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Sao Paulo Medical Journal

versión impresa ISSN 1516-3180versión On-line ISSN 1806-9460

Sao Paulo Med. J. v.117 n.4 São Paulo Jul. 1999 

Original Article

Denise Engelbrecht Zantut-Wittmann,
Luís Henrique Barbosa Boechat,
Glauce Aparecida Pinto,
Miriam Aparecida da Silva Trevisan,
José Vassallo


Autoimmune and non-autoimmune thyroid diseases have different patterns of cellular HLA class II expression
Service of Endocrinology and Department of Pathologic Anatomy, School of Medical Sciences - State University of Campinas, Campinas, Brazil




CONTEXT: Surface HLA-DR antigen is usually only expressed by antigen-presenting cells (APC). In autoimmune thyroid disease, follicle cells function as APC, thus expressing HLA-DR. However, non-autoimmune thyroid diseases may also express surface class II antigens.
OBJECTIVE: To evaluate the presence and pattern of HLA class II expression in autoimmune and non-autoimmune thyroid disorders.
DESIGN: Retrospective: histopathological and immunohistochemical analysis.
LOCATION: Referral center, university hospital.
SAMPLE: Ten histologically normal thyroids, 11 Graves’ disease, 7 Hashimoto’s thyroiditis, 10 atoxic multinodular goiter and 3 toxic adenomas were analyzed by immunohistochemistry, using a monoclonal antibody anti-HLA-DR.
MAIN MEASUREMENTS: The presence of these antigens in thyroid follicular cells and their relation to inflammatory infiltrate was evaluated. The pattern of HLA-DR expression in thyroid follicular cells was analyzed: membrane, cytoplasmic or both.
RESULTS: Although HLA-DR antigens were sparsely present in one of the 8 normal thyroids, in 6 of the 9 atoxic multinodular goiter and in 2 of the 3 toxic adenomas a net positivity could be seen in large areas. In all 5 Hashimoto’s thyroiditis and in 7 of the 10 Graves’ disease cases. This expression occurred in follicle cells either in contact with inflammatory cells or not. In non-autoimmune thyroid disease, HLA-DR positivity was essentially cytoplasmic, whereas in Graves’ disease and Hashimoto thyroiditis it was mainly in cell membranes.
CONCLUSIONS: It is suggested that the HLA class II expression on the surface of follicle cells could be related to auto-antigen presentation to the immune system by these cells, leading to inflammation.
KEY-WORDS: Graves’ disease. Autoimmune thyroiditis. HLA-DR antigens. Immunohistochemistry




Class II antigens of the human major histocompatibility complex (MHC), the human leukocyte antigens (HLA) DP, DQ and DR, are central elements in the presentation of exogenous antigens to T CD4+ lymphocytes and the regulation of immune response. Expression of these glycoproteins on the cell surface is restricted to B lymphocytes, macrophages, other antigen-presenting cells and the capillary endothelium.1 Some authors described aberrant HLA class II expression antigens in follicular thyroid cells in Graves’ disease (GD)2 and Hashimoto thyroiditis (HT)3. Cell cultures from normal thyroids or GD cases generally express HLA-DR in follicles near areas infiltrated by lymphocytes, when stimulated by g-interferon.2-4 These cells probably present self-antigens to the immune system and perpetuate the autoimmune process.5 HLA class II expression has also been observed in some normal thyroids, atoxic multinodular goiter (AMG), thyroid adenomas and carcinomas.6,7 As there has been no report on the topographical pattern of HLA-DR expression in thyrocytes, the aim of the present study was to detail this aspect in normal, autoimmune and non-autoimmune thyroid tissue, using the immunoperoxidase technique.



Thyroid surgical specimens from 8 normal individuals, 10 with GD, 5 with HT, 9 with AMG and 3 with toxic adenoma (TA) were fixed in 10% formalin, embedded in paraffin, stained with hematoxylin and eosin and selected for immunohistochemical techniques. Specific immunostaining was performed to identify HLA-DR using a modified streptavidin-biotin-peroxidase technique.8 Monoclonal primary anti-HLA-DR antibody (Dako), was diluted at 1:10 in 0.1% BSA. Detection of the streptavidin-biotin-peroxidase complex was achieved with diaminobenzidine and slight counterstaining with hematoxylin. At the end of the reaction, slides were mounted with Entellan (Merck). Immunostained sections were scored for the expression of HLA-DR using the following criteria: a) presence of HLA-DR positive follicular cells in contact with inflammatory infiltrate; b) presence of DR positive follicular cells in the absence of inflammatory infiltrate; c) presence of DR positive inflammatory infiltrate and d) presence of DR positive inflammatory infiltrate in the absence of DR positive follicular cells.

The pattern of HLA-DR expression in follicular cells was classified as: 1) restricted to the cell membrane; 2) restricted to the cytoplasm and 3) in both, cell membrane and cytoplasm.



