MATERIALS AND METHODS

Disease diagnosis was confirmed in all patients with GD (n = 6), HT (n = 1), and multinodular goitre (MNG; n = 6) by biochemical testing and histological examination. Local ethical committee approval was obtained for our study and all thyroid tissue samples were taken after informed consent. Tissue samples were either immediately snap frozen in liquid nitrogen or processed to obtain primary TFC cultures as described below. Fetal chorionic membranes were isolated as described previously.²⁴

Primary TFC cultures were prepared from GD thyroidectomy specimens using methods described elsewhere²⁵ and were grown to confluence in 25 cm² flasks before treatment for six, 12, and 24 hour periods with either 100 U/ml IL-lα (Hoffman-La Roche, Nutley, New Jersey, USA), 100 μU/ml bovine thyroid stimulating hormone (TSH; National Institute for Biological Standards and Control, Potters Bar, UK), 100 U/ml recombinant IFNγ (Roche Diagnostics GmbH, Mannheim, Germany), or 100 U/ml TNFα (Calibiochem, Nottingham, UK), as detailed previously.¹⁹

RNA was extracted from whole tissue (3 mm³ pieces), fetal chorionic membranes, and cultured thyroid cells (5 × 10⁶ cells) using TRIZOL^™ LS reagent (Life Technologies, Paisley, UK), according to the manufacturer’s protocol. RNA was checked for quality by electrophoresis in a denaturing 1.5% agarose gel. Moloney murine leukaemia virus (M-MLV) RT (Promega, Southampton, UK) was used to synthesise cDNA, as described previously.¹⁹ Briefly, 5 μg of total RNA were reverse transcribed with 200 U of M-MLV RT, 500 ng random primers (Promega), and 1mM dNTPs (Promega) in RT buffer (Promega) with 10mM dithiothreitol in a final volume of 60 μl for one hour at 37°C.

Two different oligonucleotide primer sets (Life Technologies) were designed to amplify HLA-G cDNA by PCR: DB-G1 (sense), 5‘-CCGGAGTATTGGGAAG-3‘ and DB-G2 (antisense), 5‘-CTCATAGTCAAAGACAG-3‘²⁴; G.257 (sense), 5‘-GGAAGAGGAGACACGGAACA-3‘ and G.1004 (antisense), 5‘-CCTTTTCAATCTG AGCTCTTCTTT-3‘.²³ Primers DB-G1 and DB-G2 detect the expression of membrane bound HLA-G isoforms HLA-G1 and HLA-G4 and soluble HLA-G isoform sHLA-G1 (or HLAG-5). Primers G.257 and G.1004 detect the expression of all membrane bound HLA-G isoforms HLA-G1, HLA-G2, HLAG-3, and HLA-G4 and soluble HLA-G isoforms sHLA-G1 (or HLAG-5) and sHLA-G2 (or HLA-G6). Expression of the β actin gene was used as a quantitative and qualitative control for the cDNA samples and the primer sequences for β actin amplification are detailed elsewhere.¹⁹ To provide a positive control for HLA-G PCR amplifications, cloned HLA-G cDNA was used. Control PCR amplifications without cDNA were always carried out in parallel and were consistently negative. All PCR amplifications were performed in duplicate.

Hot start PCR amplifications (three minutes at 94°C as initial denaturation) were performed in 100 μl reactions containing: 2.5 U of Taq DNA polymerase (Promega), 0.1 mM of each dATP, dGTP, dCTP, and dTTP (Promega), 2 μl of RT mixture (cDNA), 1 μM of each primer, 1.5mM MgCl₂, 10mM Tris HCl (pH 9), 50mM KCl, and RNase free water to 100 μl. Amplifications of 40 cycles were carried out in a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, Connecticut, USA) using cycles of 94°C for one minute, 55°C for two minutes, and 72°C for two minutes, followed by a final extension of 10 minutes at 72°C. Amplification of β actin was performed using 25 cycles. PCR amplification products and 100 bp molecular weight markers (Promega) were separated on 1% agarose gels and stained with ethidium bromide. The primers for HLA-G produced PCR amplification products of 426 bp²⁴ and 770 bp,²³ whereas the β actin primers gave a product of 560 bp.

RESULTS

Thyroid tissue samples from patients with MNG (n = 6), GD (n = 6), and HT (n = 1) were analysed for HLA-G and β actin gene expression by RT-PCR. The results are illustrated in fig 1 using primers DB-G1 and DB-G2 for HLA-G. β Actin transcripts were demonstrated in all samples, confirming the integrity of the cDNA preparations. In contrast, no HLA-G mRNA was detected in the thyroid tissue from patients with GD, HT, or MNG, although it was clearly evident in the PCR amplifications with fetal chorionic membrane cDNA (fig 1) and with positive control HLA-G cDNA (fig 2). Primers G.257 and G.1004 also failed to amplify HLA-G from cDNA samples derived from thyroid tissue (data not shown).

