Hydrogen peroxide-induced production of a 40 kDa immunoreactive thyroglobulin fragment in human thyroid cells: the onset of thyroid autoimmunity?

We recently reported that, during in vitro thyroid-hormone synthesis, H(2)O(2) stress cleaved thyroglobulin (Tg) into C-terminal peptides. These peptides were found to contain the immunodominant region of Tg recognized by Tg autoantibodies from patients with an autoimmune thyroid disease. To test the hypothesis that Tg fragmentation is an early upstream initiating event involved in Tg autoimmune response and the consequence of oxidative injuries, we studied the effect of H(2)O(2) stress on human thyroid cells. In culture conditions allowing Tg synthesis and iodine organification by the cells, we found that bolus addition of increasing millimolar doses of H(2)O(2) induced a dose-response appearance of floating cells in the culture medium. These cells apparently resulted from a necrotic process, and they bore iodinated Tg fragments. These fragments were found to be similar to those previously obtained in vitro from purified Tg. In both cases, Tg peptides were recognized by a well-defined monoclonal antibody directed to the immunodominant region of Tg. The smallest immunoreactive Tg peptide had a molecular mass of 40 kDa and entered human thyrocytes more efficiently than the entire Tg. These data suggest that thyrocytes exposed to locally increased H(2)O(2) doses accumulate fragmented Tg for further delivery into surrounding living thyrocytes in the course of an autoimmune response.

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.

Molecular model, calcium sensitivity, and disease specificity of a conformational thyroperoxidase B-cell epitope.

While studying the humoral mechanisms involved in thyroid autoimmunity, we located a B-cell autoepitope in the extracellular C-terminal region of human thyroperoxidase. Structural modeling showed that this region encompasses both a Sushi-like and an epidermal growth factor-like domain, the flexible arrangement of which was putatively stabilized by calcium. The recombinant peptide was found to contain the previously identified conformational thyroperoxidase autoepitope. The occurrence of a calcium-induced conformational change was confirmed using a recombinant peptide monoclonal antibody, the decrease of which in binding to calcium-saturated thyroperoxidase was reversed by a chelating agent. The disease specificity of recombinant peptide, which was more frequently recognized by Hashimoto's than by Graves' patients, adds to its potential value as a diagnostic and preventive tool in the context of B-cell autoimmunity.

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.

A conformational B-cell epitope on the C-terminal end of the extracellular part of human thyroid peroxidase.

To investigate the B-cell autoimmune epitopes on human thyroid peroxidase (TPO), we generated proteolytic peptides by enzymatic hydrolysis of TPO in nondenaturing and nonreducing conditions. The hydrolysate was chromatographed on a reverse phase column. We eluted a material immunoreactive with both a TPO monoclonal antibody recognizing a linear epitope (mAb47, amino acid 713-721) and TPO autoantibodies (aAb) from patients. The aAb immunoreactivity, but not that of mAb47, was lost after reduction. Western blots after electrophoresis without reduction showed that the aAb and mAb47 were immunoreactive with a 66-kDa band and that aAb identified a doublet at 20 kDa. For electrophoresis under reducing conditions, the 66-kDa band resolved into two peptides of 40 and 26 kDa, whereas the doublet at 20 kDa remained unchanged. None of these reduced peptides was immunoreactive with aAb, whereas the 40-kDa peptide was immunoreactive with mAb47. The 40-kDa peptide extends from amino acid 549 to 933 of TPO, and its last 192 amino acids overlap the autoimmune 20-kDa peptide. After iodine labeling, the 20-kDa peptide lost its immunoreactivity. We conclude that the C-terminal end of the extracellular part of TPO, which includes all the tyrosine residues of the 20-kDa peptide, contains at least one conformational B-cell epitope involved in autoimmune thyroid diseases.

Idiotypic study of a bispecific thyroglobulin and thyroperoxidase monoclonal antibody.

We have previously established that thyroperoxidase (TPO), a major thyroid antigen involved in autoimmune thyroid diseases, interacts with an idiotype present on human and mouse antibodies directed to thryoglobulin (TG), another thyroid autoantigen. In order to characterize the TPO-reactive idiotype, we selected a TG monoclonal antibody (mAb J7 B49.15) which bound to TPO and cross-reacted with human bispecific TG and TPO autoantibodies (TGPO aAb) for both TG and TPO binding. The TPO-reactive structure of the mAb J7 B49.15 was present on the F(ab')2, located next to the TG binding site and dependent on the association of the heavy and light chains. We found that mAb J7 B49.15 shared a TPO-reactive idiotypic structure with TG mAb from the same cluster of TG reactivity. All the mAb from this cluster were directed to an immunodominant region of TG, recognized by TG aAb. Natural anti-idiotypes from pooled normal human IgG used as a therapeutic intravenous preparation inhibited the TPO binding to mAb J7 B49.15. By homologous immunization in BALB/c mice, mAb J7 B49.15 induced an antiserum with both TG and TPO reactivities. Whereas TG reactivity decreased as early as day 14 post-immunization, TPO reactivity remained at a plateau value from day 21 to day 42. The TG and TPO reactive antisera were shown to inhibit the binding of mAb J7 B49.15 to TG and TPO, respectively. We concluded that mAb J7 B49.15 reacted with TPO through an interspecies idiotype which appeared conformational and related to the epitopic specificity of the mAb. Interestingly, this idiotype would carry the internal image of a TG structure able to induce, in homologous system, a TG antibody response followed by a TPO response without the need of any immunizing antigen.

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.

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-iodinated 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 titre. Furthermore, high TGPO autoantibody titres 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.

Immunopurification and characterization of thyroid autoantibodies with dual specificity for thyroglobulin and thyroperoxidase.

