Qualitative aspects of thyroglobulin antibodies in Hashimoto's thyroiditis and Graves' disease

We have tried to characterize the thyroglobulin antibodies (TgAb) involved in Hashimoto''s thyroiditis and Graves'' disease by studying their affinity constants and binding capacities. Two populations of antibodies were found in half of the patients with either disease, TgAb₁ with a high affinity (K₁) but a low binding capacity (B₁) and TgAb₂ with a lower affinity (K₂) but a much higher binding capacity (B₂). The mean affinity constants and binding capacities were similar in the two diseases. In the other half of the patients only one population of antibody was present, with a low affinity constant (K_t) and a very high binding capacity (B_t), thus comparable to TgAb₂. The mean K_t and B_t were similar in the two diseases. From these results it would appear that circulating thyroglobulin antibodies in Hashimoto''s thyroiditis and Graves'' disease are similar in their affinity constants and binding capacities, so that these characteristics do not reflect the different pathogenesis of each condition.     

Iodination of tyrosyls in thyroglobulin generates neoantigenic determinants that cause thyroiditis

Thyroglobulin (Tg) is unique in its ability to incorporate and store available iodine in the form of iodotyrosyl residues. Iodination of Tg has been known to increase its immunopathogenicity in experimental animals, presumably through the formation of iodine-containing neoantigenic determinants that can elicit an autoimmune response, but defined pathogenic Tg peptides carrying iodotyrosyls have not yet been identified. We report in this study that a systematic, algorithm-based search of mouse Tg has delineated three iodotyrosyl-containing peptides that activate autoreactive T cells and cause experimental autoimmune thyroiditis in normal CBA/J mice. These peptides (aa 117-132, 304-318, and 1931-1945) were not immunogenic in their native form, and iodination of tyrosyls facilitated either peptide binding to MHC or T cell recognition of the peptide. These results demonstrate that iodotyrosyl formation in normal Tg confers pathogenic potential to certain peptides that may otherwise remain innocuous and undetectable by conventional mapping methods.     

Distinct genetic pattern of mouse susceptibility to thyroiditis induced by a novel thyroglobulin peptide

Experimental autoimmune thyroiditis (EAT), induced by thuroglobulin (Tg) and adjuvant, is major histocompatibility complex-controlled and dependent on Tg-reactive T cells, but the immunopathogenic T-cell epitopes on Tg remain mostly undefined. We report here the thyroiditogenicity of a novel rat Tg peptide (TgP2; corresponding to human Tg amino acids 2695–2713), identified by algorithms as a site of putative T-cell epitope(s). TgP2 causes EAT in SJL (H-2 ^s) but not in C3H or B10.BR (H-2 ^k), BALB/c (H-2 ^d), and B10 (H-2 ^b) mice. This reveals a new genetic pattern of EAT susceptibility, since H-2 ^k mice are known to be high reponders (susceptible) after Tg challenge. Following in vivo priming with TgP2, T cells from only SJL mice proliferated significantly and consistently to TgP2 in vitro, whereas TgP2-specific IgG was observed in all strains tested. Adoptive transfer of TgP2-primed SJL lymph node cells to naive syngeneic recipients induced a pronounced mononuclear infiltration of the thyroid, which was more extensive than that observed after direct peptide challenge. TgP2 is non-immunodominant, since priming of SJL mice with rTg did not consistently elicit T-cell responses to TgP2 in vitro and a TgP2-specific T-cell hybridoma did not respond to antigen presenting cells pulsed with rTg. The data support the notion that Tg epitopes need not be either iodinated or immunodominant in order to cause severe thyroiditis and that the genetic pattern of the disease they induce can be distinct from that of Tg-mediated EAT. Correspondence to: G. Carayanniotis.     

