Hormonal change and cytokine mRNA expression in peripheral blood mononuclear cells during the development of canine autoimmune thyroiditis

Abstract

To elucidate the hormonal change and alteration in cytokine expression in peripheral blood mononuclear cells (PBMC) during the early stage of autoimmune thyroiditis, we have developed a canine model of this disease, in which normal dogs were immunized with bovine thyroglobulin (Tg) and/or canine thyroid extract. Serum samples were collected weekly, anti-canine Tg antibody was measured by enzyme-linked immunosorbent assay (ELISA) and thyroid stimulating hormone (TSH) and total T4 levels by radioimmunoassay. We also assayed T lymphocyte proliferation in response to Tg, as well as measuring cytokine mRNA by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). All six dogs immunized with bovine Tg had both canine Tg autoantibody and anti-T4 antibody. When the sample from the highest TgAA titre time-point was compared with baseline the expression of mRNA encoding the Th1-type cytokine such as interferon (IFN)-gamma, interleukin (IL)-18 and IL-15 was increased during the development of autoimmune thyroiditis. Expression of the Th2-type cytokine, IL-6 showed minimal change and IL-4 expression was not detected in any of the samples. Expression of the T suppressive cytokine, IL-10 and transforming growth factor (TGF)-beta was increased in the presence of antigen stimulation. These findings suggest that, although autoimmune thyroiditis is an organ-specific autoimmune disease, systemic cytokine mRNA expression is also changed.

Fig. 1

Detection of canine thyroglobulin autoantibody titres after immunization by the thyroid extract and/or bovine thyroglobulin. Serum samples were collected weekly and anti-canine thyroglobulin autoantibody measured by a commercially available enzyme-linked immunosorbent assay kit. The thyroglobulin autoantibody (TgAA) value was determined by calculation according to the manufacturer's instruction. The sample was considered to be positive when the value was > 25% for the TgAA assay. The value < 10% was counted as negative. Following the primer and first booster injections with bovine thyroglobulin, all seven experimental dogs [thyroid extract (Te), bovine thyroglobulin (Tg) and bovine thyroglobulin and thyroid extract (Bo) group] were positive for TgAA and maintained high antibody titres thereafter. Arrows indicate antigen injections (C: control dogs).

Fig. 2

The change of total T4 during the development of autoimmune thyroiditis. Beginning on day 24, a marked increase in total T4 was noted in five dogs [bovine thyroglobulin and thyroid extract (Bo) 1, 2 and bovine thyroglobulin (Tg) 1–3; Fig. 2]. The average of maximal total T4^a levels of these dogs was 52·2 ± 40·2 µg/dl (mean ± s.d.). Arrows indicate antigen injections. ^aReference range of T4: 0·73–2·9 µg/dl.

Fig. 3

The change of thyroid stimulating hormone (TSH) during the development of autoimmune thyroiditis. The TSH^a did not change significantly in any of the experimental groups. Arrows indicate antigen injections. (Bo: dogs immunized with bovine thyroglobulin and thyroid extract, Tg: dogs immunized with bovine thyroglobulin, Te: dog immunized with thyroid extract, Cn: normal control dog). ^aReference range of TSH: < 0·6 ng/ml.

Fig. 4

T cell proliferation against thyroglobulin. T lymphocytes obtained from dogs immunized with the thyroglobulin and/or thyroid extract (EAT) when cultured in the presence of bovine thyroglobulin showed significant proliferative response when compared with lymphocytes obtained from control dogs (P < 0·05).

Fig. 5

Comparison of cytokine mRNA expression in dogs with experimentally induced autoimmune thyroiditis. Interleukin (IL)-15 and transforming growth factor (TGF)-β were increased significantly (P < 0·05) when the RNA samples at the point of the highest thyroglobulin autoantibody (TgAA) titre were compared with baseline.

Fig. 6

Histopathology of thyroids. On day 128, the thyroid glands were resected surgically from dogs immunized with bovine thyroglobulin with/without thyroid extract and stained with haematoxylin and eosin (H&E) using routine histological technique. Immunoperoxidase staining of frozen sections was also performed. (a) Control normal gland (× 200, H&E); (b) mild infiltration of plasma cells and lymphocytes ( × 400, H&E); and (c) mild follicular atrophy (× 200, H&E) were observed in dogs with experimentally induced autoimmune thyroiditis. (d) CD4⁺ T cells infiltrating between follicles (× 400, immunoperoxidase staining); and (e) CD8⁺ T cells infiltrating between follicles (× 400, immunoperoxidase staining) were observed in dogs with experimentally induced autoimmune thyroiditis.