Journal of Neuro-Ophthalmology, June 2014, Volume 34, Issue 2

The Pathophysiology of Thyroid Eye Disease

The pathophysiology of thyroid eye disease (TED) is complex and incompletely understood. Orbital fibroblasts (OFs) seem to be the key effector cells that are responsible for the characteristic soft tissue enlargement seen in TED. They express potentially pathogenic autoantigens, such as thyrotropin receptor and insulin-like growth factor-1 receptor. An intricate interplay between these autoantigens and the autoantibodies found in Graves disease may lead to the activation of OFs, which then leads to increased hyaluronan production, proinflammatory cytokine synthesis, and enhanced differentiation into either myofibroblasts or adipocytes. Some of the OFs in TED patients seem to be derived from infiltrating fibrocytes. These cells originate from the bone marrow and exhibit both fibroblast and myeloid phenotype. In the TED orbit, they may mediate the orbital expansion and inflammatory infiltration. Last, lymphocytes and cytokines are intimately involved in the initiation, amplification, and maintenance of the autoimmune process in TED.

The Pathophysiology of Thyroid Eye Disease Shannon J. C. Shan, MD, MSc, Raymond S. Douglas, MD, PhD Abstract: The pathophysiology of thyroid eye disease (TED) is complex and incompletely understood. Orbital fibroblasts (OFs) seem to be the key effector cells that are responsible for the characteristic soft tissue enlargement seen in TED. They express potentially pathogenic autoantigens, such as thyrotropin receptor and insulin-like growth factor-1 recep-tor. An intricate interplay between these autoantigens and the autoantibodies found in Graves disease may lead to the activation of OFs, which then leads to increased hyaluronan production, proinflammatory cytokine synthesis, and enhanced differentiation into either myofibroblasts or adi-pocytes. Some of the OFs in TED patients seem to be derived from infiltrating fibrocytes. These cells originate from the bone marrow and exhibit both fibroblast and myeloid phenotype. In the TED orbit, they may mediate the orbital expansion and inflammatory infiltration. Last, lymphocytes and cytokines are intimately involved in the initiation, amplification, and maintenance of the autoim-mune process in TED. Journal of Neuro-Ophthalmology 2014;34:177-185 doi: 10.1097/WNO.0000000000000132 © 2014 by North American Neuro-Ophthalmology Society Thyroid eye disease (TED) is a vision-threatening con-dition that is most commonly associated with Graves disease (GD). Although the mechanism underlying the thyroid gland dysfunction in GD is now relatively well-characterized, the pathophysiology of TED is only begin-ning to be elucidated (Fig. 1). The common finding in TED that accounts for most of its clinical manifestations seems to be enlargement of orbital soft tissues (1). Radiographic evi-dence suggests an increase in the volume of both muscle and orbital fat (2). Histopathologic studies of the TED orbit reveal an extensive deposition of hyaluronan (a hydro-philic glycosaminoglycan) between muscle fibers, a wide-spread inflammatory infiltrate, and an overabundance of cytokines (3,4). These changes lead to interstitial edema and soft tissue expansion. Confined within the rigid orbital walls, such tissue enlargement can lead to increased intra-orbital pressure, mechanical compression of orbital tissue including the optic nerve, and further inflammation (1). ROLE OF ORBITAL FIBROBLASTS The principal cell type responsible for the enlargement of orbital soft tissues in TED seems to be the orbital fibroblast (OF) (5-10). These cells are located in the interstitial space between muscle fibers, and within orbital fat and connective tissues (11). There are 2 subpopulations of OFs, which are classified based on whether or not they express the surface marker Thy1/CD90 (8,9,12,13). Thy1-expressing (Thy1+) OFs reside in the perimysium of the extraocular muscles. When activated, they can differentiate into myofibroblasts, the contractile element found in wound healing (12). Th1-deficient (Thy12) OFs are preadipocytes found throughout the orbit and can differentiate into mature adi-pocytes (8,12,14-17). The relative proportion of activated Thy1+ and Thy12 OFs may determine whether fibrosis or adipogenesis predominates in TED (8,12). In vitro studies have demonstrated that OFs from patients with TED (TED-OFs), more than those from healthy