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Increased Serum Concentration of Soluble CD30 in Patients with Graves’ Disease and Hashimoto’s Thyroiditis¹

Department of Laboratory Medicine, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka 565, Japan

Address all correspondence and requests for reprints to: Yoh Hidaka M.D., Department of Laboratory Medicine, Osaka University Medical School, 2–2 Yamadaoka, Suita, Osaka 565, Japan.

	Abstract

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This study investigated serum levels of the soluble form of CD30 (sCD30), which is mainly secreted from T helper 2(Th2) cells, in autoimmune thyroid diseases. The possible relationship of sCD30 to autoantibody production was also evaluated. Serum levels of sCD30 were determined by an enzyme-linked immunosorbent assay in 71 patients with Graves’ disease, 37 patients with Hashimoto’s thyroiditis, and 21 normal donors. Compared with normal subjects (7.1 ± 4.5 U/mL), sCD30 was increased in patients with Graves’ disease (29.2 ± 25.2 U/mL, P < 0.0001) and in patients with Hashimoto’s thyroiditis (29.9 ± 26.9 U/mL, P < 0.0001). In Graves’ disease, sCD30 levels were higher in thyrotoxic patients (41.7 ± 31.2 U/mL, P < 0.001) than in remission patients (15.8 ± 11.0 U/mL), and a significant correlation was observed between sCD30 levels and serum activities of TSH receptor antibody (r = 0.444, P < 0.0001). In Hashimoto’s thyroiditis, sCD30 levels were higher in patients with transient destructive thyrotoxicosis caused by the aggravation of the disease (48.8 ± 34.4 U/mL, P < 0.05) than in euthyroid patients (24.2 ± 19.4 U/mL). These data suggest that serum sCD30 is a valuable marker of disease activity and support an important role of the Th2-type immune response in the pathogenesis in Graves’ disease and Hashimoto’s thyroiditis.

	Introduction

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DIFFERENTIATED human CD4⁺ T lymphocytes can be subdivided into functionally distinct subsets on the basis of their cytokine secretion profile. T helper 1(Th1) cells produce interleukin 2 (IL-2), interferon (IFN-), and lymphotoxin and induce cellular responses. Th2 cells secrete IL-4, IL-5, IL-6, IL-9, IL-10, and IL-13 and promote production of antibodies (1). Graves’ thyrotoxicosis is induced by thyroid stimulating autoantibodies to TSH receptor (2), the production of which is likely to depend upon Th2 cell function. Th2 response may play an important role in the pathophysiology of the thyroid destructive process in Hashimoto’s thyroiditis because anti-microsomal autoantibodies damage thyrocytes in vitro (3, 4), although main cytotoxic activity might be induced by cell-mediated immunity (5, 6).

The CD30 molecule, a member of the tumor necrosis factor (TNF)/nerve growth factor (NGF) receptor superfamily (7), is inducible on the membrane of some T cells that secrete Th2-type lymphokines (8). After CD30 appears on the activated T cell surface, the extracellular portion of the CD30 molecule is proteolytically cleaved to produce an 88-kDa soluble form (sCD30), which is released by cells expressing CD30 (9).

In this study, we demonstrated that increased levels of sCD30 are present in patients with Graves’ disease and Hashimoto’s thyroiditis, especially in the active stages, and we observed a significant correlation between sCD30 levels and TSH receptor antibody levels.

	Subjects and Methods

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Subjects

We studied 71 patients with Graves’ disease, 37 patients with Hashimoto’s thyroiditis, and 21 normal controls. Patients with Graves’ disease were divided into four groups; (1) 21 untreated thyrotoxic patients with positive TSH receptor antibody (TSH-R Ab) (37.2 ± 13.8 yr old, mean ± SD); (2) 8 euthyroid patients under maintenance treatment with antithyroid drugs (methimazole 5 mg or propylthiouracil 50 mg per day) with positive TSH-R Ab (39.4 ± 16.2 yr old); (3) 21 euthyroid patients under maintenance treatment with antithyroid drugs with negative TSH-R Ab (36.1 ± 12.6 yr old); and (4) 21 euthyroid patients in remission (43.1 ± 12.8 yr old). They were not treated with surgery or radioiodine. Patients with Hashimoto’s thyroiditis were divided into three groups; (1) 8 thyrotoxic patients (30.3 ± 3.8 yr old); (2) 21 untreated euthyroid patients (38.4 ± 10.1 yr old); and (3) 8 hypothyroid patients (43.8 ± 11.2 yr old). The thyrotoxicosis in patients with Hashimoto’s thyroiditis was caused by destruction of thyrocytes or thyroid follicles and was diagnosed by the increased levels of serum thyroid hormones for less than 3 months, later development of transient hypothyroidism, and/or low radioactive iodine uptake. The mean age in each group was not significantly different from that of normal controls (37.0 ± 12.0 yr old). All were Japanese females. This study was approved by the institution’s ethics committee, and all the patients gave informed consent before participation in this study.

