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Journal of Clinical Endocrinology & Metabolism, doi:10.1210/jc.2005-0679
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 10 5704-5710
Copyright © 2005 by The Endocrine Society

Human T Cell Leukemia Virus Type I-Infected Patients with Hashimoto’s Thyroiditis and Graves’ Disease

Takehiro Matsuda, Mariko Tomita, Jun-Nosuke Uchihara, Taeko Okudaira, Kazuiku Ohshiro, Takeaki Tomoyose, Tomoki Ikema, Masato Masuda, Mineki Saito, Mitsuhiro Osame, Nobuyuki Takasu, Takao Ohta and Naoki Mori

Division of Molecular Virology and Oncology (T.M., M.T., J.-N.U., T.Ok., N.M.), Graduate School of Medicine, Division of Child Health and Welfare (T.Oh.) and Division of Endocrinology and Metabolism (T.T., T.I., M.M., N.T.), Faculty of Medicine, University of the Ryukyus, Nishihara, Okinawa 903-0215, Japan; Department of Internal Medicine (K.O.), Naha Prefectural Hospital, Naha 902-8531, Japan; and Department of Neurology and Geriatrics (M.S., M.O.), Kagoshima University Graduate School of Medical and Dental Sciences, Kagoshima 890-8520, Japan

Address all correspondence and requests for reprints to: Professor Naoki Mori, M.D., Division of Molecular Virology and Oncology, Graduate School of Medicine, University of the Ryukyus, 207 Uehara, Nishihara, Okinawa, 903-0215, Japan. E-mail: n-mori{at}med.u-ryukyu.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Context: Autoimmune thyroid diseases have been reported to be associated with human T cell leukemia virus type I (HTLV-I) infection. HTLV-I proviral load is related to the development of HTLV-I-associated myelopathy/tropical spastic paraparesis and has also been shown to be elevated in the peripheral blood of HTLV-I-infected patients with uveitis, arthritis, and connective tissue disease.

Objective: The objective of the study was to evaluate the proviral load in HTLV-I-infected patients with Hashimoto’s thyroiditis (HT) or Graves’ disease (GD) and ascertain the ability of HTLV-I to infect thyroid cells.

Patients and Methods: A quantitative real-time PCR assay was developed to measure the proviral load of HTLV-I in peripheral blood mononuclear cells from 26 HTLV-I-infected patients with HT, eight HTLV-I-infected patients with GD, or 38 asymptomatic HTLV-I carriers. Rat FRTL-5 thyroid cells were cocultured with HTLV-I-infected T cell line MT-2 or uninfected T cell line CCRF-CEM. After coculture with T cell lines, changes in Tax and cytokine mRNA expression were studied by RT-PCR.

Results: HTLV-I proviral load was significantly higher in the peripheral blood of patients with HT and GD than asymptomatic HTLV-I carriers. In the peripheral blood from HTLV-I-infected patients with HT, HTLV-I proviral load did not correlate with the thyroid peroxidase antibody or thyroglobulin antibody titer. After coculture with MT-2 cells, FRTL-5 cells expressed HTLV-I-specific Tax mRNA. These cocultured FRTL-5 cells with MT-2 cells expressed IL-6 mRNA and proliferated more actively than those cocultured with CCRF-CEM cells.

Conclusion: Our findings suggest the role of the retrovirus in the development of autoimmune thyroid diseases in HTLV-I-infected patients.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
HUMAN T CELL LEUKEMIA virus type I (HTLV-I) is a human retrovirus highly endemic in southern Japan, intertropical Africa, Melanesia, Latin America, and the Caribbean basin (1). HTLV-I is the etiological agent of adult T cell leukemia (ATL) (2) and HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP), an inflammatory disease of the central nervous system (3, 4), and has also been implicated in several other inflammatory disorders, such as uveitis (5), chronic arthropathy (6), pulmonary alveolitis (7), and Sjögren’s syndrome (8). Furthermore, transgenic mice expressing Tax protein, a transactivator encoded by HTLV-I, develop proliferative synovitis (9) and exocrinopathy affecting lacrimal and salivary glands, features similar to those of Sjögren’s syndrome in humans (10).