HLA-DR expression in one of the 8 normal thyroid tissue cases, 6 of the 9 AMG and 2 of the 3 TA was restricted to small scattered groups of follicular cells. However, in 7 of the 10 GD cases and especially in all 5 HT cases, expression was more diffuse and extensive, generally observed in close relation to a lymphoplasmacytic infiltrate. In addition, in 6 GD and 4 HT cases, positive follicles were also found outside these areas.

The pattern of HLA-DR expression in follicular cells in AMG (5 of 6) and in TA (2 of 2) could only be verified restricted to the cytoplasm. In one normal thyroid that presented a small group of DR positive follicular cells, the expression was in the membrane. In GD, HLA-DR was always expressed in the membrane (7 of 7) and in 2 cases it was also observed in the cytoplasm. In 5 of the 5 HT cases, the positive membrane staining was more intense and was accompanied by cytoplasmic expression in 4 of the 5. (Figure 1, Table 1)


n4a3f1.gif (42089 bytes)

Figure 1 - A: In Graves’ disease, HLA-DR antigen is expressed on the cell membrane (anti-HLA-DR, Streptavidin-biotin peroxidase reaction, 1000x). B: In contrast, in a case of toxic adenoma cytoplasmic pattern predominates (anti-HLA-DR, StreptABP reaction, 564x).



n4a4t1.gif (3889 bytes)



This study has shown that various benign thyroid diseases may express HLA class II, in agreement with other authors.6 However, the pattern of expression was different when autoimmune and non-autoimmune thyroid diseases were compared. Only in autoimmune processes was there significant, extensive membrane expression of HLA-DR whereas in non-autoimmune processes it was generally limited to the cytoplasm.

The genes of the human MHC situated in the HLA region are related to the immune response to protein antigens. Thus, CD4+ or CD8+ T lymphocytes only recognize peptides processed by antigen-presenting cells, the macrophages, B lymphocytes, capillary endothelium and dendritic cells, when associated with products of the MHC genes expressed on the cell surface.9 In the 1980’s the inadequate expression of HLA class II in follicular cells of GD and HT cases was demonstrated, most frequently in areas close to the lymphoplasmacytic infiltrate. This suggests that these thyrocytes could play a role in the development and perpetuation of the autoimmune disturbance.6,7,10 In agreement with these data, in our study a significant number of positive cases (12 out of 21) exhibited areas of follicular cells expressing HLA-DR in the presence of inflammatory cells. In 19 of these 21 cases there was also HLA-DR expression in areas distant from the inflammatory infiltrate.

Subsequent reports have shown that follicular cells in AMG, TA and carcinomas also express HLA-DR, especially in relation to foci of inflammatory infiltrate. Studies using primary cultures of normal thyroid cells, GD and other thyroid diseases, have demonstrated that thyrocytes in autoimmune diseases could function as antigen-presenting cells, which apparently does not occur in non-autoimmune processes.6,11 In other investigations using cultures of thyroid murine cells or FRTL-5, it was observed that these cells can express the class II MHC molecules, but were unable to present antigens.12,13 These conflicting results may reflect contamination of the thyroid cell cultures by dendritic cells, which are abundant in this tissue and are powerful antigen-presenting cells.14

Our findings indicate a fundamental difference in the pattern of HLA class II expression in the follicular cells. Thus, in non-autoimmune thyroid diseases, such as AMG and TA, this expression was almost exclusively observed in cell cytoplasm, and in GD and HT, HLA-DR was expressed on the cell membrane in all cases irrespective of the presence of cytoplasmic expression. The pattern of HLA-DR expression on the cell membrane in autoimmune thyroid diseases confirms that these thyrocytes play a role in antigenic presentation. On the other hand, in cells of non-autoimmune thyroid diseases such expression may reflect a general activation of the mechanisms of cell proliferation and such activation may not result in hormone overproduction, since it was also observed in AMG.5,6,12,13,15

Our results provide evidence that, independent of the thyroid function, class II MHC products are expressed on the surface of thyrocytes in GD and HT, and that there are clear-cut differences in the patterns of HLA-DR expression in autoimmune and non-autoimmune thyroid diseases.



1- Meuer SC, Schossman SF, Reinherz EL. Clonal analysis of human cytotoxic T Lymphocytes: T4+ and T8+ effector T cells recognize products of different major histocompatibility complex region. Proc Nat Acad Sci USA 1982;79:4395-9.        [ Links ]

2- Hanafusa T, Pujol-Borrel R, Chiovato L, Russel RCG, Doniach D, Bottazzo GF. Aberrant expression of HLA-DR antigen on thyrocytes in Graves’ disease: relevance for autoimmunity. Lancet 1983;2:1111-5.        [ Links ]

3- Aichinger G, Fill H, Wick G. In situ immune complexes, lymphocyte subpopulations, and HLA-DR-positive epithelial cells in Hashimoto thyroiditis. Lab Invest 1985;52:132-40.        [ Links ]

4- Iwatani Y, Gerstein HC, ltaka M, Row W, Volpé R. Thyrocyte HLA-DR expression and interferon-gamma production in autoimmune thyroid disease. J Clin Endocrinol Metab 1986;63:695-708.        [ Links ]

