BACKGROUND Thyroid-associated ophthalmopathy, a condition commonly associated with Graves' diseas... more BACKGROUND Thyroid-associated ophthalmopathy, a condition commonly associated with Graves' disease, remains inadequately treated. Current medical therapies, which primarily consist of glucocorticoids, have limited efficacy and present safety concerns. Inhibition of the insulin-like growth factor I receptor (IGF-IR) is a new therapeutic strategy to attenuate the underlying autoimmune pathogenesis of ophthalmopathy. METHODS We conducted a multicenter, double-masked, randomized, placebo-controlled trial to determine the efficacy and safety of teprotumumab, a human monoclonal antibody inhibitor of IGF-IR, in patients with active, moderate-to-severe ophthalmopathy. A total of 88 patients were randomly assigned to receive placebo or active drug administered intravenously once every 3 weeks for a total of eight infusions. The primary end point was the response in the study eye. This response was defined as a reduction of 2 points or more in the Clinical Activity Score (scores range from 0 to 7, with a score of ≥3 indicating active thyroid-associated ophthalmopathy) and a reduction of 2 mm or more in proptosis at week 24. Secondary end points, measured as continuous variables, included proptosis, the Clinical Activity Score, and results on the Graves' ophthalmopathy-specific quality-of-life questionnaire. Adverse events were assessed. RESULTS In the intention-to-treat population, 29 of 42 patients who received teprotumumab (69%), as compared with 9 of 45 patients who received placebo (20%), had a response at week 24 (P<0.001). Therapeutic effects were rapid; at week 6, a total of 18 of 42 patients in the teprotumumab group (43%) and 2 of 45 patients in the placebo group (4%) had a response (P<0.001). Differences between the groups increased at subsequent time points. The only drug-related adverse event was hyperglycemia in patients with diabetes; this event was controlled by adjusting medication for diabetes. CONCLUSIONS In patients with active ophthalmopathy, teprotumumab was more effective than placebo in reducing proptosis and the Clinical Activity Score. (Funded by River Vision Development and others; ClinicalTrials.gov number, NCT01868997.
The thyroid gland secretes primarily tetraiodothyronine (T4), and some triiodothyronine (T3). Und... more The thyroid gland secretes primarily tetraiodothyronine (T4), and some triiodothyronine (T3). Under normal physiological circumstances, only one-fifth of circulating T3 is directly released by the thyroid, but in states of hyperactivation of thyroid-stimulating-hormone receptors (TSHRs), patients develop a syndrome of relative T3-toxicosis. Thyroidal T4 production results from iodination of thyroglobulin (TG) at residues Tyr5 and Tyr130, whereas thyroidal T3 production may originate in several different ways. In this study, the data demonstrate that within the carboxyl-terminal portion of mouse TG, T3 is formed de novo independently of deiodination from T4. We found that upon iodination in vitro, de novo T3 formation in TG was decreased in mice lacking TSHRs. Conversely, de novo T3 that can be formed upon iodination of TG secreted from PCCL3 (rat thyrocyte) cells was augmented from cells previously exposed to increased TSH, a TSHR agonist, a cAMP analog, or a TSHR-stimulating antibo...
Thyroid-associated ophthalmopathy, a condition commonly associated with Graves' disease, rema... more Thyroid-associated ophthalmopathy, a condition commonly associated with Graves' disease, remains inadequately treated. Current medical therapies, which primarily consist of glucocorticoids, have limited efficacy and present safety concerns. Inhibition of the insulin-like growth factor I receptor (IGF-IR) is a new therapeutic strategy to attenuate the underlying autoimmune pathogenesis of ophthalmopathy. We conducted a multicenter, double-masked, randomized, placebo-controlled trial to determine the efficacy and safety of teprotumumab, a human monoclonal antibody inhibitor of IGF-IR, in patients with active, moderate-to-severe ophthalmopathy. A total of 88 patients were randomly assigned to receive placebo or active drug administered intravenously once every 3 weeks for a total of eight infusions. The primary end point was the response in the study eye. This response was defined as a reduction of 2 points or more in the Clinical Activity Score (scores range from 0 to 7, with a sc...
The thyroid gland is a butterfly-shaped endocrine gland that is located in the lower front of the... more The thyroid gland is a butterfly-shaped endocrine gland that is located in the lower front of the neck. The thyroid makes thyroid hormones, which are secreted into the blood and then carried to every tissue in the body. Thyroid hormones help the body use energy, stay warm and keep the brain, heart, muscles, and other organs working appropriately. WHAT IS GRAVES' DISEASE? Graves' disease is an autoimmune disease that leads to a generalized overactivity of the entire thyroid gland (hyperthyroidism). It is the most common cause of hyperthyroidism in the United States. It is named after Robert Graves, an Irish physician, who described this form of hyperthyroidism about 150 years ago. It is 7-8 times more common in women than men.
A hallmark of regulatory B cells is IL-10 production, hence their designation as IL-10+ B cells. ... more A hallmark of regulatory B cells is IL-10 production, hence their designation as IL-10+ B cells. Little is known about the ability of self-antigens to induce IL-10+ B cells in Graves' disease (GD), Hashimoto's thyroiditis (HT), or other autoimmune disease. Here we pulsed purified B cells from 12 HT patients, 12 GD patients, and 12 healthy donors with the thyroid self-antigen, thyroglobulin (TG) and added the B cells back to the remaining peripheral blood mononuclear cells (PBMCs). This procedure induced IL-10+ B-cell differentiation in GD. A similar tendency was observed in healthy donors, but not in cells from patients with HT. In GD, B cells primed with TG induced IL-10-producing CD4+ T cells. To assess the maximal frequency of inducible IL-10+ B cells in the three donor groups PBMCs were stimulated with PMA/ionomycin. The resulting IL-10+ B-cell frequency was similar in the three groups and correlated with free T3 levels in GD patients. IL-10+ B cells from both patient gr...