The possibility that HTLV-I may cause thyroid diseases was initially raised by reports of Hashimoto’s thyroiditis (HT) in HTLV-I carriers and patients with HAM/TSP (11, 12). Graves’ disease (GD) has also been observed in HTLV-I carriers (13, 14). Epidemiological studies have demonstrated that HTLV-I seropositivity is a risk factor for thyroid disorders in Japan. Kawai et al. (12) reported that the prevalence of HTLV-I antibody in HT patients resident of Tokushima and Kochi prefectures, Japan, was 6.3%, which was significantly higher than the expected frequency of 2.2%. Mizokami et al. (15) also reported that the prevalence of HTLV-I antibody was significantly higher in patients with either antithyroid antibody-positive chronic thyroiditis or GD than the expected frequency in Fukuoka prefecture, Japan. Mine et al. (16) found that the frequency of antithyroid antibodies in blood donors with HTLV-I antibody was significantly higher than that in control donors without the antibody. Akamine et al. (17) also found a high prevalence of positivity for thyroid autoantibodies in ATL patients and HTLV-I carriers.

Several findings support the hypothesis of the etiopathogenic role of HTLV-I in thyroid diseases: HTLV-I envelope protein and Tax mRNA have been detected in follicular epithelial cells of the thyroid tissues of a patient with HT (18); Tax mRNA was also found in infiltrating lymphocytes in the interfollicular space (18); and HTLV-I proviral DNA and HTLV-I have been detected in thyroid tissues of patients with HT and GD (18, 19).

T lymphocytes, especially CD4+ T cells, are the main target of HTLV-I in vivo and carry the majority of the HTLV-I proviral load (20). The HTLV-I proviral load in peripheral blood mononuclear cells (PBMCs) is higher in patients with HAM/TSP than asymptomatic HTLV-I carriers (21), and the equilibrium set point of the proviral load is suspected to determine the development of the disease (22). We postulated that HTLV-I proviral load also influences the initiation and course of autoimmune thyroid diseases. To test our hypothesis, we measured this marker in PBMCs from HTLV-I-infected patients with HT and GD. To better understand the pathogenic mechanisms of HTLV-I-associated thyroid disorders, we determined whether HTLV-I could infect thyroid cells, and we characterized cell proliferation and cytokine gene expression in these cells after HTLV-I infection, using FRTL-5 rat thyroid cells.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Clinical samples

Blood samples were collected from 116 HTLV-I-infected patients, 38 asymptomatic carriers (33 females and five males, 21–79 yr old), 26 patients with HT (19 females and seven males, 37–80 yr old), eight patients with GD (seven females and one male, 40–59 yr old), 21 patients with HAM/TSP (17 females and four males, 31–74 yr old), and 23 patients with ATL (18 females and five males, 44–87 yr old). The diagnosis of HT was based on the presence of positive thyroid autoantibodies [thyroid peroxidase (TPO) and/or thyroglobulin (Tg)] and at least one of two additional criteria (hypothyroidism and/or goiter). Antibodies to TPO and Tg were determined by RIAs using commercially available kits (Cosmic, Tokyo, Japan). The patients with HT were treated with L-thyroxine. GD was diagnosed on the basis of history and signs of hyperthyroidism with diffuse goiter and the laboratory findings, including elevated serum T4 and T3 concentrations, undetectable serum TSH, and positive TSH receptor antibody (TRAb). TRAb was measured as TSH binding inhibitory Ig. One patient had ophthalmopathy. The patients with GD were treated with methimazole or propylthiouracil. Diagnosis and classification of the clinical subtypes of ATL were made based on the criteria of the Lymphoma Study Group (23) and were then confirmed in all cases by Southern blot hybridization analysis with detection of monoclonal integration of HTLV-I provirus into the genome. Diagnosis of HAM/TSP was based on the World Health Organization diagnosis guidelines (24). PBMCs donated by HTLV-I-seronegative healthy individuals (one female and two males, 25–29 yr old) served as normal controls. These control subjects did not have a history of thyroid or autoimmune diseases. PBMCs were isolated from heparinized blood by density gradient centrifugation. Seropositivity for HTLV-I was obtained by ELISA and particle agglutination assays. The screening of serum HTLV-I antibody was studied in all patients who visited our clinic at the University of the Ryukyus. All patients, HTLV-I asymptomatic carriers, and HTLV-I-seronegative healthy controls were Japanese, and they were living in Okinawa and Kagoshima prefectures (HTLV-I endemic areas), Japan. All individuals gave written informed consent for their participation.

