Josée-Martine Durand-Gorde

Production of Immunoreactive Thyroglobulin C-Terminal Fragments during Thyroid Hormone Synthesis

Here, we studied the fragmentation of the prothyroid hormone, thyroglobulin (Tg), which occurs during thyroid hormone synthesis, a process which involves iodide, thyroperoxidase, and the H2O2-generating system, consisting of glucose and glucose oxidase. Various peptides were found to be immunoreactive to autoantibodies to Tg from patients and monoclonal antibodies directed against the immunodominant region of Tg. The smallest peptide (40 kDa) bore thyroid hormones and was identified at the C-terminal end of the Tg molecule, which shows homologies with acetylcholinesterase. Similar peptides were obtained by performing metal-mediated oxidation of Tg via a Fenton reaction. It was concluded that the oxidative stress induced during hormone synthesis generates free radicals, which, in turn, cleave Tg into immunoreactive peptides.

Significance of thyroglobulin antibodies cross-reactive with thyroperoxidase (TGPO antibodies) in individual patients and immunized mice

Thyroglobulin (TG) and thyroperoxidase (TPO), both involved in thyroid hormone synthesis, represent major autoantigens in thyroid autoimmune disease. Despite numerous studies, the emergence, pathophysiological significance and role of autoantibodies to TG and TPO remain elusive. The recent identification of a new category of thyroid‐specific autoantibody interacting with both TG and TPO (TGPO autoantibodies) offers a new opportunity in the study of thyroid autoimmunity. To gain a better insight into the significance of these TGPO autoantibodies, measurement in individual samples appeared necessary. The unique property of TGPO autoantibodies, simultaneous binding to TG and TPO, was used to set up a sandwich method which combined coated TG and radio‐iodinatcd TPO. This method was found to be strictly specific for TGPO autoantibodies and sensitive enough to assay TGPO autoantibodies in serum. In humans, TGPO autoantibodies were found in most of the sera with high TG and TPO autoantibody titres, but not in sera negative for TG autoantibodies, whatever the TPO autoantibody litre. Furthermore, high TGPO autoantibody litres were found in sera strongly cytotoxic for cultured porcine thyroid cells. However, significant correlation of TGPO autoantibody titre was observed neither with TG and TPO autoantibody titres (n=48) nor with complement‐dependent cytotoxicity (n= 50). TGPO antibody assay was also performed in individual plasma of CBA/J mice immunized with either human TG (n= 6) or human TPO (n= 6). Immunization with TG induced high levels of not only TG but also TGPO antibodies, which exhibited a strong reactivity for TPO and whose binding to TG and TPO was fully inhibited by TG. In contrast, immunization with TPO induced high levels of only specific TPO antibodies accompanied by low levels of specific TG antibodies. In this case TGPO antibodies were not detected. Of note, TG‐ and TPO‐immunized mice mounted an immune response against their own TG, but did not exhibit histological signs of thyroiditis. Large panels of TG and TPO MoAbs were also investigated with this method: 18/25 TG MoAbs and only 1/13 TPO MoAbs were found cross‐reactive. Taken together, these data provide evidence that TGPO antibodies are effectively present in individual patients and TG‐immunized mice, are different from specific TG and TPO antibodies, and may derive from natural B cell repertoire by autoimmune processes involving TG and not TPO.

Autoantibodies and Monoclonal Antibodies Directed to an Immunodominant Antigenic Region of Thyroglobulin Interact with Thyroperoxidase Through an Interspecies Idiotype

We investigated whether thyroglobulin (TG) autoantibodies (aAb) cross-react with thyroperoxidase (TPO) through an idiotypic structure using pooled normal human IgG (NhlgG) as a natural anti-idiotype reagent. Affinity-purified TG aAb from pooled IgG of patients with autoimmune thyroid disease were chromatographed on Sepharose-bound NhlgG. About one fourth of the loaded material bound to and eluted from the coupled gel. Eluted TG aAb were found reactive to TG and TPO and their TPO but not TG binding was strongly inhibited by molar excess of NhlgG. These TG aAb appeared to be mainly directed to an immunodominant TG antigenic region defined by TG monoclonal antibodies (mAb) from a single cluster of reactivity. These TG mAb were also found to recognize TPO and their binding to TPO but not TG was inhibited by molar excess of NhlgG as already observed with TG aAb. Taken together, these results indicated that TPO interacts with an idiotype present on human TG aAb and mouse TG mAb displaying a similar epitopic specificity; this interspecies idiotype is recognized by anti-idiotype antibodies present in NhlgG. Our results suggest that thyroid autoimmunity can be envisaged, at least in part, as a disturbance in interconnected idiotypic networks.

Antigenicity and immunogenicity of the C-terminal peptide of human thyroglobulin

Thyroglobulin (Tg) is cleaved into several peptides during thyroid hormone synthesis, an oxidative process. P40, an iodinated C-terminal peptide from human Tg, has a molecular weight of about 40 kDa and contains two hormonogenic sites. P40 is the smallest peptide that is still recognized by monoclonal antibodies from mice immunized with human Tg directed against its immunodominant region. Since P40 also contains several T-cell epitopes, it is a good candidate for studying the primary events involved in the process of hormone synthesis leading to thyroid autoimmunity. The present results show that P40 is recognized by Tg antibodies from patients with thyroid disorders and induces Tg antibodies in CBA mice. P40 may therefore be involved in the autoimmune process, thus providing a useful tool for diagnostic and therapeutic purposes.

Thyroglobulin monoclonal antibody cross-reacting with thyroperoxidase induces in syngeneic mice anti-idiotypic monoclonal antibodies with dual autoantigen binding properties. The intertope hypothesis

Autoimmune thyroid diseases are characterized by antibodies (Ab) directed to thyroglobulin (Tg) and thyroperoxidase (TPO). Some of them, TGPO Ab, are Tg Ab with an interspecies idiotype (Id) reacting with TPO. Taking advantage of a carefully studied TGPO monoclonal antibody (mAb), we examined the basis of the hypothesis that TPO Ab would ultimately derive from TGPO Ab through idiotypic induction. We repeatedly immunized naive, syngeneic mice with the TGPO mAb and we derived three novel mAb directed to both Tg and TPO. The most reactive of them, mAb 4F8, was further purified, radiolabeled and its binding properties studied by radioimmunoassay. mAb 4F8 bound to Tg, TPO, the immunogen Ab1 and even to itself, being thus considered as a self‐binding Ab2 . Competitive binding inhibition experiments demonstrated that Tg, TPO, Ab1 and Ab2 cross‐reacted for Ab2 binding to Tg, TPO and Ab1. Fine specificity mapping using panels of specific mAb revealed that Ab1 and Ab2 were similar because they were directed against the same immunodominant regions on Tg and TPO. We propose that unique Id of TGPO Ab resemble dominant epitopes of Tg as well as paratopes of Ab directed against dominant TPO epitopes. This category of Id that we called intertopes may induce TPO‐monospecific Ab from TGPO Ab by idiotypically driven somatic mutations.