sCD30 assay

Detection of sCD30 was performed on serum samples kept frozen at -20 C, using a sandwich enzyme-linked immunosorbent assay (CD30, Ki-1 antigen, ELISA: DAKO, Glostrup, Denmark), which is based on the use of two monoclonal antibodies reacting with two different epitopes on the 88-kDa soluble form of the CD30 molecule, as previously described (10). Briefly, predicted sCD30 calibrators, a curve control, and patient specimens were added to peroxidase-conjugated mouse anti-CD30 in polystyrene microtiter wells precoated with another CD30 monoclonal antibody. After a 2-h incubation and washing to remove unbound material, a chromogenic substrate was added to the wells. The reaction was then stopped, and absorbance at 450 nm was measured. The standard curve was prepared from six sCD30 calibrators (0, 5, 16, 41, 101, 238 U/mL), and the concentration of sCD30 in serum samples was determined by interpolation. The intraassay coefficient of variation was 2.4%, and the interassay coefficient of variation was 3.7%. The detection limit of the assay was 5 U/mL.

Thyroid autoantibodies

Serum levels of TSH-R Ab were measured by a radioreceptor assay with a commercial kit (Cosmic Corp., Tokyo, Japan) (11). The results were expressed as percent inhibition of binding of labeled TSH. The normal value was less than 12%. Serum levels of antibodies to thyroperoxidase (TPO) and thyroglobulin (TG) were measured using commercial enzyme immunoassay kits (Boehringer Mannheim, Mannheim, Germany). Normal values of TPO antibody and TG antibody were less than 16.0 IU/mL and less than 86.4 IU/mL, respectively.

Statistical methods

Five U/mL were assigned to the value less than 5 U/mL in the statistical calculation of sCD30. The Mann-Whitney test was used to compare sCD30 levels between different groups. Linear correlation analysis was used to examine the correlation between sCD30 levels and other immunological parameters.

	Results

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sCD30 serum levels

Serum levels of sCD30 were increased in patients with Graves’ disease (n = 71, 29.2 ± 25.2 U/mL, mean ± SD, P < 0.0001), and Hashimoto’s thyroiditis (n = 37, 29.9 ± 26.9 U/mL, P < 0.0001) compared with those in normal controls (n = 21, 7.1 ± 4.5 U/mL). In Graves’ disease, the mean value of sCD30 in 21 thyrotoxic patients was 41.7 ± 31.2 U/mL, that in 8 euthyroid patients with positive TSH-R Ab was 38.4 ± 26.6 U/mL, that in 21 euthyroid patients with negative TSH-R Ab was 26.6 ± 22.1 U/mL, and that in 21 remission patients was 15.8 ± 11.0 U/mL (Fig. 1). Serum levels of sCD30 in thyrotoxic patients (P < 0.001) and in euthyroid patients with positive TSH-R (P < 0.05) were significantly higher than those in remission patients. In Hashimoto’s thyroiditis, the mean value of sCD30 in 8 thyrotoxic patients was 48.8 ± 34.4 U/mL, and that in 21 euthyroid patients was 24.2 ± 19.4 U/mL, and that in 8 hypothyroid patients was 25.8 ± 30.5 U/mL (Fig. 1). Serum levels of sCD30 in thyrotoxic patients were significantly (P < 0.05) higher than those in euthyroid patients.

View larger version (20K): [in this window] [in a new window]   Figure 1. Serum levels of sCD30 in patients with Graves’ disease and Hashimoto’s thyroiditis. The patients with Graves’ disease were divided into four groups; (1) untreated thyrotoxic patients; (2) euthyroid patients under antithyroid drug therapy with positive TSH-R Ab; (3) euthyroid patients under antithyroid drug therapy with negative TSH-R Ab; (4) euthyroid patients in remission. The patients with Hashimoto’s thyroiditis were divided into three groups; (1) thyrotoxic patients; (2) euthyroid patients; (3) hypothyroid patients. *** P < 0.0001, ** P < 0.001, * P < 0.05.

Significant correlation was observed between individual values of serum sCD30 and serum activities of TSH receptor antibody (r = 0.444, P < 0.0001) in 71 patients with Graves’ disease (Fig. 2). There was no significant correlation between individual values of serum sCD30 and serum titers of TPO antibody nor TG antibody in 37 patients with Hashimoto’s thyroiditis (data not shown).

View larger version (19K): [in this window] [in a new window]   Figure 2. Correlation between individual values of serum sCD30 and serum TSH receptor antibody (TSH-R Ab) in 71 patients with Graves’ disease.