controls, are prone to activation, leading to proliferation, hyaluronan secretion, and soft tissue expansion (18-28). As will be dis-cussed below, some OFs have robust expression of thyrotro-pin receptor (TSHR) and insulin-like growth factor-1 receptor (IGF-1R), 2 autoantigens that are thought to con-tribute to the activation of OFs in TED. Finally, TED-OFs are both more capable of secreting and responding to inflam-matory cytokines compared with controls, possibly leading to amplification of the disease process (8,21,29-34). ROLE OF FIBROCYTES Fibrocytes are bone marrow-derived, fibroblast-like progen-itor cells that circulate in the peripheral blood and may play a role in the pathogenesis of TED. They express the Wilmer Eye Institute (SJCS), The Johns Hopkins University School of Medicine, Baltimore, Maryland; and Kellogg Eye Center (RSD), University of Michigan, Ann Arbor, Michigan. The authors report no conflicts of interest. Address correspondence to Raymond S. Douglas, MD, PhD, Kellogg Eye Center, University of Michigan, 1000 Wall Street, Ann Arbor, MI 48105; E-mail: [e-mail] Shan and Douglas: J Neuro-Ophthalmol 2014; 34: 177-185 177 State-of-the-Art Review Section Editors: Val erie Biousse, MD Steven Galetta, MD Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. hematopoietic stem cell marker CD34 and the leukocyte common antigen CD45, and also various fibroblast proteins such as alpha-smooth muscle actin, collagen I and III, fibro-nectin, and vimentin (35). Fibrocytes are capable of migrat-ing to sites of injury and differentiating into fibroblasts or adipocytes, participating in tissue remodeling and induction of T-cell proliferation (36,37). With both tissue-remodeling properties of fibroblasts, and proinflammatory properties of macrophages, fibrocytes have been implicated in various inflammatory or autoimmune-related fibrotic processes (38). Fibrocytes are significantly more abundant in the periph-eral circulation of patients with GD compared with healthy controls (39). Moreover, they have been shown to infiltrate both the orbital and thyroid tissues in GD patients (39,40). Within the orbit, fibrocytes exhibits remarkable plasticity and, similar to OFs, can differentiate into adipocytes or myofibroblasts (41). Furthermore, fibrocytes can be activated to produce cytokines in a similar manner as OFs. Patients with TED have markedly increased prevalence of CD40+ fibrocytes (42), which, in response to CD40 ligand, can pro-duce interleukin (IL)-6, IL-8, macrophage chemoattractant protein 1, chemokine ligand 5 (CCL5), and tumor necrosis factor alpha (TNF-a), a profile very similar to that of acti-vated OFs (42). Fibrocytes also resemble OFs in that they express both TSHR and IGF-1R on their surface. The poten-tial functional relevance of this finding is discussed below. In FIG. 1. Pathophysiology of thyroid eye disease (TED). Self-tolerance to thyrotropin receptor (TSHR) and insulin-like growth factor-1 receptor (IGF-1R) is lost for unclear reasons. Antigen-presenting cells internalize TSHR and IGF-1R and present them to helper T cells, which become activated and may either induce B cells to produce autoantibodies isolated from serum of GD patients (GD-IgGs), or become autoreactive T cells. GD-IgGs interact with TSHR on thyroid follicular epithelial cells, leading to follicular hyperplasia and hypertrophy. Autoreactive CD4 T cells can travel to orbital tissues in response to T-cell chemoattractants and interact with orbital fibroblasts (OFs). This interaction leads to the mutual activation of both cell types. Various inflammatory cytokines are secreted by T cells, B cells and OFs. Each of these cell types also overexpress IGF-1R, which can interact with GD-IgGs, resulting in cellular activation. On the surface of OFs, IGF-1R and TSHR form a physical and functional complex that interacts with GD-IgGs. Some of the OFs in TED patients may be from infiltrating fibrocytes derived from the bone marrow. Activated OFs can differentiate into either adipocytes or myofibroblasts, and have increased hyaluronan synthesis. Together, these processes lead to the expansion of orbital soft tissues in TED. 178 Shan and Douglas: J Neuro-Ophthalmol 2014; 34: 177-185 State-of-the-Art Review Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. aggregate, the above evidence introduces fibrocytes as a poten-tial player in the pathogenesis of TED. Studies to further delineate the precise role of fibrocytes