• Download figure
• Open in new tab
• Download powerpoint

Figure 1

RT-PCR analysis of HLA-G and β actin gene expression in thyroid tissue samples from patients with autoimmune thyroid disease. The cDNA samples were from: fetal chorionic membranes (lane 1); thyroid tissue from a patient with Graves’disease (lane 2); thyroid tissue from a patient with Hashimoto’s thyroiditis (lane 3); thyroid tissue from a patient with multinodular disease (lane 4); and negative control without cDNA (lane 5).

• Download figure
• Open in new tab
• Download powerpoint

Figure 2

Reverse transcription polymerase chain reaction analysis of HLA-G and β actin gene expression in thyroid cell culture. The cDNA samples were from: HLA-G cDNA in pcDNA3 (lane 1); unstimulated thyroid follicular cells (TFCs) (lane 2); TFCs stimulated with: interleukin 1α (lane 3), interferon γ (IFNγ; lane 4), thyroid stimulating hormone (lane 5), tumour necrosis factor α (TNFα; lane 6) and TNFα plus IFNγ (lane 7); and negative control without cDNA (lane 8).

Figure 2 shows the results of experiments to detect HLA-G mRNA in cultured thyroid cells by RT-PCR using primers DB-G1 and DB-G2. The β actin gene was detected in all the cDNA samples from primary thyroid cell cultures but the expression of HLA-G could not be demonstrated. The effects of TSH, IL-1α, IFNγ, TNFα, and TNFα plus IFNγ upon HLA-G gene expression were analysed by RT-PCR following stimulation for six, 12, and 24 hour periods (fig 2). HLA-G mRNA was not detectable in the stimulated cultured thyroid cells. The same results were obtained for HLA-G expression in TFCs using primers G.257 and G.1004 (data not shown).

DISCUSSION

According to previous reports,^2,14–17 HLA-G might act to protect tissues from damage by infiltrating cytotoxic T cells during the process of inflammation. The characteristic T cell infiltration of the thyroid in patients with autoimmune thyroid disease can eventually lead to tissue destruction, and HLA-G expression might reduce this effect. However, our findings suggest that HLA-G is not expressed in either thyroid tissue or in thyrocytes derived from patients with autoimmune thyroid disease: HLA-G transcripts could not be detected by PCR amplification using two different sets of HLA-G specific primers, even at a high cycle number. This is contrary to a recent study that detailed HLA-G expression in TFCs obtained from patients with GD.²⁰ Here, expression of full length membrane bound HLA-G1 and its membrane bound short forms—HLA-G2, HLA-G3, and HLA-G4—was demonstrated. The functions of the short forms of HLA-G have yet to be clarified, although their physiological role is thought to be extremely limited,¹ and how their expression might relate to thyroid autoimmunity is unknown. The study also reported that the soluble forms of HLA-G—sHLA-G1 and sHLA-G2—were not expressed in patient TFCs,²⁰ a finding that does reflect our results. Soluble HLA-G1 molecules have been proposed to suppress activated CD8⁺ T cells⁸ and help in the control of inflammation.¹⁷ Absence of these sHLA-G1 functions may be responsible in part for the ongoing inflammation that is seen in GD.²⁰ The differences seen in the expression of HLA-G in TFCs between our study and that of Castro and colleagues²⁰ may reflect variations in the groups of patients with GD analysed, particularly with respect to any drug treatments that they might have received during the course of the disease.

“Soluble HLA-G1 molecules have been proposed to suppress activated CD8⁺ T cells and help in the control of inflammation”

Stimulation of primary thyrocytes with several proinflammatory cytokines, including IFNγ, failed to induce HLA-G expression. These results contrast with several studies detailing IFNγ stimulated expression of HLA-G in both normal and malignant cells,^21,22 but they do reflect the findings of Frumento et al,²³ who failed to stimulate HLA-G expression in melanoma cell lines using IFNγ.

From our failure to detect HLA-G transcripts in either thyroid tissue or TFCs from patients with GD we conclude that HLA-G expression does not occur in the thyroid of patients with this autoimmune disorder and that HLA-G does not play a pathophysiological role in GD. Although the expression of HLA-G was not detected in the thyroid sample of the patient with HT, a greater sample size would be required to conclude that HLA-G does not have a part to play in this autoimmune thyroid disease.