Measurement of HTLV-I proviral load

DNA was prepared from each sample using a blood and tissue genomic DNA minikit, according to the protocol recommended by the manufacturer (Viogene-Biotek Corp., Hsichih, Taiwan) and stored at –80 C until use. The concentration of extracted DNA was adjusted to 10 ng/µl of the working solution. A quantitative real-time PCR assay was developed to measure the proviral load of HTLV-I in PBMCs. The HTLV-I copy number was referenced to the actual amount of cellular DNA by quantification of ß-actin gene. The forward and reverse primers used for HTLV-I pX region were 5'-CAAACCGTCAAGCACAGCTT-3' positioned at 7140–7159 and 5'-TCTCCAAACACGTAGACTGGGT-3' positioned at 7362–7341. The internal HTLV-I pX TaqMan probe (5'-TTCCCAGGGTTTGGACAGAGTCTTCT-3') was located between positions 7307 and 7332 of the genome, and carried a 5' reporter dye FAM (6-carboxy fluorescein) and a 3' quencher dye TAMRA (6-carboxy tetramethyl rhodamine). To quantify the human ß-actin gene, the forward and reverse primers 5'-TCACCCACACTGTGCCCATCTACGA-3' positioned at 2141–2165 and 5'-CAGCGGAACCGCTCATTGCCAATGG-3' positioned at 2435–2411, and the ß-actin TaqMan prove (5'-ATGCCCTCCCCCATGCCATCCTGCGT-3' positioned at 2171–2196) were used. PCR was performed with 5 µl DNA template with the use of the TaqMan universal PCR master mix (Applied Biosystems, Foster City, CA) and target gene assay mix containing each respective forward and reverse primer and TaqMan probe. The PCR conditions were as follows: 1 cycle at 50 C for 2 min and 95 C for 10 min and 45 cycles of denaturation at 95 C for 15 sec and annealing/extension at 58 C for 1 min. PCR was carried out in triplicate for each sample. HTLV-I provirus DNA cloned into the plasmid served as the control template and the ß-actin gene as the internal control. Data were quantified as mean values from the relative standard curve according to the instructions provided by the manufacturer (Applied Biosystems). Cycle numbers obtained at the log-linear phase of the reaction were plotted against a standard curve prepared with serially diluted control samples. The amount of HTLV-I proviral DNA was calculated by the following formula: copy number of HTLV-I (pX) per 1 x 104 PBMCs = [(copy number of pX)/(copy number of ß-actin/2)] x 104.

Cell culture and HTLV-I infection in vitro

FRTL-5 cells are a continuous line of rat thyroid cells and were grown in the Coon’s modified Ham’s F-12 medium containing 5% fetal bovine serum (FBS) (JRH Biosciences, Lenexa, KS) with the addition of a mixture of six hormones: bovine thyroid-stimulating hormone (10 mU/ml), transferrin (5 µg/ml), somatostatin (10 ng/ml), glycyl-L-histidyl-L-lysine acetate (10 ng/ml), hydrocortisone (10 nM), and insulin (10 µg/ml). All hormones were purchased from Sigma-Aldrich (St. Louis, MO). MT-2 cells, obtained by coculturing peripheral leukemic cells from an ATL patient with normal umbilical cord leukocytes (25), were used as an HTLV-I-infected T cell line. MT-2 cells contain proviral HTLV-I DNA and produce viral particles. CCRF-CEM cells were used as the uninfected T cell line. These T cells were treated with 100 µg/ml mitomycin C (MMC) for 1 h at 37 C. After washing three times with PBS, they were cultured with an equal number of FRTL-5 cells in Coon’s modified Ham’s F-12 medium containing 5% FBS. The culture medium was changed on the third day after coculture. FRTL-5 cells were harvested at 3 and 7 d, followed by RNA extraction as described below.