in TED are ongoing. ROLE OF AUTOANTIGENS OFs become abnormally activated in TED in both an antigen-dependent and antigen-independent manner. Sev-eral potential autoantigens have been identified in TED, although very few show any correlation with the presence or severity of TED (43-49). Nonetheless, 2 proteins that show significant promise as pathogenic autoantigens in TED are TSHR and IGF1-R. Role of Thyrotropin Receptor Thyrotropin receptor and its autoantibodies have a well-established role in the pathogenesis of GD (50). Accumu-lating evidence indirectly implicates them in the pathogen-esis of TED. Autoantibodies against TSHR can be detected in up to 98% of patients with TED (51). Titers of the 2 subtypes of TSHR antibodies, thyroid stimulating immu-noglobulins (TSI), which directly activate TSHR, and TSHR binding inhibitory immunoglobulins (TBII), which prevent TSH from binding TSHR, are both positively cor-related with the clinical activity and severity of TED (47,48,51-53). It is unclear how these autoantibodies with seemingly opposite mechanisms of action on TSHR would positively correlate with each other and with the severity of disease. One hypothesis is that they may serve as a nonspe-cific marker of the B-cell-mediated autoimmune response. The expression of TSHR, once thought to be limited to thyrocytes, has now been reported in a variety of cell types throughout the body, albeit at very low levels (54). Orbital tissues and primary cultures of OFs from patients with TED have increased TSHR expression compared with those from healthy controls (55-58). Moreover, TSHR expres-sion in TED orbital tissues is higher in active disease com-pared with inactive disease (59). Although fibrocytes from both TED patients and healthy controls express TSHR, the fraction of TSHR-expressing fibrocytes is also significantly increased in the peripheral circulation of patients with TED (42). These TSHR-expressing fibrocytes from TED patients have an extremely high expression of TSHR per cell, rival-ing thyrocytes (39,40,42). The mechanism underlying TSHR overexpression in OFs and fibrocytes remains unclear, but the above correlational evidence suggests the possibility that TSHR and its autoantibodies are involved in the pathogenesis of TED. In vitro studies with cultured OFs yield further evidence that TSHR is a pathogenic autoantigen in TED. Treating Thy1-OFs (preadipocytes) with TSH or a stimulatory TSHR antibody, M22, leads to enhanced adipocyte differentiation as evidenced by increased expression of late-adipocyte genes adiponectin and leptin (60). On differ-entiation into mature adipocytes, these cells further increase the expression of TSHR, more so in TED-OFs than con-trols, which may contribute to the maintenance of disease (8,14-16,55,60-62). This TSHR-antibody-mediated acti-vation of TED-OFs can be attenuated by a small molecule antagonist of the TSHR (63). Conversely, OFs transfected with a constitutively active TSHR mutant construct show stimulated hyaluronan production and early differentiation into adipocytes (64,65). The upregulation of TSHR in fibrocytes also seems to have a functional significance, as treatment of these cells with TSH leads to the production of the proinflammatory cytokines, TNF-a and IL-6 (39,40,42). The collective in vitro evidence above suggests that TSHR is a key pathogenic autoantigen in TED. Several in vivo models of GD have been developed in recent decades, using various means to immunize mice with TSHR and induce TSHR antibody production (66-70). Although these models were able to produce hyperthyroid-ism, the orbital soft tissue changes as seen in TED were either not assessed or not present (66-70). An animal model with orbital features analogous to TED was recently re-ported. This model was generated by immunizing female BALB/c mice by in vivo muscle electroporation with the extracellular ligand-binding domain of TSHR (71,72). All immunized mice produced measurable TSHR antibodies, although most produced TBIIs rather than TSIs, and the mice developed hypothyroidism rather than hyperthyroid-ism (71). Nevertheless, immunized mice developed orbital changes that clinically, radiographically, and pathologically resembled those observed in humans with TED (71). This study provides the strongest in vivo evidence to-date supporting an integral role of TSHR in the pathogenesis of TED. Role of IGF-1R Another potentially pathogenic autoantigen in TED is the