The sites of thyroid hormone formation in rabbit thyroglobulin

Rabbit thyroglobulin (Tg) was labeled in vivo with 125I and purified by gel filtration. Separation by high performance liquid chromatography (HPLC) of tryptic digests of S-cyanoethylated Tg yielded four major iodothyronine-containing peaks, designated A, B, C, and D. These were further purified on HPLC and sequenced for identification of amino acid residues and for location of the iodothyronine by 125I counting. The published primary structure for bovine Tg, derived from cDNA sequencing of the Tg gene (Mercken, L., Simons, M.J., Swillens, S., Massaer, M., and Vassart, G. (1985) Nature 316, 647-651), permitted tentative location of the rabbit hormonogenic peptides within the Tg polypeptide chain. Site A, corresponding to bovine residue 5, contained 44% of Tgs [125I]T4 (thyroxine) and 25% of its [125I]T3 (triiodothyronine); its specific activity of iodine was higher than that for other sites, indicating priority of iodination. Site B, containing 24% of Tgs [125I]T4 and 18% of its [125I]T3, corresponded to bovine residue 2555. Site C, at the third residue from the C terminus (bovine residue 2748), was the major T3 site, accounting for over 50% of Tgs [125I]T3. The amino acid sequence around this site shows less homology among different animal species than do those flanking the other hormonogenic sites. Site D accounted for 17% of Tgs [125I]T4 and corresponded to bovine Tyr-1291, in the midportion of Tgs polypeptide chain. The three major T4-forming sites had the sequence Asp-Tyr (sites B and D) or Glu-Tyr (site A), while the sequence Ser-Tyr-Ser appeared to favor T3 synthesis (site C), suggesting an important influence of primary structure on hormonogenesis. We conclude that site A is the major T4-forming site and site C the major T3-forming one, but others are available and offer the opportunity for flexibility in meeting different demands for hormone formation.     

Pathogenic human thyroglobulin peptides in HLA-DR3 transgenic mouse model of autoimmune thyroiditis

To identify pathogenic epitopes on human thyroglobulin (hTg), a homodimer of 660kDa, we have applied a computer-based algorithm to predict potential HLA-DR3-binding peptides and have tested them in DR3-transgenic mice. Of the 39 peptides selected, four stimulated a proliferative response from hTg-primed cells of DR3+ mice, but not DQ8+ mice. Of the four peptides, one, hTg2079, was consistently pathogenic. Thyroiditis was not only produced by adoptive transfer of hTg-primed, hTg2079-activated cells but also by direct immunization with the peptide. These results demonstrate the utility of using this computer-based algorithm with synthetic peptides to help identify pathogenic T cell epitopes on hTg.     

Analyses of the serum concentrations and antigenic determinants of thyroglobulin in chickens susceptible to autoimmune thyroiditis

These studies revealed an abnormal elevation in serum thyroglobulin (Tg) concentrations of the thyroiditis-prone OS chicks. Their serum levels, measured by a sensitive and specific RIA, remained high for the first 2 wk of age, a time preceding significant lymphoid infiltration of their thyroid glands. The elevated serum Tg levels probably resulted from, or were related to, hyperactivity of the OS thyroid gland. Reducing the activity of the OS thyroid gland by exogenous administration of T4 caused a temporary but significant reduction in thyroidal infiltration and the synthesis of Tg antibodies. In addition to the analysis of serum concentrations of Tg, a competitive binding RIA was developed to determine whether unique antigenic determinants exist on Tg isolated from OS thyroid glands. This was proved to be unlikely because OS chicks produced autoantibodies that fully cross-reacted with Tg isolated from normal chicken thyroid glands. The relationships between intrinsic thyroid hyperactivity, high serum Tg levels, the sensitization of OS thymocytes, and thyroiditis are discussed.     