RT-PCR

Total RNA was extracted with Trizol (Invitrogen, Carlsbad, CA) according to the protocol provided by the manufacturer, and the amount of total RNA was determined by measuring absorbance at 260 nm. First-strand cDNA was synthesized from 5 µg total cellular RNA in a 20-µl reaction volume using an RNA PCR kit (Takara Shuzo, Kyoto, Japan) with random primers. Thereafter cDNA was amplified using a multiplex PCR kit for rat inflammatory cytokine gene (Maxim Biotech, Inc., San Francisco, CA) according to the instructions provided by the manufacturer. Product sizes were 351 bp for TNF{alpha}, 294 bp for IL-1ß, 453 bp for IL-6, 250 bp for TGFß, and 532 bp for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The expression levels of Tax and ß-actin mRNAs were analyzed as described previously (26). Product sizes were 203 bp for Tax and 548 bp for ß-actin. PCR products were fractionated on 2% agarose gels and visualized by ethidium bromide staining.

Cell proliferation assay

FRTL-5 cells (1 x 104 cells/well) were cultured with or without MMC-treated MT-2 or CCRF-CEM (1 x 104 cells/well) cell line in 96-well culture plates in Coon’s modified Ham’s F-12 medium containing 5% FBS for 1, 3, or 5 d. The data were obtained by triplicate experiments. Four hours before terminating the culture, 10 µl of the cell proliferation reagent water-soluble tetrazolium salt (WST)-8, a tetrazolium salt (Wako Chemicals, Osaka, Japan) were added to each well. At the end of incubation, absorbance at 450 nm was measured using an automated microplate reader. Measurement of the mitochondrial dehydrogenase-mediated cleavage of WST-8 to formazan dye indicates the level of proliferation.

Statistical analysis

Data are expressed as mean ± SD. Statistical significance was analyzed by Mann-Whitney U test. The Spearman’s rank correlation coefficient was used to describe the association between different variables. The Student’s t test was performed for comparisons of growth of uninfected FRTL-5 cells and that of HTLV-I-infected FRTL-5 cells.


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Quantification of HTLV-I proviral DNA in asymptomatic HTLV-I carriers, HTLV-I-infected patients with HT or GD, HAM/TSP, and ATL

As shown in Fig. 1Go, we estimated the absolute copy number of HTLV-I proviral DNA per 104 PBMCs. First, proviral load was quantified in three healthy volunteers (seronegative), 21 HAM/TSP patients, and 23 ATL patients. The provirus was undetectable in all healthy noncarriers (Fig. 1BGo), whereas HAM/TSP and ATL patients were positive for HTLV-I with a proviral load of 1986 ± 198 copies (range 879-4137 copies) and 2791 ± 320 copies (range 874-6175 copies), respectively (Fig. 1AGo). The provirus loads in smoldering-, chronic-, acute-, and lymphoma-type ATL patients were 1561 ± 268, 2683 ± 782, 3098 ± 468, and 3248 ± 893, respectively. The copy numbers in asymptomatic carriers varied from 0.4 to 347, those of HTLV-I-infected patients with HT varied from 2 to 1076, and those of HTLV-I-infected patients with GD varied from 29 to 1222 (Fig. 1BGo). The mean ± SD and median of the copy number was 60 ± 11 and 39 in asymptomatic carriers. With regard to HTLV-I-infected patients with HT and GD, the values were 276 ± 53 (median 199) and 303 ± 137 (median 200), respectively. The median copy number of HTLV-I-infected patients with HT and GD was about 5-fold higher than that of asymptomatic carriers. The differences were statistically significant between asymptomatic carriers and HTLV-I-infected patients with HT and between asymptomatic carriers and HTLV-I-infected patients with GD, respectively (Mann-Whitney U test) (Fig. 1BGo). There was no significant correlation between copy number of HTLV-I proviral DNA and antibody titer of either Tg (P = 0.6535) or TPO (P = 0.4703) in HTLV-I-infected patients with HT (Spearman’s rank correlation) (Fig. 2Go). Among the HTLV-I-infected patients with GD, the correlation between copy number of HTLV-I proviral DNA and TRAb titer was not observed (data not shown).