The presence of autoantibodies (aAbs) to thyroglobulin (TG) and thyroperoxidase (TPO) in most of the patients with autoimmune thyroid disease is now well documented. Studies of these aAbs suggested that some, termed TGPO aAbs, could interact with both TG and TPO. This hypothesis was investigated using IgG fraction from a pool of 25 patients' sera with high TG and TPO aAb titres. Immunopurification of TG, TPO and TGPO aAbs was carried out by sequential affinity chromatography using a large quantity of highly purified human TG and TPO. TGPO aAbs, obtained absorption-elution of affinity purified TG aAbs onto a TPO column, were found to represent about 20% of the TG reactive aAbs and 0.23% of the total amount of IgG. Purified TGPO aAbs were characterized and compared to specific TG and TPO aAbs. In contrast to TG and TPO aAbs which recognized only their target antigen, TGPO aAbs showed high affinity interactions with both TG and TPO. As compared to TG aAbs, TGPO aAbs displayed similar affinity for native TG and higher affinity for denatured TG. Compared to TPO aAbs, TGPO aAbs showed lower affinity for both native and denatured TPO. TGPO aAbs also differed from specific TG and TPO aAbs with regard to IgG subclass distribution and antigen fine specificities as determined by monoclonal antibody assisted mapping of TG and TPO surface epitopes. Taken together, these data indicate that TGPO aAbs are effectively present in the serum of patients with autoimmune thyroid disease. TGPO aAbs may be considered as a subpopulation of TG aAbs with the unique property to cross-react with TPO. The existence of aAbs cross-reacting with these functionally and antigenically related thyroid molecules could lead to a re-examination of the emergence of thyroid autoimmunity.

Relationship between immunological structure and biochemical properties of human thyroid peroxidase.

Although the primary structure of human thyroid peroxidase (hTPO) has been recently deciphered, little is known about its spatial conformation. Such information is of crucial importance in any attempt to relate the structure with the function of hTPO. To probe the antigenic surface of hTPO and to correlate its immunological structure to its biochemical properties, we used 13 monoclonal antibodies (mAb) displaying various affinity for hTPO. Criss-cross experiments showed 7 clusters of reactivity which were interpreted as reflecting 7 epitopes on the surface of the hTPO molecule. Extending our analysis to partial and nonsymmetrical cross-reactivities, these epitopes were shown to be localized in 4 antigenic domains of the hTPO. We further investigated the nature of these 7 hTPO epitopes by testing mAb binding to peroxidases from various origins and chemically modified hTPO; 3 epitopes were shown to be evolutionary conserved, and 5 resistant to reduction and denaturation. We also analyzed the role of the hTPO epitopes in the enzymatic activity and autoimmune targeting of the molecule. Nine epitopes were shown to be localized at the vicinity of both catalytic sites as the binding of their respective mAb modulated the enzyme activity. Autoantibodies from patients presenting with autoimmune thyroid disorders were essentially directed to epitopes similar or adjacent to those recognized by 8 of the 13 mAb and present on only 2 antigenic domains of hTPO. Taken together these data allowed us to propose a tentative map of the surface of the hTPO molecule which associates its epitopic structure with its biochemical functions.

Changes in tyrosine hydroxylase, glutamic acid decarboxylase and choline acetyltransferase after local microinoculation of scrapie agent into the nigrostriatal system of the golden hamster.

A microinjection of a homogenate of scrapie agent-infected brain (strain 263 K) into the nigrostriatal system in the golden hamster is followed by the progressive development of the disease which terminates by the death of animals around the 4th month postinoculation. These intracerebral inoculations induce more rapid changes in neuronal activity which can be revealed by the assessment of the specific synthesizing enzymes of neurotransmitter systems. The microinoculation of a homogenate of an infected brain unilaterally into the substantia nigra (SN) provokes a decrease in tyrosine hydroxylase (TH), the synthesizing enzyme for dopamine in the dopaminergic neurones, in the striatum ipsilateral to the injected SN. This biochemical response, specifically induced by the active pathogen, is detectable as soon as the 5th day postinoculation and is detectable towards the 80th day. A return of TH levels to control values is detected after this period. At the end of the incubation period and towards the death of the animals, TH is not different from control TH measured from intact animals. The decrease in TH is concomitant with an increase in striatal glutamic acid decarboxylase (GAD), the synthesizing enzyme for gamma-aminobutyric acid (GABA), measured 20 days postinoculation with no change in choline acetyltransferase (ChAT), the synthesizing enzyme for acetylcholine. Studies of the biochemical responses associated locally to the scrapie agent inoculation have been performed at the striatal level. The intrastriatal administration of the infective agent induces 20 days postinoculation an increase in GAD with no change in TH and ChAT. Ninety days postinoculation, a decrease in GAD was detected associated with an increase in TH with no change in ChAT.(ABSTRACT TRUNCATED AT 250 WORDS)

Early changes in tyrosine hydroxylase and glutamate decarboxylase activity in the golden hamster striatum after intracerebral inoculation of the nigrostriatal system with scrapie agent (strain 263 K).

Unilateral inoculation of hamster substantia nigra (SN) with scrapie agent led to an early decrease in tyrosine hydroxylase (TH) activity in the corresponding striatum, which was detectable by the 5th day. This decrease was accompanied by an increase in glutamate decarboxylase (GAD) observed on the 20th day. Local phenomena related to administration of the agent were investigated by intrastriatal inoculation followed by local measurement of TH, GAD and choline acetyltransferase (ChAT) activities. A rise in GAD activity was observed 20 days later. The decrease in TH activity which occurred 5 days after inoculation of the substantia nigra with scrapie agent constitutes an extremely early indication in hamsters of the slow pathological processes at work: at clinical and behavioural levels, these can be detected at best only 80 days after the intracerebral inoculation.