The induction of tolerance to thyroglobulin significantly reduces the severity of thyroiditis in obese strain chickens

Obese strain chickens develop severe spontaneous autoimmune thyroiditis several weeks after hatching, characterized by mononuclear cell infiltration and antibodies to thyroglobulin (Tg). The presence of antibodies to Tg suggests that Tg is an important antigen in this disease, but it does not provide definitive evidence. To clarify this point, Obese strain chicks were tolerized at hatching with Tg and then examined up to 6 wk later for antibodies to Tg, thyroid pathology, and function. Various tolerance regimens were tested. The optimal conditions were i.v. injection of Tg within 24 hr of hatching, and injection of at least 1 mg. Tg isolated from normal thyroid glands was satisfactory, and it did not have to be deaggregated. Tolerance induced by the above procedure significantly retarded all parameters of autoimmunity, although by 6 wk of age some of the tolerized chicks had severe thyroiditis. Multiple weekly injections of Tg were no more effective than a single injection at hatching. Interestingly, a single injection at hatching was very effective, yet it was cleared from the circulation within 24 hr. In summary, tolerance induced with Tg had a profound effect on the disease and thus provides good evidence for the role of Tg in this disease.     

Delineation of five thyroglobulin T cell epitopes with pathogenic potential in experimental autoimmune thyroiditis

Experimental autoimmune thyroiditis (EAT) is a T cell-mediated disease that can be induced in mice after challenge with thyroglobulin (Tg) or Tg peptides. To date, five pathogenic Tg peptides have been identified, four of which are clustered toward the C-terminal end. Because susceptibility to EAT is under control of H-2A(k) genes, we have used an algorithm-based approach to identify A(k)-binding peptides with pathogenic potential within mouse Tg. Eight candidate synthetic peptides, varying in size from 9 to 15 aa, were tested and five of those (p306, p1579, p1826, p2102, and p2596) were found to induce EAT in CBA/J (H-2(k)) mice either after direct challenge with peptide in adjuvant or by adoptive transfer of peptide-sensitized lymph node cells (LNCs) into naive hosts. These pathogenic peptides were immunogenic at the T cell level, eliciting specific LNC proliferative responses and IL-2 and/or IFN-gamma secretion in recall assays in vitro, but contained nondominant epitopes. All immunogenic peptides were confirmed as A(k) binders because peptide-specific LNC proliferation was blocked by an A(k)-specific mAb, but not by a control mAb. Peptide-specific serum IgG was induced only by p2102 and p2596, but these Abs did not bind to intact mouse Tg. This study reaffirms the predictive value of A(k)-binding motifs in epitope mapping and doubles the number of known pathogenic T cell determinants in Tg that are now found scattered throughout the length of this large autoantigen. This knowledge may contribute toward our understanding of the pathogenesis of autoimmune thyroiditis.     

T lymphocyte line specific for thyroglobulin produces or vaccinates against autoimmune thyroiditis in mice

We investigated Ly-1+ T lymphocyte line cells specifically reactive to thyroglobulin (Tg) that were isolated from mice primed with mouse Tg in adjuvant. Intravenous inoculation of as few as 10(5) line cells was sufficient to cause severe and prolonged thyroiditis in recipient mice that were intact, irradiated, or athymic nudes. Disease was independent of circulating Tg antibodies, suggesting that anti-Tg T lymphocytes could cause thyroiditis unaided by antibodies. Thyroiditogenic T lymphocytes could be isolated as cell lines from apparently healthy mice that had been immunized with non-thyroiditogenic bovine Tg in adjuvant, which indicates that autoimmune effector T lymphocytes may develop covertly in the course of immunization with foreign antigens. Finally, a single i.v. inoculation of anti-Tg T lymphocyte line cells attenuated by irradiation vaccinated mice completely against subsequent development of autoimmune thyroiditis produced either by active immunization to Tg or by passive transfer of intact line cells. Vaccinated mice that were protected from inflammatory lesions of thyroiditis still produced high titers of Tg antibodies in response to active immunization. Thus, vaccination specifically inhibited thyroiditogenic T lymphocytes but not helper T lymphocytes required for the production of Tg autoantibodies.     