	Discussion

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Many autoimmune endocrinopathies appear to be directly mediated by autoreactive CD8⁺ cytotoxic T lymphocytes with help from Th1 CD4⁺ cells, while others appear to be mediated by autoantibodies to cellular receptors that arise as part of the Th2 response. Mullins et al. (18) reported that TSH receptor-specific T cell clones, made from the intrathyroidal lymphocytes of a Graves’ patient, showed Th0/Th2 characteristics. Heuer et al. (19) reported that the thyroid tissue from Graves’ patients showed a shift to a more Th2-driven cytokine pattern as determined by reverse transcriptase polymerase chain reaction. Recently, we found that allergic rhinitis not only aggravated Graves’ disease but also induced the clinical onset of Graves’ thyrotoxicosis, suggesting that Th2-derived cytokines, produced in type I allergy, help to produce TSH-R antibody (20, 21). This study shows that serum levels of sCD30 were increased in Graves’ disease and that significant correlation was observed between serum levels of sCD30 and TSH receptor antibody. Del Prete et al. (8) showed that CD30 is preferentially expressed on Th cells producing Th2-type cytokines, and that CD30 acts as a costimulatory molecule whose cross-linking is able to promote both expansion and effector function of Th2-like cells (22). These findings therefore suggest the in vivo involvement of Th2 cells in the regulation of immunological processes in Graves’ disease. The natural ligand for CD30 (CD30L) has been cloned (23), and the blockage of CD30L-CD30 interaction by anti-CD30L favored the development of higher numbers of Ag-specific Th cells showing the opposite (Th1-like) phenotype (22). This kind of molecule might be used to down-regulate undesired Th2 responses in Graves’ disease, and other autoimmune diseases.

Cytotoxic T lymphocytes specific for thyroid epithelial cells can be cloned from intrathyroidal lymphocytes in Hashimoto’s thyroiditis (5). The proportion of intrathyroidal CD8⁺ CD11b^- cytotoxic T cells is high in Hashimoto’s thyroiditis (6). These findings suggest a potential role of cytotoxic T lymphocytes in the thyroid damage caused by Hashimoto’s thyroiditis. On the contrary, anti-microsomal autoantibodies may damage thyrocytes via activation of the complement system and antibody-dependent cell-mediated cytolysis (ADCC) (3, 4). This study has also shown that serum sCD30 increased in Hashimoto’s thyroiditis, especially in patients with active Hashimoto’s thyroiditis who show transient thyrotoxicosis caused by destruction of thyrocytes or thyroid follicles by immunological cytotoxic factors (destructive thyrotoxicosis). These findings suggest that not only Th1 cells but also Th2 cells are involved in the destruction of the thyroid gland in Hashimoto’s thyroiditis.

	Footnotes

 
¹ This study was supported by grants from a Grant-in-Aid for Scientific Research (to Dr. Amino; No. 07457616) from the Ministry of Education, Science, and Culture of Japan; grants from the Japan Foundation for Health Science and from the Intractable Disease Division of the Public Health Bureau, Ministry of Health and Welfare; and (to Dr. Hidaka) a grant from the Senri Life Science Foundation and the Clinical Pathology Research Foundation of Japan.

Received December 16, 1996.

Revised February 13, 1997.

