Spreading of antibody reactivity to non-thyroid antigens during experimental immunization with human thyroglobulin.

Intermolecular spreading of antibody reactivity has been implicated in the evolution of autoimmune disease. In this study, spreading of antibody reactivity to non-thyroid autoantigens after experimental immunization with thyroglobulin (Tg) was investigated. For this purpose, two rabbits were injected with human Tg six times (stages 1-6) every 3 weeks. Animals were also bled before priming. Antisera were tested by enzyme-linked immunosorbent assay (ELISA) for reactivity to several non-thyroid antigens: bovine serum albumin (BSA), native DNA (nDNA), human myosin, human globular (G) and filamentous (F) actin and porcine tubulin. Tg-immunized animals developed the following serological reactivity pattern: (a) high reactivity to myosin from stage 2 onward, (b) significant reactivity to F-actin, remaining high up to stage 6, (c) reactivity to BSA with a peak at stage 3, (d) a small increase of reactivity to G-actin at stage 3 and (e) no increase of reactivity to nDNA and tubulin. The study of affinity-purified anti-Tg antibodies and the use of competitive assays revealed that reactivity to F-actin was not due to cross-reaction with Tg. On the contrary, reactivity to myosin during the first stages of immunization was due to cross-reaction with Tg, while at stage 6 it became myosin-specific. Reactivity to BSA at stage 3 was also due to cross-reaction with Tg. We conclude that at least part of the induced anti-Tg antibodies may result from the expansion of B cell clones producing polyreactive natural autoantibodies, and polyreactivity of anti-Tg antibodies during the first stages of Tg-immunization may be responsible for the intermolecular spreading of antibody response.

Induction of hyperthyroidism in mice by intradermal immunization with DNA encoding the thyrotropin receptor.

Intramuscular injection with plasmid DNA encoding the human thyrotropin receptor (TSHR) has been known to elicit symptoms of Graves' disease (GD) in outbred but not inbred mice. In this study, we have examined, firstly, whether intradermal (i.d.) injection of TSHR DNA can induce hyperthyroidism in BALB/c mice and, secondly, whether coinjection of TSHR- and cytokine-producing plasmids can influence the outcome of disease. Animals were i.d. challenged at 0, 3 and 6 weeks with TSHR DNA and the immune response was assessed at the end of the 8th or 10th week. In two experiments, a total of 10 (67%) of 15 mice developed TSHR-specific antibodies as assessed by flow cytometry. Of these, 4 (27%) mice had elevated thyroxine (TT4) levels and goitrous thyroids with activated follicular epithelial cells but no evidence of lymphocytic infiltration. At 10 weeks, thyroid-stimulating antibodies (TSAb) were detected in two out of the four hyperthyroid animals. Interestingly, in mice that received a coinjection of TSHR- and IL-2- or IL-4-producing plasmids, there was no production of TSAbs and no evidence of hyperthyroidism. On the other hand, coinjection of DNA plasmids encoding TSHR and IL-12 did not significantly enhance GD development since two out of seven animals became thyrotoxic, but had no goitre. These results demonstrate that i.d. delivery of human TSHR DNA can break tolerance and elicit GD in inbred mice. The data do not support the notion that TSAb production is Th2-dependent in murine GD but they also suggest that codelivery of TSHR and Th1-promoting IL-12 genes may not be sufficient to enhance disease incidence and/or severity in this model.

Assessment of the frequency of mutant (hprt-) T lymphocytes from peripheral blood of patients with Hashimoto's thyroiditis.

A salient feature of Hashimoto's thyroiditis (HT) is the T-cell-mediated destruction of the thyroid gland leading to hypothyroidism. In HT, as in other autoimmune diseases, a central premise has been that autoreactive T cells must be dividing in response to autoantigens, accumulating random spontaneous mutations during the activation process. Here, we have examined this hypothesis by using as monitor of somatic cell mutation the hprt gene, encoding the salvage pathway enzyme hypoxanthine-guanine phosphoribosyl transferase. Eleven newly diagnosed patients with HT and 10 patients with chronic disease were selected for the study, whereas 10 healthy individuals were used as controls. Peripheral T cells were cultured under limiting dilution conditions in the presence of 6-thioguanine and the frequency (MF) of surviving mutant hprt(-) T cells was calculated by Poisson statistics. It was observed that the mean MF value of either patient group (6.6 +/- 5.8 per 10(6) cells for the newly diagnosed, and 8.8 +/- 4.0 per 10(6) cells for the patients with chronic disease) was not significantly different (p > 0.05) from that of the control group (6.8 +/- 6.4 per 10(6) cells). These data do not support the concept that patients with HT have an increased number of actively dividing T cells in the circulation compared to healthy controls. Autoreactive T cells may be activated mainly in situ or home readily to the thyroid in the early stages of the disease and reach a nonexpansion stage as the chronic disease is stabilized.