In vitro T-lymphocyte proliferative response to mouse thyroglobulin in experimental autoimmune thyroiditis

The in vitro lymphocyte proliferative response to mouse thyroglobulin (MTg) was studied in good and poor responder mice in relationship to in vivo antibody formation and thyroid infiltration. CBA(H-2^k) and BALB/c(H-2^d) mice were immunized in the hind footpads with MTg incorporated into complete Freund's adjuvant (CFA). At weekly intervals up to 28 days, groups of mice were sacrificed. Their popliteal lymph nodes were cultured in vitro for proliferative response to MTg and their antibody levels and thyroid involvement determined. In good responder CBA mice, the proliferative responses to MTg were strongest on Days 8 to 14, where they were 9- to 14-fold over control levels, depending on the day of harvest. The response declined to 2- to 4-fold over background on Days 21 to 28, although high antibody levels were present throughout this period. The proliferative response was abrogated by anti-Thy-1 treatment, indicating its dependence on T cells. In poor responder BALB/c mice, no significant proliferative responses to MTg were observed at any time, although the animals displayed moderate levels of MTg antibody. The responses to PPD, in contrast, were similar in both strains, usually being 4- to 7-fold above background. Thyroid infiltration, like the proliferative response to MTg, was observed only in CBA mice. Thus lymphocyte proliferation at 8 to 14 days represents a reliable, early in vitro correlate of autoimmune thyroiditis induced with CFA as adjuvant.     

Induction of lordosis behavior in female rats by intravenous administration of progestins

Progesterone is rapidly metabolized by neural cells in the rat. Progesterone could, therefore, act as a “prohormone,” stimulating lordosis behavior in estrogen-primed rats only after metabolic conversion. Were such the case, one might expect one or more of the naturally occurring metabolites of progesterone to be more potent than the parent compound. Estradiol benzoate-primed rats were therefore administered intravenously 200 μg of progesterone or one of five immediate metabolites of progesterone. The steroid 20α-dihydroprogesterone was found to be more potent than progesterone. Both 20α-hydroxy-5α-pregnan-3-one and 3α-hydroxy-5α-pregnan-20-one were less potent than progesterone, but more potent than the vehicle propylene glycol. Neither 5α-pregnane-3α, 20α-diol nor 5α-pregnane-3,20-dione (dihydroprogesterone, DHP) differed from the vehicle in potency. The data suggest that 20α-dihydroprogesterone, which is secreted at high levels during the estrous cycle, could play a role in the regulation of sexual receptivity. The data also suggest that 5α-reduction is probably not crucial for progesterone's action.     

Suppression in experimental autoimmune thyroiditis: the role of unique and shared determinants on mouse thyroglobulin in self-tolerance.

Previous studies have shown that T cells from mice genetically susceptible to experimental autoimmune thyroiditis (EAT) recognize determinants shared between mouse thyroglobulin (Tg) and heterologous Tgs. Some shared determinants are thyroiditogenic; lymphocytes from mice immunized with mouse Tg (MTg) or human Tg (HTg) and reciprocally restimulated in vitro with either Tg can transfer EAT. Studies on the mechanisms of self-tolerance have shown that pretreatment with soluble MTg suppresses in vitro proliferation to MTg and EAT induction with MTg. To determine the role of share epitopes in maintaining tolerance, mice were pretreated with soluble HTg and immunized with HTg or MTg and adjuvant. Cells from HTg-pretreated. HTg-immunized mice showed suppressed in vitro proliferative response to HTg. Following MTg immunization, the cells showed suppressed in vitro response to MTg. However, in contrast to MTg pretreatment, the subsequent development of EAT in vivo was unaltered in severity following HTg pretreatment. Thus, determinants shared between HTg and MTg can induce suppression of in vitro responses to HTg and MTg, but not inhibit the onset of thyroiditis, suggesting that T cells recognizing MTg-unique epitopes expanded to mediate thyroiditis. We conclude that recognition of both unique epitopes expanded to mediate thyroiditis. We conclude that recognition of both unique and shared epitopes on MTg are essential for the overall maintenance of self-tolerance.