Elsevier

Clinical Immunology

Volume 121, Issue 1, October 2006, Pages 108-117
Clinical Immunology

By-stander activation in autoimmune thyroiditis: Studies on experimental autoimmune thyroiditis in the GFP+ fluorescent mouse

https://doi.org/10.1016/j.clim.2006.03.011Get rights and content

Abstract

We have taken advantage of GFP+ fluorescent protein (GFP) tagged lymphocytes to examine by-stander activity in experimental autoimmune thyroiditis in the mouse. To generate GFP-positive EAT-susceptible CBA/J mice (H-2k) (GFP-CBA/J mice), we backcrossed CBA/J (H-2k) with heterozygous GFP+ transgenic mice (C57Bl/6; H-2b). I-Ak and GFP expression on peripheral lymphocytes was used to select the resulting progeny up to the N7 generation. Mixed lymphocyte reactions using spleen cells from N7 GFP-CBA/J mice showed negative responses to spleen cells from CBA/J confirming the inbreeding and with marked reactivity to cells from C57BL/6. Immunization with human thyroglobulin (hTg) in GFP-CBA/J mice induced thyroiditis in 50% of the animals and high titers of Tg antibodies in all the animals. In addition, priming of GFP+ spleen cells in vitro with hTg induced a marked proliferative response (mean stimulation index = 24.7), These proliferating spleen cells were then transferred to CBA/J recipients. Fourteen days after transferring 30 × 106 Tg-primed GFP+ spleen cells into irradiated (500 rad) normal syngeneic hosts, a GFP+ lymphocytic infiltration was seen within their thyroid glands along with a GFP lymphocytic infiltration arising from the host. This suggested that the hTg-specific transferred cells had initiated by-stander activation of naive host lymphocytes. This model of bystander cell detection confirmed that such an effect occurs in EAT and adds weight to the importance of this phenomenon in the initiation of autoimmune thyroid disease.

Introduction

Experimental autoimmune thyroiditis (EAT) in the mouse has been well characterized over many years and has served as a useful model for human autoimmune thyroid disease [1], [2]. The most explored model has been that initiated by immunization with the major thyroid protein-thyroglobulin (Tg) [1], [2], although systems using thyroid peroxidase or the TSH receptor have been described [3], [4], [5]. EAT is characterized by a marked intrathyroidal lymphocytic infiltrate which reaches its peak some 4 weeks after immunization of a susceptible strain [1] and subsequently subsides unless immunization is continued. The mixed lymphocytic infiltrate has been shown to have a predominant CD8+ cytotoxic T cell component which results in widespread thyroid cell apoptosis as seen in human disease [6], [7], [8]. Such cytotoxic T cells have been isolated and shown to be able to transfer the disease to naive recipients. However, it has not been possible to examine the naive host bystander-response to such transferred Tg-specific immune cells. Since bystander effects are likely to be of importance in the development of autoimmune disease [9], [10], [11] we have developed a model of EAT utilizing transgenic mice with enhanced GFP expression under the control of an actin promoter. This transgenic mouse, first reported by Okabe et al. [12], exhibits fluorescence by all actin expressing cells. We have previously used such animals in our studies of fetal microchimerism [13] and have now developed an EAT model in these mice.

We first bred the GFP transgene onto an H-2k susceptible strain (CBA/J) and confirmed the onset of thyroiditis following immunization with Tg. We then transferred GFP-expressing (GFP+), Tg-specific, immune cells to naive CBA/J hosts and were able to observe the developing thyroiditis. Histological examination revealed intense GFP+ lymphocyte infiltration within the thyroid glands of the naive hosts along with a secondary non-GFP expressing bystander infiltration initiated by the transferred cells. This model of bystander cell detection confirmed that such an effect occurs in EAT and adds weight to the importance of this phenomenon in autoimmune thyroid disease.

Section snippets

Animals

Female CBA/J (H-2k) and C57Bl/6 (H-2b) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Heterozygous, enhanced GFP+ fluorescent protein-transgenic (GFP-t−) mice [C57Bl/6 (H-2b) Transgenic-15 (act-EGFP) Osb1] were kindly provided by Dr. Masaru Okabe (Osaka University, Osaka, Japan) [12]. Male heterozygous GFP-transgenic mice were backcrossed for up to 7 generations with CBA/J mice. In the third generation, we selected I-Ak positive/I-A b negative GFP+ mice (N3 GFP-CBA/J mice) to

Transfer of the GFP transgene to an I-Ak background

The existence of the GFP transgene in progeny of GFP-t− mice was easily assessed with a hand illuminator. To examine I-A gene expression in GFP positive progeny, the expression of the I-A haplotype by peripheral mononuclear cells (PMNCs) was studied. The PMNCs of N3 mice showed the same high I-Ak expression as control CBA/J mice while detectable I-Ab expression was already lost (data not shown). GFP expression was also well maintained in these mice. These I-Ak positive/I-Ab negative GFP+ mice

Discussion

In these studies, we developed a model of EAT utilizing mice transgenic for enhanced GFP expression. Moreover, we were able to directly demonstrate GFP host cell infiltration within the thyroid initiated by the transferred cells using the adoptive transfer model of Tg-primed GFP-CBA/J mice.

EAT can be induced in susceptible mice after immunization with Tg. This susceptibility to EAT induction is H-2 linked and the immune response gene is located in the I-A subregion [18]. Therefore, to produce

Acknowledgments

This work was supported by the NIH, the David Owen Segal Fund, and the Sinkoff Endowment. We are most thankful to Dr Genichiro Sano for advising us on flow cytometric analysis of intrathyroidal cells and Dr. Rauf Latif for critical review and advice. We dedicate this paper to the late Ryohei Arata, husband of NA, who died during the completion of this work.

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