Autoimmune and non-autoimmune thyroid diseases have different patterns of cellular HLA class II expression

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 antigenpresenting cells and the capillary endothelium. 1


INTRODUCTION
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 antigenpresenting cells and the capillary endothelium.

METHODS
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. 8Monoclonal 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.

RESULTS
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)

DISCUSSION
This study has shown that various benign thyroid diseases may express HLA class II, in agreement with other authors. 6However, 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 nonautoimmune 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. 9In 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. 6,7,10In 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. 12,13These 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 clearcut differences in the patterns of HLA-DR expression in autoimmune and non-autoimmune thyroid diseases.