: 14 Graves' disease, 2 Hashimoto's disease and 3 endemic goiters. One patient underwent surgery of the paraglands
because
of primary hyperparathyroidism
("healthy thyroid AB."). In order to facilitate surgical preparation of the thyroid gland, patients received 400 mg iodide intravenously (ENDOJODIN®, Bayer, FRG) once a day for four days before surgery. Prior to iodide application treatment consisted of varying concentrations of antithyroid drugs with or without thyroid hormones.
Downloaded by: Boston University. Copyrighted material.
Department of Clinical Endocrinology, Hannover Medical School, Hannover/Germany
F. Schuppert et al., TSH-Receptor and Adhesion Molecules in ATD
The following plasmids were used for probe preparation: hTSH-
R: human TSH-R cDNA in pUC 13 (kindly provided by Dr. N. E. Heldin, Heldin and Westermark, unpublished); MHC I: human HLA-B 8 cDNA in pUC9 (Pohla et al., kindly provided by Dr. E. H. Weiss); MHCII: human HLA-DR alpha (DR 1) cDNA in pcDV l-pL2 (Sekaly et al., 1986; ATCC); ICAM-1: human ICAM-1 in pCDM8 (Simmons et al., 1989; British
ELAM-]: human ELAM-1 in pCDM8 (Bevilacqua et al., 1989, British Biotechnology). As a calibration standard a 32P labelled oligo 28S rRNA probe was used Biotechnology);
(Gonzalez et al., 1985; Barbu and Dautry, 1989; MedProbe AS., Norway). 100 ng of cDNA was labelled using a random primed DNA labelling kit (Boehringer Mannheim, Mannhein, FRG Feinberg and Vogelstein, 1983; Feinberg and Vogelstein, 1984]) and alpha-[32P]-dCTP (Amersham, FRG). 1 sg of the oligonucleotide was labelled using T4 polynucleotide kinase (Bethesda Research Laboratories, USA) and gamma-[32P]-ATP (Amersham, FRG). Total RNA was isolated from thyroid fragments which had been frozen in liquid nitrogen immediately after surgical removal. Total RNA was extracted following the method as described by Chirgwin and co-workers (Chirgwin et al., 1979). Northern blot analysis was performed by separating 20 sg of denatured total RNA per lane on a formaldehyde 1.5 % agarose gel. Integrity of RNA was controlled after staining with
ethidium bromide under UY light before transfer. After an overnight transfer onto ZETAPROBE® nylon filters (Bio-Rad, FRG) and U.V cross-linking filters were prehybridized at 42 oc for 4 hours with 5 mL of hybridization fluid. After overnight hybridization at 42 °C filters were washed at increasing temperatures up to 60°C. Filters were exposed to Kodak X-OMAT
AR films (Kodak, USA) at 80°C using LIGHTNING PLUS CRONEX® XTRA LIFE intensifying screens (Du Pont, USA). Nylon filters were stripped and rescreened.
560 bp Xba-Pst I fragment from ELAM- 1. The probe was label-
led with 35S-UTP to a specific activity of approximately l0 cpmIsg using an in vitro transcription kit (Stratagene, FRG). Hybridization preceeded with 40 pL probe at 50°C overnight. As a control no labelling was seen when thyroid tissue sections were hybridized with 35S-labeled sense probe. Non-hybridized single-stranded RNA probe was then digested with RNase A (40 sg/mL) in ribonuclease buffer (0.5 mol!L NaCI, 10 mmol/L Tris pH 8.0, 1 mmol/L EDTA) at 37 °C for 30 min. Slides were washed at increasing temperatures in SSC of decreasing concentrations, finally twice at 60 oc in 0.1 X SSC for 15 min. The slide preparations were autoradio-
graphed by dipping into autoradiography emulsion (LM 1, Amersham, FRG) which was diluted 1: 1 with 0.6 mol!L ammonium acetate. Exposure preceeded at 4 °C in light tight boxes. Slides were developed with D19 developer (Kodak, USA) and counterstained with hematoxylin. Photographs were made on a Zeiss Axiomat photomicroscope (Zeiss, FRG).
Results Northern Blot Analysis Thyroids investigated by Northern blot analysis displayed a wide variation of TSH-R mRNA levels. A band of 4.4
to 4.6 kilobase (kb) of size was generated which is in accordance with previous studies using human thyroid tissue (Huber et al., 1991; Maenhaut et al., 1991). In some of the thyroids two faint bands above 7.5 kb of size could be visualized in addition. Expression of MHC I and
II was found in all thyroid glands including healthy thyroid tissue and closely correlated with each other (r = 0.886, p < 0.001). ICAM-1 expression was absent in some Graves' thyroids but overall positively correlated
to MHC I (r = 0.62, p < 0.05) and MHC II (r = 0.731,
Analysis of autoradio grap hic signals and statistical analysis Autoradiographic signals were quantified by laser scanning densitometry (Pharmacia LKB, FRG) and compared to signals
of oligo 28S rRNA in order to calibrate for potential RNA loading differences (e.g. TSH-R mRNA!28S rRNA ratio vs MHC II mRNA/28S rRNA ratio). Values of the healthy thyroid
p < 0.01) transcript levels. There was a tendency towards an inverse relation between MHC I, MHC II and ICAM- I
expression on one hand and TSH-R expression on the other. In contrast, ELAM-1 expression which was increased in some Graves' thyroids and one Hashimoto's thyroid showed no relation to expression of the MHC I, MHC II, TCAM-1 or TSH-R gene.
tissue were arbitrarily assigned as unity value. Correlation analysis of expression levels was performed and p