IGF-1R. This receptor tyrosine kinase and its signaling pathway have a wide spectrum of functions in tissue growth and development, and may participate in the pathogenesis of several metabolic, neoplastic, and immunologic diseases (73-77). The expression of IGF-1R is increased in TED-OFs compared with that in controls (78). The fraction of IGF-1R-expressing fibrocytes also seems to be increased in TED (39). When TED-OFs, but not control OFs, are treated with IGF1, they become activated and upregulate hyaluronan synthesis, similar to the response observed in these cells when treated with TSHR antibodies (25,79). This raises that possibility that IGF1 and TSHR antibodies may act through the same pathway. In addition, after the addition of IGF-1 or autoantibodies isolated from serum of GD patients (GD-IgG), TED-OFs, but not the control OFs, produce 2 powerful T-cell chemoattractants, IL-16 and CCL5 (80,81). On the contrary, recombinant human TSH could not induce this particular response in TED-OFs (80). This suggests that the GD-IgGs may be capable of Shan and Douglas: J Neuro-Ophthalmol 2014; 34: 177-185 179 State-of-the-Art Review Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. activating the TED-OFs through a pathway independent of the TSHR, namely, the IGF-1R pathway (80). There is increasing in vitro evidence now supporting the role of the IGF-1R pathway in the pathophysiology of TED. GD-IgGs can displace IGF1 from its high-affinity binding site on the cell surface of OFs (82). Although this binding site has not been confirmed to be a part of the IGF- 1R, its dissociation constant is similar to that previously reported for IGF-1R (82-85). This suggests that GD-IgGs have an IGF-1R binding component. When IGF-1R function is disrupted in TED-OFs, either through the treat-ment with an IGF-1R blocking antibody or transfection with a dominant negative mutant IGF-1R, the GD-IgG- induced activation of TED-OFs is attenuated (81). There-fore, it seems that the GD-IgGs exert their effects on the TED-OFs at least in part through the IGF-1R pathway. The exact component of GD-IgGs that may be interacting with the IGF-1R is unknown. Autoantibodies against IGF- 1R have been identified but have similar prevalence in TED patients and healthy controls, and the antibody concentra-tion does not correlate with TED severity (86). Two mouse models of GD reported that some mice developed low titers of IGF-1R antibodies after immunization with TSHR (71,87). Interestingly, mice immunized with IGF-1R do not develop any obvious pathology, suggesting the impor-tance of the thyroid autoantigens (71). Finally, it is possible that TSHR autoantibodies are the entities in GD-IgGs that are cross-reacting with IGF-1R. IGF-1R and TSHR may work in a concerted fashion in the pathogenesis of TED. The IGF-1 and TSH have long been known to exert synergistic regulatory influences on target T-cell function, growth, and proliferation (74,88-90). This may in part be explained by the close physical rela-tionship between the 2 receptors (78,91). Immunofluores-cence staining shows that IGF-1R and TSHR colocalize to the perinuclear, cytoplasmic, and plasma membrane com-partments in thyrocytes and OFs (78). Antibodies against either IGF-1R or TSHR can immunoprecipitate both pro-teins (78). Furthermore, an IGF-1R-blocking antibody can also block the signaling initiated by TSH, TSHR stimulating antibody, and GD-IgGs in TED-OFs (78,92).Whether or not these findings are due to antibody cross-reactivity between the 2 receptors is unclear. Studies to further characterize the physical and functional relation-ship between IGF-1R and TSHR and its implications in TED are ongoing. ROLE OF LYMPHOCYTES It is not known what initiates the immune response against autoantigens in autoimmune diseases. Factors such as susceptible genetic polymorphisms and environmental triggers such as infection have been proposed to contribute to this process in TED, but none have been definitively proven (93). Both T cells and B cells are intimately involved in the autoimmune response. Antigen-presenting cells pres-ent a pathogenic epitope of an autoantigen to CD4+ helper T cells, leading to T-cell activation and proliferation. The activated T cells may then either induce and sustain B cells to produce antibodies against the autoantigen, or be involved directly as autoreactive T cells in inflammation and/or cellular destruction (94). The tissue damage in auto-immune