Monoclonal Pathogenic Antibodies to the Thyroid-Stimulating Hormone Receptor in Graves’ Disease with Potent Thyroid-Stimulating Activity but Differential Blocking Activity Activate Multiple Signaling Pathways

Abstract

The thyroid target Ag for disease-inducing autoantibodies in Graves’ disease is the receptor for thyroid-stimulating hormone (TSH), but little is known about the molecular basis of this pathogenic Ab response. We describe the characteristics of two high- affinity mAbs developed from an experimental murine model of hyperthyroid Graves’ disease that exhibit potent thyroid-stimulating activity. Nanogram concentrations of the IgG mAbs KSAb1 and KSAb2 and their Fab induce full stimulation of the TSH receptor that is matched by the ligand TSH and, thus, act as full agonists for the receptor. However, KSAb1 and KSAb2 display differential activities in their ability to block TSH-mediated stimulation of the receptor, indicating subtle differences in their biological properties. In displacement studies, IgG and Fabs of KSAb1 and KSAb2 compete with Graves’ disease autoantibodies as well as thyroid-blocking Abs present in some hypothyroid patients, indicating a close relationship between these autoimmune determinants on the receptor. In passive transfer studies, single injections of microgram quantities of KSAb1 or KSAb2 IgG led to rapid elevation of serum thyroxine and a hyperthyroid state that was maintained for a number of days. The thyroid glands showed evidence of cell necrosis, but there was no accompanying mononuclear cell infiltrate. In studying their receptor activation pathways, both KSAb1 and KSAb2 provoked phosphorylation of the intracellular ERK1/2 pathway in primary thyrocytes, indicating that multiple signaling pathways may participate in the pathogenesis of Graves’ disease. In summary, our findings emphasize the similarities of the experimental mouse model in reproducing the human disorder and provide improved means for characterizing the molecular basis of this pathogenic response.

Yersinia enterocolitica Provides the Link between Thyroid-Stimulating Antibodies and Their Germline Counterparts in Graves’ Disease

“…Recent genetic studies in inbred mice strains have implicated the IGH V region gene locus in TSAb development (5,6). Monoclonal TSAbs with powerful agonist activity have been characterized from mice undergoing experimental Graves' disease following immunization by genetic delivery of plasmid or adenovirus encoding TSHR or the extracellular region of the receptor, known as TSHR A-subunit (7)(8)(9). The monoclonal TSAbs derived from experimental models of Graves' disease have been shown to be pathogenic in vivo, confirming their role in disease pathogenesis (8)(9)(10).…”

“…Monoclonal TSAbs with powerful agonist activity have been characterized from mice undergoing experimental Graves' disease following immunization by genetic delivery of plasmid or adenovirus encoding TSHR or the extracellular region of the receptor, known as TSHR A-subunit (7)(8)(9). The monoclonal TSAbs derived from experimental models of Graves' disease have been shown to be pathogenic in vivo, confirming their role in disease pathogenesis (8)(9)(10). Importantly, human monoclonal TSAbs from patients with Graves' disease have also been derived, providing detail into their molecular and biochemical properties, and pathogenicity in vivo (11)(12)(13).…”

G13-dependent Activation of MAPK by Thyrotropin

“…Additionally, the potential engagement of mitogen-activated protein kinases (MAPK), especially p44/42 MAPK, in TSH signaling has been evaluated in several studies. However, the results obtained are inconsistent, since three of six reports demonstrated TSH-mediated p44/42 MAPK activation (23)(24)(25) whereas no specific, TSH-promoted activation of p44/42 MAPK was observed in the remaining investigations (26 -28). Based on this inconsistency, which might be due to different cellular model systems used in these studies, the importance of this pathway for TSH-dependent regulation of gene expression still remains under debate.…”

“…TSH-mediated p44/42 MAPK activation has been demonstrated in several previous studies (23)(24)(25). Surprisingly, other reports questioned such a TSH-promoted activation of p44/42 MAPK (26 -28).…”

“…This discrepancy could be explained by the different cellular models used. For example, it appears that TSH-promoted activation of p44/42 MAPK is not detectable in dog thyrocytes (27,28), whereas TSH activated MAPK in studies in human thyroid cells (23,25). Interestingly, in contrast to the human TSHR that activates G q/11 proteins, the dog TSHR failed to do so (8).…”