5- Faure GC, Bensoussan-Lyzerowicz D, Bene MC, Aubert V, Leclere J. Coexpression of CD40 and class II antigen HLA-DR in Graves’ disease thyroid epithelial cells. Clin Immunol Immunopathol 1997;84:212-5.        [ Links ]

6- Grubeck-Loebenstein B, Londei M, Greenall C, Pirich K, Kassai H, Waldhäusi W, Feldmann M. Pathogenetic relevance of HLA class II expressing thyroid follicular cells in nontoxic goiter and Graves’ disease. J Clin Invest 1988;81:1608-14.        [ Links ]

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8- Hsu SM, Raine L, Fanger H. Use of avidin-biotin-peroxidase complex (ABC) in immunoperoxidase techniques: A comparison between ABC and unlabelled antibody (PAP) procedure. J Histochem Cytochem 1981;29:557-80.        [ Links ]

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11- Pujol-Borrel R, Lucas Martin A, Fox M, Todd I, Bottazzo GF. Inappropriate HLA class II expression in a wide variety of thyroid diseases. J Clin Invest 1986;9:71-8.        [ Links ]

12- Ebner SA, Stein ME, Minami M, Dorf ME, Stadecker MJ. Murine thyroid follicular cells can be induced to express class II (Ia) gene products but fail to present antigen in vitro. Cell Immunol 1987;104:154-68.        [ Links ]

13- Minami M, Ebner AS, Stadecker MJ, Dorf ME. The effects of phorbol ester on alloantigen presentation. J Immunol 1987;138:393-400.        [ Links ]

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15- Weetman AP. New aspects of thyroid immunity. Horm Res 1997;48(suppl 4):51-4.        [ Links ]



Denise Engelbrecht Zantut-Wittmann - Doutora em Medicina Interna - Disciplina de Endocrinologia – FCM-UNICAMP

Luís Henrique Barbosa Boechat - Mestre em Clínica Médica

Glauce Aparecida Pinto - Doutora em Ciências- Departamento de Anatomia Patológica - FCM-UNICAMP

Miriam Aparecida da Silva Trevisan - Livre-Docente - Departamento de Anatomia Patológica - FCM-UNICAMP

José Vassallo - Doutor em Medicina - Departamento de Anatomia Patológica - FCM-UNICAMP


Sources of Funding: Not declared
Conflict of interest: Not declared
Last received: 18 January 1999
Accepted: 8 February 1999
Address for correspondence:
Denise Engelbrecht Zantut-Wittmann
Disciplina de Endocrinologia - Departamento de Clínica Médica - Faculdade de Ciências Médicas da Universidade Estadual de Campinas - PO Box 6111
Campinas/SP - Brasil - CEP 13083-970




CONTEXTO: Normalmente, apenas células apresentadoras de antígenos expressam o HLA-DR em sua superfície. Na doença tiroidiana auto-imune, as células foliculares adquirem o papel de apresentar antígenos e assim expressam o HLA-DR. Entretanto, doenças tiroidianas não auto-imunes também podem expressar antígenos de classe II. OBJETIVO: Avaliar a presença e o padrão de expressão do HLA de classe II em tiróides de pacientes com doenças tiroidianas auto-imunes e não auto-imunes. TIPO DE ESTUDO: Retrospectivo histopatológico e imunohistoquímico. LOCAL: Centro de referência, hospital universitário. AMOSTRA: 10 tiróides histologicamente normais, 11 com doença de Graves, 7 com tiroidite de Hashimoto, 10 com bócio multinodular atóxico e 3 com adenoma tóxico foram analisadas através de imunohistoquímica usando um anticorpo monoclonal anti-HLA-DR. VARIÁVEIS ESTUDADAS: Avaliamos a presença desses antígenos na célula folicular tiroidiana, sua relação com o infiltrado inflamatório e o padrão de expressão do HLA-DR nas células foliculares, se presente na membrana, no citoplasma ou em ambos. RESULTADOS: A expressão do HLA-DR ocorreu focalmente em uma das 8 tiróides normais, 6 dos 9 casos de bócio multinodular atóxico e em 2 dos 3 adenomas tóxicos. Entretanto, foi nítida sua positividade em áreas extensas de células foliculares tiroidianas nos 5 casos de tiroidite de Hashimoto e, em menor grau, em 7 dos 10 casos de doença de Graves, quer em associação ao infiltrado inflamatório, longe dele ou apenas nas células linfóides. Nas doenças tiroidianas não auto-imunes, o padrão de expressão do HLA-DR foi essencialmente citoplasmático, enquanto que na doença de Graves e tiroidite de Hashimoto esta foi verificada na membrana celular, muitas vezes associada à citoplasmática. CONCLUSÕES: Estes fatos sugerem que a expressão do HLA de classe II na superfície das células foliculares estaria relacionada à apresentação dos auto-antígenos ao sistema imunológico por estas células, levando ao processo inflamatório.

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