diseases arise from either direct attack by autoanti-bodies or autoreactive T cells, immune complex formation, or from local inflammation (94). Autoantibodies may also bind to receptors on target cells, causing enhanced activa-tion or suppression of their signaling pathways (e.g., TSHR antibodies), leading to cellular dysfunction (94). All of the aforementioned autoimmune processes likely partake in the pathophysiology of TED. Current evidence sheds light on a few more specific ways in which the T cells and TED-OF interact. Activated TED-OFs can produce potent T-cell chemoattractants, IL-16 and CCL5, facilitat-ing the recruitment of T cells to the orbit (80,81). Once there, the T cells can reciprocate and activate TED-OFs either through cell-cell interaction or through diffusible cytokines. For example, the CD4+ T cells express CD145 (also known as CD40 ligand) on their cell surface. This ligand binds to CD40, a T-cell costimulatory protein ex-pressed on the surface of TED-OFs in a higher amount as compared with controls (33). When treated with the cyto-kine interferon g (IFN g), TED-OFs increase their expres-sion of CD40 even further (33,95). The binding of CD145 to CD40 triggers the activation of both the T cells and the TED-OFs. Activation of the T cells allows for the develop-ment of their effector functions including induction of B-cell differentiation and activation of monocytes and mac-rophages (96). The CD40-CD154-induced activation of TED-OFs lead to cell proliferation (97), increased synthesis of hyaluronan and prostaglandins (21), and production of proinflammatory cytokines including IL-6, IL-8, and mac-rophage chemoattractant protein-1 (33,95). These T-cell- mediated events contribute to the soft tissue remodeling and local inflammatory response in TED. The principal functions of B cells include antibody production, antigen presentation, and cytokine production. B-cell-deficient mice fail to generate T-cell-mediated responses after immunization with TSHR (98). Therefore, B cells are indispensible to the initiation of the autoimmune process in GD. Rituximab is a monoclonal antibody that binds to the B-cell surface antigen CD20. The treatment of B cells with rituximab leads to the attenuation of CD20- dependent B-cell maturation, and reduced B-cell-mediated antigen presentation and cytokine production (99). More nonrandomized trials and case series have suggested that rituximab can induce lasting clinical improvement in TED (100-106). This confirms the critical role of B cells in the pathogenesis of TED. Several randomized controlled clinical trials are underway to further assess the efficacy and safety of this novel therapy for TED. 180 Shan and Douglas: J Neuro-Ophthalmol 2014; 34: 177-185 State-of-the-Art Review Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. The expression of IGF-1R on T cells and B cells may further contribute to the autoimmune response against OFs in TED. A significantly higher fraction of the T-cells and B cells in GD patients expresses IGF-1R as compared with controls (107,108). This is evident in T cells from the blood and orbit (107), and in B cells from the blood, orbit, and bone marrow in these patients (108). Studies on discordant monozygotic twins show that this increase in the fraction of IGF-1R+ T cells and B cells in GD is not due to genetic determinants (109). For the T cells, display of IGF-1R protects these cells from apoptosis and promotes survival (107). For the B cells, increased IGF-1R display facilitates clonal expansion and propagates antibody production (108). These IGF-1R-mediated effects may contribute to the maintenance of the autoimmune response in TED. ROLE OF CYTOKINES Cytokines are small secreted proteins that are responsible for modulating the immune system. They are produced by each of the cell types discussed in this review. The aberrantly abundant expression of cytokines plays a central role in the pathogenesis of TED (110-116). There are 2 groups of cytokines: Th1-type (also known as type-1) cytokines produce proinflammatory responses, whereas Th2-type (also known as type-2) cytokines are essentially anti-inflammatory but can influence the production of antibodies (117). Different cyto-kine profiles are found in orbital tissues from TED patient with different stages of disease (111,118). In the early active stage of TED, Th1 cytokines such as IL-2, IFN-g, TNF-a predominate (119). These proinflammatory cyto-kines can