Abstract
Background: Experimental autoimmune thyroiditis (EAT) is induced in genetically susceptible mice by immunization with thyroglobulin (Tg) with adjuvant. Granulomatous EAT (G-EAT) is obtained by adoptive transfer of splenocytes from mouse Tg-primed mice, restimulated with mTg, plus anti-IL-2R or anti-IFN-gamma antibodies and IL-12.
Aim: Our aim was to evaluate whether hydrophobic domains exposed during human Tg unfolding may shift the cytokine milieu of the adaptive response to hTg towards inflammation and its histopathology towards G-EAT.
Methods: Unfolded hTg was prepared by denaturation in 0.120 M Tris/HCl, 3.5 M urea, pH 9.0, yielding a mixture of partially unfolded monomers (UM), exposing a hydrophobic core domain, as shown by 8-anilino-1-naphthalensulfonic acid binding, in equilibrium with non-native dimers (NND), reassociating by virtue of a hydrophobic effect, which persisted after partial renaturation upon removal of urea, and were separated by sucrose gradient centrifugation. Groups of 6 female CBA/J(H-2k) mice were immunized with 100 μg of native hTg, UM and NND in CFA on day 0, followed by 50 μg IFA on day 8. After sacrifice on day 35, we evaluated thyroid histology and secondary proliferative and IFN-γ and IL-12 secretory responses of splenocytes.
Results: Immunization with native hTg was associated with low-grade, focal infiltration of thyroids with mononuclear cells. Instead, aspects of granulomatous infiltration, with widespread follicle destruction and fibrosis were apparent in animals immunized with hTg NND and, particularly, UM. Secondary proliferative and IL-12 and IFN-gamma secretory responses were maximal in mice immunized with hTg UM.
Conclusions: These data represent the first demonstration that unfolded hTg has a markedly stronger ability, compared with native hTg, to promote adaptive self responses, culminating in thyroid tissue damage, and shed light onto the mechanisms by which the unmasking of unfolded intracellular Tg, as a consequence of thyroid cell damage, may incite thyroid autoimmunity.