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  		FIG. 1. HTLV-I proviral load in the peripheral blood of HAM/TSP and ATL (A) and healthy individuals without HTLV-I, asymptomatic carriers, and HTLV-I-infected patients with HT or GD (B). Data are HTLV-I copy number per 104 PBMCs.

 


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  		FIG. 2. Correlation between HTLV-I proviral load and antibody titer of Tg (A) or TPO (B) in HTLV-I-infected patients with HT. There was no significant correlation between copy number of HTLV-I proviral DNA and antibody titer of either Tg (P = 0.6535) or TPO (P = 0.4703).

 
Detection of HTLV-I Tax mRNA in FRTL-5 cells cocultured with HTLV-I infected T cells

FRTL-5 cells were cocultured with either MT-2 or CCRF-CEM cells. After cocultivation for 3 d, FRTL-5 cells were washed extensively and exchanged with fresh medium. After the cells were cultured for further 4 d, they were washed thoroughly. At 3 and 7 d after cocultivation, FRTL-5 cells were harvested for assessment by RT-PCR for expressing HTLV-I viral antigen. Because T cell lines were pretreated extensively with MMC, these MMC-treated T cells could not proliferate, as determined by WST-8 assay. These specimens of FRTL-5 cell at 3 and 7 d of culture contained no viable MT-2 cells. As shown in Fig. 3AGo, FRTL-5 cells cocultured with MT-2 cells showed strong expression of Tax mRNA. In contrast, FRTL-5 cells cocultured with CCRF-CEM cells did not express Tax mRNA. To determine whether the Tax cDNA sequence was amplified from residual MT-2 cells that had been added after MMC treatment, PCR amplification of a human PTHrP exon 3 sequence was done, using these DNA samples. The human PTHrP sequence was amplified from MT-2 DNA by PCR. However, the human PTHrP sequence was not detected in any of the cocultured rat FRTL-5 cells, which suggests that residual MT-2 cells in these samples were not amplified (data not shown). These results suggest that the HTLV-I can be transmitted into FRTL-5 cells from HTLV-I producing MT-2 cells.



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  		FIG. 3. HTLV-I can infect FRTL-5 cells and induce gene expression of IL-6. A, Detection of HTLV-I Tax mRNA in FRTL-5 cells by RT-PCR. FRTL-5 cells were cocultured with MMC-treated MT-2 or CCRF-CEM cells. At 3 and 7 d after cocultivation, FRTL-5 cells were harvested and then Tax mRNA expression was analyzed. Lane 1, cultured FRTL-5 cells at 3 d; lanes 2 and 3, FRTL-5 cells cocultured with MT-2 and CCRF-CEM cells at 3 d; lane 4, cultured FRTL-5 cells at 7 d; lanes 5 and 6, FRTL-5 cells cocultured with MT-2 and CCRF-CEM cells at 7 d. Human ß-actin mRNA was used as a control. B, Induction of expression of IL-6 gene in FRTL-5 cells. Lane 1, cultured FRTL-5 cells at 3 d; lanes 2 and 3, FRTL-5 cells cocultured with MT-2 and CCRF-CEM cells at 3 d; lane 4, positive control. Rat GAPDH mRNA was used as a control.

 
Cytokine expression in FRTL-5 cells cocultured with MT-2 or CCRF-CEM cells

Tax activates not only the transcription of the viral genome but also the expression of various cellular genes. It is now clear that HTLV-I-infected T cells are capable of producing various cytokines through the transactivation of cytokine genes by the Tax protein (27). HTLV-I-infected nonlymphoid cells have also been reported to express various types of cytokines (28, 29). Therefore, we investigated the expression of inflammatory cytokines in FRTL-5 cells cocultured with MT-2 or CCRF-CEM cells by RT-PCR. RT-PCR was carried out with primer sets for IL-1ß, IL-6, TNF{alpha}, and TGFß as well as rat GAPDH. As shown in Fig. 3BGo, low levels of expression of IL-6 and TGFß mRNA were detected in control FRTL-5 cells. The level of expression of IL-6 was increased in FRTL-5 cells cocultured with MT-2 cells but not in FRTL-5 cells cocultured with CCRF-CEM cells. Transcripts of IL-1ß and TNF{alpha} were not detected in any of the samples.