Accepted February 27, 1997.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

1. Mosmann TR, Sad S. 1996 The expanding universe of T-cell subsets: Th1, Th2 and more. Immunol Today. 17:138–146.[CrossRef][Medline]
2. Burman KD, Baker JJ. 1985 Immune mechanisms in Graves’ disease. Endocr Rev. 6:183–232.[Medline]
3. Chiovato L, Bassi P, Santini F, et al. 1993 Antibodies producing complement-mediated thyroid cytotoxicity in patients with atrophic or goitrous autoimmune thyroiditis. J Clin Endocrinol Metab. 77:1700–1705.[Abstract]
4. Bogner U, Schleusener H, Wall JR. 1984 Antibody-dependent cell mediated cytotoxicity against human thyroid cells in Hashimoto’s thyroiditis but not Graves’ disease. J Clin Endocrinol Metab. 59:734–738.[Abstract]
5. MacKenzie WA, Schwartz AE, Friedman EW, Davies TF. 1987 Intrathyroidal T cell clones from patients with autoimmune thyroid disease. J Clin Endocrinol Metab. 64:818–824.[Abstract]
6. Iwatani Y, Hidaka Y, Matsuzuka F, Kuma K, Amino N. 1993 Intrathyroidal lymphocyte subsets, including unusual CD4+ CD8+ cells and CD3loTCR alpha beta lo/-CD4-CD8- cells, in autoimmune thyroid disease. Clin Exp Immunol. 93:430–436.[Medline]
7. Durkop H, Latza U, Hummel M, Eitelbach F, Seed B, Stein H. 1992 Molecular cloning and expression of a new member of the nerve growth factor receptor family that is characteristic for Hodgkin’s disease. Cell. 68:421–427.[CrossRef][Medline]
8. Del Prete G, De Carli M, Almerigogna F, et al. 1995 Preferential expression of CD30 by human CD4+ T cells producing Th2-type cytokines. FASEB J. 9:81–86.[Abstract]
9. Falini B, Pileri S, Pizzolo G, et al. 1995 CD30 (Ki-1) molecule: a new cytokine receptor of the tumor necrosis factor receptor superfamily as a tool for diagnosis and immunotherapy. Blood. 85:1–14.[Free Full Text]
10. Pizzolo G, Vinante F, Morosato L, et al. 1994 High serum level of the soluble form of CD30 molecule in the early phase of HIV-1 infection as an independent predictor of progression to AIDS. AIDS. 8:741–745.[Medline]
11. Tamaki H, Amino N, Aozasa M, Mori M, Tanizawa O, Miyai K. 1987 Serial changes in thyroid-stimulating antibody and thyrotropin binding inhibitor immunoglobulin at the time of postpartum occurrence of thyrotoxicosis in Graves’ disease. J Clin Endocrinol Metab. 65:324–330.[Abstract]
12. Schwab U, Stein H, Gerdes J, et al. 1982 Production of a monoclonal antibody specific for Hodgkin and Sternberg-Reed cells of Hodgkin’s disease and a subset of normal lymphoid cells. Nature. 299:65–67.[CrossRef][Medline]
13. Nawrocki JF, Kirsten ES, Fisher RI. 1988 Biochemical and structural properties of a Hodgkin’s disease-related membrane protein. J Immunol. 141:672–680.[Abstract]
14. Josimovic-Alasevic O, Durkop H, Schwarting R, Backe E, Stein H, Diamantstein T. 1989 Ki-1 (CD30) antigen is released by Ki-1-positive tumor cells in vitro and in vivo. I. Partial characterization of soluble Ki-1 antigen and detection of the antigen in cell culture supernatants and in serum by an enzyme-linked immunosorbent assay. Eur J Immunol. 19:157–162.[Medline]
15. Nadali G, Vinante F, Ambrosetti A, et al. 1994 Serum levels of soluble CD30 are elevated in the majority of untreated patients with Hodgkin’s disease and correlate with clinical features and prognosis. J Clin Oncol. 12:793–797.[Abstract]
16. Gerli R, Muscat C, Bistoni O, et al. 1995 High levels of the soluble form of CD30 molecule in rheumatoid arthritis (RA) are expression of CD30+ T cell involvement in the inflamed joints. Clin Exp Immunol. 102:547–550.[Medline]
17. Caligaris-Cappio F, Bertero MT, Converso M, et al. 1995 Circulating levels of soluble CD30, a marker of cells producing Th2-type cytokines, are increased in patients with systemic lupus erythematosus and correlate with disease activity. Clin Exp Rheumatol. 13:339–343.[Medline]
18. Mullins RJ, Cohen SB, Webb LM, et al. 1995 Identification of thyroid stimulating hormone receptor-specific T cells in Graves’ disease thyroid using autoantigen-transfected Epstein-Barr virus-transformed B cell lines. J Clin Invest. 96:30–37.
19. Heuer M, Aust G, Ode-hakim S, Scherbaum WA. 1996 Different cytokine mRNA profiles in Graves’ disease, Hashimoto’s thyroiditis, and nonautoimmune thyroid disorders determined bu quantitative reverse transcriptase polymerase chain reaction (RT-PCR). Thyroid. 6:97–106.[Medline]
20. Hidaka Y, Amino N, Iwatani Y, Itoh E, Matsunaga M, Tamaki H. 1993 Recurrence of thyrotoxicosis after attack of allergic rhinitis in patients with Graves’ disease. J Clin Endocrinol Metab. 77:1667–1670.[Abstract]
21. Hidaka Y, Masai T, Sumizaki H, Takeoka K, Tada H, Amino N. 1996 Onset of Graves’ thyrotoxicosis after an attack of allergic rhinitis. Thyroid. 6:347–351.
22. Del Prete G, De Carli M, D’Elios MM, et al. 1995 CD30-mediated signaling promotes the development of human T helper type 2-like T cells. J Exp Med. 182:1655–1661.[Abstract/Free Full Text]
23. Smith CA, Gruss HJ, Davis T, et al. 1993 CD30 antigen, a marker for Hodgkin’s lymphoma, is a receptor whose ligand defines an emerging family of cytokines with homology to TNF. Cell. 73:1349–1360.[CrossRef][Medline]

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