Building the Case for Insulin-Like Growth Factor Receptor-I Involvement in Thyroid-Associated Ophthalmopathy.
ABSTRACT: The pathogenesis of orbital Graves' disease (GD), a process known as thyroid-associated ophthalmopathy (TAO), remains incompletely understood. The thyrotropin receptor (TSHR) represents the central autoantigen involved in GD and has been proposed as the thyroid antigen shared with the orbit that could explain the infiltration of immune cells into tissues surrounding the eye. Another cell surface protein, insulin-like growth factor-I receptor (IGF-IR), has recently been proposed as a second antigen that participates in TAO by virtue of its interactions with anti-IGF-IR antibodies generated in GD, its apparent physical and functional complex formation with TSHR, and its necessary involvement in TSHR post-receptor signaling. The proposal that IGF-IR is involved in TAO has provoked substantial debate. Furthermore, several studies from different laboratory groups, each using different experimental models, have yielded conflicting results. In this article, we attempt to summarize the biological characteristics of IGF-IR and TSHR. We also review the evidence supporting and refuting the postulate that IGF-IR is a self-antigen in GD and that it plays a potentially important role in TAO. The putative involvement of IGF-IR in disease pathogenesis carries substantial clinical implications. Specifically, blocking this receptor with monoclonal antibodies can dramatically attenuate the induction by TSH and pathogenic antibodies generated in GD of proinflammatory genes in cultured orbital fibroblasts and fibrocytes. These cell types appear critical to the development of TAO. These observations have led to the conduct of a now-completed multicenter therapeutic trial of a fully human monoclonal anti-IGF-IR blocking antibody in moderate to severe, active TAO.
Project description:Graves' disease (GD) and thyroid-associated ophthalmopathy (TAO) are thought to result from actions of pathogenic antibodies mediated through the thyrotropin receptor (TSHR). This leads to the unregulated consequences of the antibody-mediated receptor activity in the thyroid and connective tissues of the orbit. Recent studies reveal antibodies that appear to be directed against the insulin-like growth factor-I receptor (IGF-IR). Areas covered: In this brief article, I attempt to review the fundamental characteristics of the TSHR, its role in GD and TAO, and its relationship to IGF-IR. Strong evidence supports the concept that the two receptors form a physical and functional complex and that IGF-IR activity is required for some of the down-stream signaling initiated through TSHR. Recently developed small molecules and monoclonal antibodies that block TSHR and IGF-IR signaling are also reviewed in the narrow context of their potential utility as therapeutics in GD and TAO. The Pubmed database was searched from its inception for relevant publications. Expert opinion: Those agents that can interrupt the TSHR and IGF-IR pathways possess the potential for offering more specific and better tolerated treatments of both hyperthyroidism and TAO. This would spare patients exposure to toxic drugs, ionizing radiation and potentially hazardous surgeries.
Project description:Thyroid-associated ophthalmopathy (TAO) is a vexing and undertreated ocular component of Graves disease in which orbital tissues undergo extensive remodelling. My colleagues and I have introduced the concept that fibrocytes expressing the haematopoietic cell antigen CD34 (CD34(+) fibrocytes), which are precursor cells of bone-marrow-derived monocyte lineage, express the TSH receptor (TSHR). These cells also produce several other proteins whose expression was traditionally thought to be restricted to the thyroid gland. TSHR-expressing fibrocytes in which the receptor is activated by its ligand generate extremely high levels of several inflammatory cytokines. Acting in concert with TSHR, the insulin-like growth factor 1 receptor (IGF-1R) expressed by orbital fibroblasts and fibrocytes seems to be necessary for TSHR-dependent cytokine production, as anti-IGF-1R blocking antibodies attenuate these proinflammatory actions of TSH. Furthermore, circulating fibrocytes are highly abundant in patients with TAO and seem to infiltrate orbital connective tissues, where they might transition to CD34(+) fibroblasts. My research group has postulated that the infiltration of fibrocytes into the orbit, their unique biosynthetic repertoire and their proinflammatory and profibrotic phenotype account for the characteristic properties exhibited by orbital connective tissues that underlie susceptibility to TAO. These insights, which have emerged in the past few years, might be of use in therapeutically targeting pathogenic orbit-infiltrating fibrocytes selectively by utilizing novel biologic agents that interfere with TSHR and IGF-1R signalling.