Proliferation of FRTL-5 cells

It was reported previously that HTLV-I could infect synovial cells, resulting in their active proliferation (28). Finally, to investigate the relation of thyroid cell proliferation and HTLV-I infection, the proliferative response of FRTL-5 cells was examined by cocultivation with MT-2 cells and compared with that of FRTL-5 cells cocultured with CCRF-CEM cells using the WST-8 assay as an index of cell number. The proliferation of FRTL-5 cells at d 1, 3, and 5 was significantly increased by coculture with HTLV-I-infected T cells (Fig. 4Go). It was noted that MMC-treated MT-2 and CCRF-CEM cells could not proliferate.



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  		FIG. 4. Proliferation of FRTL-5 cells cocultured with MT-2 or CCRF-CEM cells. FRTL-5 cells were cultured in the presence or absence of MMC-treated MT-2 or CCRF-CEM cells for the indicated time periods. Four hours before terminating the culture, WST-8 was added to each well, and absorbance at 450 nm was measured. Data are expressed as percentage growth, compared with the uninfected FRTL-5 cells and represent the mean ± SD of triplicate measurements. MMC-treated MT-2 and CCRF-CEM cells could not proliferate.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
Although the etiology of autoimmune thyroid diseases has yet to be established, it appears to result from complex interactions between host genetic and environmental factors. The involvement of viral infection, particularly retrovirus infection, in the pathogenesis of autoimmune thyroid diseases has been demonstrated in animals and humans (30, 31, 32, 33, 34). However, the role of infection in precipitating autoimmune thyroid diseases remains largely hypothetical (34). Ciampolillo et al. (31) reported the presence of a HIV type 1-related DNA sequence in the thyroid and PBMCs of the patients with GD, but this finding was disputed by Humphrey et al. (35) and Tominaga et al. (36). Jaspan et al. (37) reported that over 85% of patients with GD have detectable serum antibodies against a human intracisternal type A retroviral particle. Furthermore, data from 35 members of three kindreds suggest that both human leukocyte antigen susceptibility and exposure to the retroviral particle are necessary for the development of GD (38). HTLV-I is considered to be implicated in the pathogenesis of autoimmune thyroid diseases in Japan, where this retrovirus is endemic, and epidemiological studies have shown an association between HTLV-I infection and thyroid disorders in Japan (12, 15, 16, 17).

The present study provides biological data suggesting the contribution of HTLV-I in the development of autoimmune thyroid diseases. Our results showed that: 1) the circulating HTLV-I proviral load was higher in HTLV-I-seropositive patients with HT or GD than asymptomatic HTLV-I carriers and lower than that in patients with HAM/TSP or ATL; 2) HTLV-I can be transmitted into thyroid cells from an HTLV-I-producing T cell line; 3) HTLV-I infection induced expression of IL-6 gene but not IL-1ß, TNF{alpha}, and TGF-ß in thyroid cells; and 4) HTLV-I-infected thyroid cells proliferated more actively than control cells.

The HTLV-I proviral load is thought to be a major determinant of HTLV-I-associated diseases. The proviral load is higher in the peripheral blood of patients with HAM/TSP than blood of asymptomatic carriers (21), as confirmed in the present study. It is also higher in the peripheral blood of patients with HTLV-I-associated uveitis and HTLV-I-seropositive patients with arthritis or connective tissue disease than asymptomatic carriers (39, 40). Similarly, we observed a significantly higher proviral load in HTLV-I-infected patients with either HT or GD than in HTLV-I asymptomatic carriers. Thus, a high proviral load might be involved in the pathogenesis of several other HTLV-I-associated inflammatory disorders in addition to HAM/TSP.