recruit more immune cells and amplify the immune response within the orbit (59,118,120). In the later, more inactive, stage of TED, Th2 cytokines such as IL-4, IL-6, IL-10, IL-13 predominate, and collectively, they stimulate B-cell proliferation and maturation into plasma cells, increas-ing antibody production (111,118,119). Furthermore, Th2-dominanted inflammatory responses have been well-documented to lead to fibrosis in various tissues including the heart, liver, and lungs (121-128). Thus, the overexpres-sion of Th2 cytokines may also contribute to the fibrotic changes seen in the later-stage of TED. An integral event in the pathogenesis of TED is cytokine-mediated activation of the OFs. Interestingly, OFs in culture exhibit a phenotype distinct from fibroblasts derived from other tissues such as the skin or lung: they show more exaggerated inflammatory response to cytokines (8,10,97,129). The TED-OFs and normal OFs have simi-larly exuberant response to activation by proinflammatory cytokines. Their activation by IL-1a, TGF-b, and leukor-egulin leads to drastically increased synthesis of hyaluronan (19,20,24,27) and prostaglandins (29,30). Other activating effects of the cytokines on normal and TED-OFs have also been demonstrated. TGF-b can stimulate the differentia-tion of the Thy1+ subgroup OFs into myofibroblasts (12). IL-6 can promote adipogenesis and increase the expression of TSHR on Thy1+ OFs (61). Nevertheless, normal and TED-OFs have been shown to respond differently to a few cytokines. The proliferative capacity of TED-OFs was enhanced significantly more than normals in response to cytokines IL-1, IL-4, IGF-1, TGF-b, and platelet-derived growth factor (28). Furthermore, IL-1 stimulated hyalur-onan secretion much more in TED-OFs than in normal OFs (26). Thus, TED-OFs may be even more sensitized to activational cues from cytokines than the already highly responsive normal OFs. Another unique phenotype of the OFs is that on activation, they are fully capable of cytokine expression. TED-OFs produce higher levels of the proinflammatory cytokine IL-1 and lower levels of the neutralizing interleukin-1-receptor antagonist compared with normal OFs (130). This imbalance may lead to poorly opposed IL-1 signaling and an exaggerated inflammatory response. Other proinflammatory cytokines produced by activated TED-OFs include IL-6, IL-8, and macrophage chemoat-tractant protein-1 (8,33). Moreover, TED-OFs, but not normal OFs, express high levels of T-cell chemoattractants IL-16 and CCL5 when activated by treatment with IL-1 (34) or GD-IgGs (80), recruiting more inflammatory cells to the local tissue. Last, when treated with leukoregulin, IL-1, or recombinant CD40 ligand, TED-OFs are induced to express extremely high levels of prostaglandin E2, which is a potent mediator of inflammation (21,29-32). Collec-tively, this evidence solidifies the role of the OFs as the key effector cells in TED with a pronounced ability to respond to activating signals and a propensity to produce more proinflammatory signals of their own, hence generating the vicious cycle where inflammation begets more inflammation. CONCLUSIONS TED is an enigmatic vision-threatening autoimmune condition. Although our understanding of its pathophysi-ology has grown significantly in recent years, much remains to be discovered. Current evidence supports a central role of the OFs in the pathogenesis of TED. They become aberrantly and robustly activated in TED. The mechanism underlying this activation likely involves the autoantigens TSHR and IGF-1R and the GD autoantibodies, as well as interaction with immune cells and proinflammatory cyto-kines. Fibrocytes are progenitor cells that infiltrate the orbit and differentiate into OFs in TED, contributing to the pathogenesis of TED. As the disease mechanisms of TED continue to become elucidated, therapies that alter the course of disease can be developed. REFERENCES 1. Bahn RS. Graves' ophthalmopathy. N Engl J Med. 2010;362:726-738. Shan and Douglas: J Neuro-Ophthalmol 2014; 34: 177-185 181 State-of-the-Art Review Copyright © North American Neuro-Ophthalmology Society. Unauthorized reproduction of this article is prohibited. 2. Forbes G, Gorman CA, Brennan MD, Gehring DG, Ilstrup DM, Earnest F. Ophthalmopathy of Graves' disease: computerized volume measurements of the orbital fat and muscle. AJNR Am J Neuroradiol. 1986;7:651-656. 3. 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