Project description:Graves' disease (GD) is a common autoimmune condition. At its core, stimulatory autoantibodies are directed at the thyroid-stimulating hormone receptor (TSHR), resulting in dysregulated thyroid gland activity and growth. Closely associated with GD is the ocular condition known as thyroid-associated ophthalmopathy (TAO). The pathogenesis of TAO remains enigmatic as do the connections between the thyroid and orbit. This review highlights the putative molecular mechanisms involved in TAO and suggests how these insights provide future directions for identifying therapeutic targets. Genetic, epigenetic, and environmental factors have been suggested as contributory to the development of GD and TAO. Thyroid-stimulating hormone receptor and insulin-like growth factor receptor (IGF-1R) are expressed at higher levels in the orbital connective tissue from individuals with TAO than in healthy tissues. Together, they form a functional complex and appear to promote signaling relevant to GD and TAO. Orbital fibroblasts display an array of cell surface receptors and generate a host of inflammatory molecules that may participate in T and B cell infiltration. Recently, a population of orbital fibroblasts has been putatively traced to bone marrow-derived progenitor cells, known as fibrocytes, as they express CD45, CD34, CXCR4, collagen I, functional TSHR, and thyroglobulin (Tg). Fibrocytes become more numerous in GD and we believe traffic to the orbit in TAO. Numerous attempts at developing complete animal models of GD have been largely unsuccessful, because they lack fidelity with the ocular manifestations seen in TAO. Better understanding of the pathogenesis of TAO and development of improved animal models should greatly accelerate the identification of medical therapy for this vexing medical problem.
Project description:IL-6 plays diverse roles in normal and disease-associated immunity such as that associated with Graves' disease (GD). In that syndrome, the orbit undergoes remodeling during a process known as thyroid-associated ophthalmopathy (TAO). Recently, CD34(+) fibrocytes were found to infiltrate the orbit in TAO where they transition into CD34(+) orbital fibroblasts. Surprisingly, fibrocytes display high levels of functional thyrotropin receptor (TSHR), the central antigen in GD. We report here that TSH and the pathogenic anti-TSHR antibodies that drive hyperthyroidism in GD induce IL-6 expression in fibrocytes and orbital fibroblasts. Unlike TSHR signaling in thyroid epithelium, that occurring in fibrocytes is completely independent of adenylate cyclase activation and cAMP generation. Instead TSH activates PDK1 and both AKT/PKB and PKC pathways. Expression and use of PKCβII switches to that of PKCµ as fibrocytes transition to TAO orbital fibroblasts. This shift is imposed by CD34(-) orbital fibroblasts but reverts when CD34(+) fibroblasts are isolated. The up-regulation of IL-6 by TSH results from coordinately enhanced IL-6 gene promoter activity and increased IL-6 mRNA stability. TSH-dependent IL-6 expression requires activity at both CREB (-213 to -208 nt) and NF-κB (-78 to -62 nt) binding sites. These results provide novel insights into the molecular action of TSH and signaling downstream for TSHR in non-thyroid cells. Fibrocytes neither express adenylate cyclase nor generate cAMP and thus these findings are free from any influence of cAMP-related signaling. They identify potential therapeutic targets for TAO.
Project description:The molecular basis for anatomically dispersed clinical manifestations in Graves' disease (GD) eludes our understanding. Bone marrow-derived, pluripotent fibrocytes represent a subset of peripheral blood mononuclear cells and infiltrate the orbital and thyroid tissues in GD. These cells may be involved in the pathogenesis of thyroid-associated ophthalmopathy (TAO).The objective of the study was to quantify fibrocyte display of functional cell surface TSH receptor (TSHR), identify the profile of chemokines they express after TSHR activation, and determine whether circulating TSHR(+) peripheral blood fibrocytes are more frequent in situ in patients with TAO.Using a newly developed technique, fibrocytes were directly identified in peripheral blood from 31 patients with TAO and 19 healthy subjects receiving care at a multidisciplinary academic center.The frequency in situ of fibrocytes (collagen 1(+), CD45(+), CD34(+), CD14(+), CD86(+) peripheral blood mononuclear cells) was assessed by multiparameter flow cytometry and correlated to clinical disease activity and smoking status. Levels of TSHR-displaying fibrocytes and their response to TSH and TSHR-activating antibody, M22, were measured by flow cytometry, Luminex, and real-time PCR.The levels of TSHR expression by fibrocytes are substantially higher than those found in orbital fibroblasts. Moreover, the frequency of TSHR(+) fibrocytes in patients with TAO was greater than that in healthy subjects in situ. Their abundance is not influenced by disease activity or smoking history. These cells produce high levels of several cytokines and chemokines including IL-8, regulated upon activation, normal T cell expressed and secreted, and monocyte chemoattractant protein-1 when treated with TSH or M22. TSH induces IL-8 production at the pretranslational level. This induced cytokine can be detected in intact fibrocytes ex vivo.Frequency of circulating TSHR(+) fibrocytes is markedly increased in patients with TAO, and they express proinflammatory chemokines in response to TSH. Because they infiltrate both orbit and thyroid in GD, they may represent the link between systemic immunoreactivity and organ-specific autoimmunity.
