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

Abstract

SummaryTo 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)-g, 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)-b 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.

The effect of gene therapy using CTLA4Ig/silica‐nanoparticles on canine experimental autoimmune thyroiditis

“…According to a previous study on cytokine mRNA expression from fresh PBMC, mRNA expression of TGF‐β1 and Th 1‐type cytokines such as IL‐18, IFN‐γ and IL‐15 increases during the development of canine autoimmune thyroiditis 3. The CTLA4Ig‐induced suppression of Th1‐type cytokines found in the present in vivo study is relatively more significant than it appears because autoimmune thyroiditis is a Th1‐polarized disease not only in the thyroid, but also in peripheral blood (i.e.…”

“…We induced experimental autoimmune thyroiditis in six normal dogs by injecting them with bovine Tg according to methods described previously 3.…”

“…Total RNA from 2‐day‐cultured PBMC was extracted by TRIzol (Invitrogen) and reverse transcriptase‐polymerase chain reaction (RT‐PCR) was carried out using a RT‐PCR PreMix Kit (iNtRON Biotechnology, Sungnam, South Korea). The primers sequences and optimal PCR annealing temperatures and cycle numbers used were validated for use in our previous study 3. The amount of DNA was quantitated as band intensities using GelDoc software from Bio‐Rad (Hercules, CA, USA).…”

“…Thyroid glands were removed from dogs with EAT on day 42 after gene therapy or no therapy and cryosectioned and immunostained for CD4 and CD8 or sectioned and stained with hematoxylin and eosin (H&E), and then examined with the aid of light microscope 3.…”

“…Hypothyroidism primarily results from the individual's own immune system attacking the tissues of their thyroid gland, a condition known as autoimmune thyroiditis. We have examined the hormonal changes and cytokine profiles of autoimmune thyroiditis using a canine model of the condition, which we established by immunizing normal dogs with bovine thyroglobulin (Tg) 3. It has been reported that CTLA4, a gene that encodes a negative regulator of the T‐lymphocyte immune response, is associated with autoimmune thyroid disease 4.…”