The unusually high proviral loads in HTLV-I infection results mainly from the Tax-driven activation and expansion of infected cells (41). The HTLV-I targets are mainly CD45RO-expressing CD4+ T lymphocytes, and the proviral load is reported to correlate with the number of memory T cells (42). Migration of HTLV-I-infected CD4+ T cells and HTLV-I-specific CD8+ cytotoxic T lymphocytes into the central nervous system is a critical step in the pathogenesis of HAM/TSP (43). Similarly, infiltration of lymphocytes plays a central role in the initiation and perpetuation of autoimmune thyroid diseases. Previous studies showed a good correlation between the degree of intrathyroidal lymphocytic infiltration and antithyroid antibody titer not only in HT (44) but also in GD (45). Although the accumulation of HTLV-I-infected T cells in the thyroid remains uncertain, HTLV-I proviral load did not correlate with antibody titer of either TPO or Tg in our study with HT. Further research using thyroid tissue from HTLV-I-infected patients is needed to support the hypothesis of the pathogenic involvement of HTLV-I-infected T lymphocytes.

HTLV-I might be transmitted from infiltrated lymphocytes to thyrocytes. We obtained evidence that thyroid cells can be infected by HTLV-I and that this infection induced gene expression of inflammatory cytokine IL-6 in vitro. HTLV-I Tax mRNA was detected in the FRTL-5 cells cocultured with MT-2 cells. Transcription of IL-6 is regulated by Tax protein in T cells and synovial cells (46, 47). Although the precise role of IL-6 in the pathogenesis of thyroid diseases is unknown, these results suggest the involvement of IL-6 expression in thyroid cells, which is related to Tax, in the development of inflammatory lesions caused by HTLV-I infection in the thyroid. To clarify the pathological association of thyroiditis with HTLV-I, we are attempting to detect HTLV-I proviral DNA and viral gene expression in the tissue of HTLV-I-associated thyroiditis.

The effect of HTLV-I infection on FRTL-5 growth was assessed by the WST-8 assay. Coculture with MT-2 cells increased the rate of cell proliferation. Because these effects were not observed in FRTL-5 cells cocultured with CCRF-CEM cells, they support the specific effect of HTLV-I infection on thyroid cell growth. Although several cytokines are known to modulate the proliferation of FRTL-5 cells, IL-6 had no significant effects on the cell growth (48). Because Tax can stimulate cell growth, the active proliferation of HTLV-I-infected thyroid cells may be related to Tax expression, and goiter in patients with autoimmune thyroid diseases may be regulated by HTLV-I infection.

HTLV-I might cause a systemic immune-mediated inflammatory disease potently involving tissues other than the central nervous system, HAM/TSP being only the major syndrome. The pathological association of HTLV-I with autoimmune thyroid diseases in HTLV-I carriers still remains to be clarified. It should be noted that HTLV-I infection is not the sole cause of autoimmune thyroid diseases because HTLV-I antibody was not present in the majority of the cases. Genetic factors, involved in autoimmune thyroid diseases, include human leukocyte antigen and cytotoxic T lymphocyte antigen-4 (CTLA-4) (49, 50). It has been shown that HTLV-I infection is not associated with CTLA-4 polymorphisms in either HT or controls (51). HTLV-I infection is not regulated by genetic factor such as CTLA-4 and may affect occurrence of HT as an independent, purely environmental factor. Further studies on the effects of HTLV-I infection of thyroid tissues should help elucidate the pathobiology and pathogenesis of HTLV-I-associated thyroid diseases.


   Acknowledgments
 
We are indebted to the HTLV-I carriers, patients with HT, GD, HAM/TSP, and ATL, and the control subjects who provided blood samples for these studies. We also thank Dr. Hiroyuki Namba, Nagasaki University (Nagasaki, Japan) for providing the FRTL-5 cell line.


   Footnotes
 
This work was supported in part by a Grant-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science.

First Published Online August 2, 2005

Abbreviations: ATL, Adult T cell leukemia; CTLA-4, cytotoxic T lymphocyte antigen-4; FBS, fetal bovine serum; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GD, Graves’ disease; HAM/TSP, human T cell leukemia virus type I-associated myelopathy/tropical spastic paraparesis; HT, Hashimoto’s thyroiditis; HTLV-I, human T cell leukemia virus type I; MMC, mitomycin C; PBMC, peripheral blood mononuclear cell; Tg, thyroglobulin; TPO, thyroid peroxidase; TRAb, TSH receptor antibody; WST, water-soluble tetrazolium salt.

Received March 28, 2005.

Accepted July 25, 2005.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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