Project description:CONTEXT:Thyroid-associated ophthalmopathy (TAO) is the component of Graves' disease characterized by orbital inflammation and connective tissue remodeling. The IGF-1 receptor (IGF-1R) and TSH receptor (TSHR) form a physical and functional complex in orbital fibroblasts. A subset of these fibroblasts is derived from infiltrating CD34(+) fibrocytes. Teprotumumab (RV 001, R1507) is a human monoclonal anti-IGF-1R blocking antibody currently undergoing a phase 2 clinical trial in patients with active TAO. OBJECTIVE:To determine whether teprotumumab inhibits the induction by TSH of IL-6 and IL-8 in fibrocytes. DESIGN:Fibrocytes were treated without or with teprotumumab in combination with IGF-1 or TSH. MAIN OUTCOME MEASURES:IL-6 and IL-8 mRNA expression and protein production were analyzed by real-time PCR and Luminex, respectively. Phosphorylated Akt (S473) levels were analyzed by Western blot. TSHR and IGF-1R display was assessed by flow cytometry. RESULTS:Fibrocyte display of IGF-1R and TSHR was reduced with teprotumumab, as were IGF-1- and TSH-dependent phosphorylated Akt levels. TSH induction of IL-6 and IL-8 mRNA and protein was also reduced by the monoclonal antibody. CONCLUSIONS:Teprotumumab attenuates the actions of both IGF-1 and TSH in fibrocytes. Specifically, it blocks the induction of proinflammatory cytokines by TSH. These results provide, at least in part, the molecular rationale for interrogating the therapeutic efficacy of this antibody in TAO.
Project description:Human CD34+ fibrocytes, circulating monocyte lineage progenitor cells, have recently been implicated in thyroid-associated ophthalmopathy (TAO), the ocular manifestation of Graves' disease (GD). Fibrocytes express constitutive MHC class II (MHC-2) and, surprisingly, thyroglobulin (Tg) and functional thyrotropin (TSH) receptor (TSHR). Underlying expression of these thyroid proteins is the autoimmune regulator protein (AIRE). Fibrocytes respond robustly to TSH and thyroid-stimulating Igs by generating extremely high levels of inflammatory cytokines, such as IL-6. In TAO, they appear to infiltrate the orbit, where they transition to CD34+ orbital fibroblasts (OF). There, they coexist with CD34- OF as a mixed fibroblast population (GD-OF). In contrast to fibrocytes, GD-OF express vanishingly low levels of MHC-2, Tg, TSHR, and AIRE. Further, the amplitude of IL-6 induction by TSH in GD-OF is substantially lower. The molecular basis for this divergence between fibrocytes and CD34+ OF remains uncertain. In this article, we report that Slit2, an axon guidance glycoprotein, is constitutively expressed by the CD34- OF subset of GD-OF. Culture conditioned medium (CM) generated by incubating with GD-OF and CD34- OF substantially reduces levels of MHC-2, Tg, TSHR, and AIRE in fibrocytes. Expression can be restored by specifically depleting CM of Slit2. The effects of CD34- OF CM are mimicked by recombinant human Slit2. TSH induces Slit2 levels in GD-OF by enhancing both Slit2 gene transcription and mRNA stability. These findings suggest that Slit2 represents a TSH-inducible factor within the TAO orbit that can modulate the inflammatory phenotype of CD34+ OF and therefore may determine the activity and severity of the disease.
