Analysis of the initiation mechanisms in human organ specific autoimmune disease using the chicken model for spontaneous autoimmune thyroiditis

Hashimoto`s thyroiditis, a prototype human organ specific autoimmune disease, affects people of all ages, with the peak occurence in women of 30 to 50 years of age. This long timespan makes it thus virtually impossible to analyze the initiating mechanism in humans. The Obese strain (OS) of chickens is an excellent animal model of this human disease in most clinical, histological, immunological and endocrinological aspects. All chickens of this strain develop symptoms of hypothyroidism due to spontaneous autoimmune thyroiditis (SAT) and the first lymphocytes infiltrating the thyroid are observed as early as 1-2 weeks after hatching. This short interval between the maturation of the immune system and the onset of the thyroid infiltrates implicates the importance of genetic factors for this disease, and reduces the importance of environmental factors. The results of our experiments indicate that CD4+ TCR2 (V beta 1)+ lymphocytes and monocytes/macrophages are important in SAT of OS chickens. While CD4+ cells are important in the disease process through cytokine release, the role of macrophages in SAT might be even more complex, They can function in initiating the immune response, cooperating with T cells, or as effector cells and the progression from autoimmune response to autoimmune disease may thus not proceed directly, but rather by activation of macrophages, The focus of this project is the definition of the role of monocytes/macrophages in the initiation phase of SAT, and on their cooperation with lymphocytes. Our experiments will adress the following hypotheses: macrophage activity is an important factor for the initiation of autoreactivity of the immune system, the role of macrophages as antigen presenting cells in the initiation of lymphocyte autoreactivity in SAT, genetic conditions regulating the level of intrinsic macrophage activity and the role of the expression of nonspecific esterase in thyroid folicular epithelium in the initiation of the thyroid infiltration, By a combination of immunological, immunogenetic and molecular biological techniques, we hope to define more precisely the etiologically important factors in SAT of OS chickens, The results obtained from our animal model, in addition to their basic scientific value, will have a practical impact on treatment and prevention of Hashimoto`s thyroiditis in humans,

Hashimotos thyroiditis, a prototypic human organ specific autoimmune disease, affects people of all ages, with the peak occurrence in women of 30 to 50 years of age. The Obese Strain (OS) of chickens is an excellent animal model of this human disease in most clinical, histological, immunological and endocrinological aspects. All chickens of this strain develop symptoms of hypothyroidism due to spontaneous autoimmune thyroiditis (SAT), characterised by massive mononuclear cell infiltration of the thyroid, starting already seven days after hatching. We have focused our experiments on the analysis of the onset of SAT by means of two different types of methods, namely immunobiological and molecular biological. In immunological response cytokines (defined as proteins released by cells) are able to communicate among cells and regulate their response. Progress in avian cytokines enable us to analyse their role in the reactivity of chicken immune system to body own antigens. Gene structure information makes it possible to specifically quantify mRNA levels using real-time quantitative RT-PCR and to investigate the role of cytokines in driving the lymphoid infiltration of the thyroid seen in OS birds from the 7 post-hatch. We characterised the expression of IFN-gamma, IL-1beta, IL-2, IL-6, IL-8, IL-15 and IL-18 in thyroids from OS birds, and control CB line birds, both in the embryo just prior to hatch (E20) and at 3 and 5 days post-hatch. All of these cytokines were upregulated, as compared to levels in CB thyroids, at least at some time- points, with some evidence for coordination of regulation, e.g. for the pro-inflammatory cytokines IL-1beta and IL-8. Only IL-15 was upregulated at all time-points. This could explain the general immune system hyperreactivity exhibited by OS line chickens, and may be a factor driving the lymphoid infiltration of the thyroid. We analysed the genetic regulation of SAT by means of crosses between OS chickens with the disease, and healthy inbred chickens of line CB. Mononuclear cell infiltration was used as a criterion for the disease. We confirmed the existence of one recessive gene, regulating the susceptibility of the thyroid gland to autoimmune attack. From the frequency of progeny with the thyroid infiltration in backcross and F2 generations, we presume the existence of one or two additional dominant genes coding for abnormal reactivity of the immune system. The total number of genes regulating SAT in OS is therefore a maximum of three. We attempted to identify disease-specific transcripts responsible for the initiation of the disease using suppression subtractive hybridisation of RNA prepared from OS and CB thyroid lobes, obtained from 3-day-old chicks. From forward and reverse subtractions, we recovered a fragment mixture in the range of 300 bp to 1.5 kb. In total, 768 clones were screened and 9 were sequenced. Four of them represent unknown sequences. Two, specific for OS thyroid, correspond to envelope genes of avian endogenous viruses (ev) -1, -3 and -6. Two thyroid specific clones had significant alignment to human thyroglobulin, and one clone to human coatomer protein. Further experiments will be required to analyse the function of these transcripts. The identified sequences specific for the disease can be used for the comparative screening of the human genome and for identification of predisposed individuals to Hashimotos disease. The lower level of iodine in the diet in these individuals can at least reduce the severity of the disease.