Project description:A relationship between the actions of TSH and IGF-1 was first recognized several decades ago. The close physical and functional associations between their respective receptors (TSHR and IGF-1R) has been described more recently in thyroid epithelium and human orbital fibroblasts as has the noncanonical behavior of IGF-1R. Here we report studies conducted in lung fibroblasts from female wild-type C57/B6 (TSHR(+/+)) mice and their littermates in which TSHR has been knocked out (TSHR(-/-)). Flow cytometric analysis revealed that cell surface IGF-1R levels are substantially lower in TSHR(-/-) fibroblasts compared with TSHR(+/+) fibroblasts. Confocal immunofluorescence microscopy revealed similar divergence with regard to both cytoplasmic and nuclear IGF-1R. Western blot analysis demonstrated both intact IGF-1R and receptor fragments in both cellular compartments. In contrast, IGF-1R mRNA levels were similar in fibroblasts from mice without and with intact TSHR expression. IGF-1 treatment of TSHR(+/+) fibroblasts resulted in reduced nuclear and cytoplasmic staining for IGF-1R?, whereas it enhanced the nuclear signal in TSHR(-/-) cells. In contrast, IGF-1 enhanced cytoplasmic IGF-1R? in TSHR(-/-) fibroblasts while increasing the nuclear signal in TSHR(+/+) cells. These findings indicate the intimate relationship between TSHR and IGF-1R found earlier in human orbital fibroblasts also exists in mouse lung fibroblasts. Furthermore, the presence of TSHR in these fibroblasts influenced not only the levels of IGF-1R protein but also its subcellular distribution and response to IGF-1. They suggest that the mouse might serve as a suitable model for delineating the molecular mechanisms overarching these two receptors.
Project description:CONTEXT:Graves' disease (GD) is caused by persistent, unregulated stimulation of thyrocytes by thyroid-stimulating antibodies (TSAbs) that activate the TSH receptor (TSHR). We previously reported the first small-molecule antagonist of human TSHR and showed that it inhibited receptor signaling stimulated by sera from four patients with GD. OBJECTIVE:Our objective was to develop a better TSHR antagonist and use it to determine whether inhibition of TSAb activation of TSHR is a general phenomenon. DESIGN:We aimed to chemically modify a previously reported small-molecule TSHR ligand to develop a better antagonist and determine whether it inhibits TSHR signaling by 30 GD sera. TSHR signaling was measured in two in vitro systems: model HEK-EM293 cells stably overexpressing human TSHRs and primary cultures of human thyrocytes. TSHR signaling was measured as cAMP production and by effects on thyroid peroxidase mRNA. RESULTS:We tested analogs of a previously reported small-molecule TSHR inverse agonist and selected the best NCGC00229600 for further study. In the model system, NCGC00229600 inhibited basal and TSH-stimulated cAMP production. NCGC00229600 inhibition of TSH signaling was competitive even though it did not compete for TSH binding; that is, NCGC00229600 is an allosteric inverse agonist. NCGC00229600 inhibited cAMP production by 39 ± 2.6% by all 30 GD sera tested. In primary cultures of human thyrocytes, NCGC00229600 inhibited TSHR-mediated basal and GD sera up-regulation of thyroperoxidase mRNA levels by 65 ± 2.0%. CONCLUSION:NCGC00229600, a small-molecule allosteric inverse agonist of TSHR, is a general antagonist of TSH receptor activation by TSAbs in GD patient sera.
Project description:Graves' disease (GD) involves the presence of agonistic auto-antibodies against the thyrotropin receptor (TSHR), which are responsible for the clinical symptoms. While failure of TSHR tolerance is central to GD pathogenesis, the process leading to this failure remains poorly understood. Two mechanisms intimately linked to tolerance have been proposed to explain the association of SNPs located in TSHR intron 1 to GD: (1) differential alternative splicing in the thyroid; and (2) modulation of expression in the thymus. To elucidate the relative contribution to these two mechanisms to GD pathogenesis, we analyzed the level of full-length and ST4 and ST5 isoform expression in the thyroid (n = 49) and thymus (n = 39) glands, and the influence of intron 1-associated SNPs on such expression. The results show that: (1) the level of flTSHR and ST4 expression in the thymus was unexpectedly high (20% that of the thyroid); (2) while flTSHR is the predominant isoform, the levels are similar to ST4 (ratio flTSHR/ST4 = 1.34 in the thyroid and ratio flTSHR/ST4 in the thymus = 1.93); (3) next-generation sequencing confirmed the effect of the TSHR intron 1 polymorphism on TSHR expression in the thymus with a bias of 1.5 ± 0.2 overexpression of the protective allele in the thymus compared to the thyroid; (4) GD-associated intron 1 SNPs did not influence TSHR alternative splicing of ST4 and ST5 in the thyroid and thymus; and (5) three-color confocal imaging showed that TSHR is associated with both thymocytes, macrophages, and dendritic cells in the thymus. Our findings confirm the effect of intron 1 polymorphisms on thymic TSHR expression and we present evidence against an effect on the relative expression of isoforms. The high level of ST4 expression in the thymus and its distribution within the tissue suggest that this would most likely be the isoform that induces central tolerance to TSHR thus omitting most of the hinge and transmembrane portion. The lack of central tolerance to a large portion of TSHR may explain the relatively high frequency of autoimmunity related to TSHR and its clinical consequence, GD.