The IGF-1R is required for optimal growth in vivo and in vitro; it plays a crucial role in the es... more The IGF-1R is required for optimal growth in vivo and in vitro; it plays a crucial role in the establishment and maintenance of the transformed phenotype and it protects tumor cells from apoptosis both in vivo and in vitro.
Graves' disease, an autoimmune process associated with thyroid dysfunction, can also manifest as ... more Graves' disease, an autoimmune process associated with thyroid dysfunction, can also manifest as remodeling of orbital connective tissue. Affected tissues exhibit immune responses that appear to be orchestrated by resident cells and those recruited from the bone marrow through their expression and release of cytokines and surface display of cytokine receptors. Cytokines are small molecules produced by many types of cells, including those of the ''professional'' immune system. Aberrant cytokine expression appears to play an important role in the pathogenesis of many human diseases, including thyroid autoimmunity. The skewed pattern of cytokine expression in the thyroid, including the T helper cell bias, may condition the response to apoptotic signals and determine the characteristics of an autoimmune reaction. Furthermore, chemoattractant cytokines, including IL16, RANTES, and CXCL10, elaborated by resident cells in the thyroid and orbit may provoke mononuclear cell infiltration. Other cytokines may drive cell activation and tissue remodeling. Thus cytokines and the signaling pathways they activate represent attractive therapeutic targets. Interruption of these might alter the natural course of Graves' disease and its orbital manifestations.
Purpose-To study the effectiveness of anti-CD20 (Rituximab, RTX, Rituxan®, Genentech Inc. USA) th... more Purpose-To study the effectiveness of anti-CD20 (Rituximab, RTX, Rituxan®, Genentech Inc. USA) therapy in patients with severe, corticosteroid (CS)-resistant thyroid-associated ophthalmopathy (TAO). Design-Retrospective interventional case series Participants-Six consecutive subjects with severe, progressive TAO unresponsive to CS. Methods-Electronic medical record review of consecutive patients receiving RTX during the previous 18 months. Responses to therapy were graded using standard clinical assessment and flowcytometric analysis of peripheral lymphocytes. Main outcome measures-Clinical activity score (CAS), proptosis, strabismus, treatment sideeffects, and quantification of regulatory T cells. Results-Six patients were studied. Systemic CS failed to alter clinical activity in all patients (CAS= 5.3 + 1.0 (mean + standard deviation) before vs 5.5 + 0.8 during therapy for 7.5+ 6.4 months,
The thyroid target Ag for disease-inducing autoantibodies in Graves’ disease is the receptor for ... more 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 ...
Human orbital fibroblasts exhibit a unique inflammatory phenotype. In the present study, we repor... more Human orbital fibroblasts exhibit a unique inflammatory phenotype. In the present study, we report that these fibroblasts, when treated with IL-1β, express high levels of IL-6, a cytokine involved in B cell activation and the regulation of adipocyte metabolism. The magnitude of this induction is considerably greater than that in dermal fibroblasts and involves up-regulation of IL-6 mRNA levels. IL-1β activates both p38 and ERK 1/2 components of the MAPK pathways. Disrupting these could attenuate the IL-6 induction. The up-regulation involves enhanced IL-6 gene promoter activity and retardation of IL-6 mRNA decay by IL-1β. Dexamethasone completely blocked the effect of IL-1β on IL-6 expression. Orbital fibroblasts also express higher levels of IL-6R than do skin-derived cells. When treated with rIL-6 (10 ng/ml), STAT3 is transiently phosphorylated. Thus, the exaggerated capacity of orbital fibroblasts to express high levels of both IL-6 and its receptor in an anatomic site-selective ...
Graves’ disease (GD) is associated with T cell infiltration, but the mechanism for lymphocyte tra... more Graves’ disease (GD) is associated with T cell infiltration, but the mechanism for lymphocyte trafficking has remained uncertain. We reported previously that fibroblasts from patients with GD express IL-16, a CD4-specific chemoattractant, and RANTES, a C-C chemokine, in response to GD-specific IgG (GD-IgG). We unexpectedly found that these responses result from a functional interaction between GD-IgG and the insulin-like growth factor (IGF)-I receptor (IGF-IR). IGF-I and the IGF-IR-specific IGF-I analog, des(1–3), mimic the effects of GD-IgG. Neither GD-IgG nor IGF-I activates chemoattractant expression in control fibroblasts from donors without GD. Interrupting IGF-IR function with specific receptor-blocking Abs or by transiently transfecting fibroblasts with a dominant negative mutant IGF-IR completely attenuates signaling provoked by GD-IgG. Moreover, GD-IgG displaces specific 125I-labeled IGF-I binding to fibroblasts and attenuates IGF-IR detection by flow cytometry. These findi...
We have reported recently that IgG from patients with Graves’ disease (GD) can induce the express... more We have reported recently that IgG from patients with Graves’ disease (GD) can induce the expression of the CD4-specific T lymphocyte chemoattractant, IL-16, and RANTES, a C-C chemokine, in their fibroblasts. This induction is mediated through the insulin-like growth factor-1 receptor (IGF-1R) pathway. We now report that Abs from individuals with active rheumatoid arthritis (RA-IgG) stimulate in their synovial fibroblasts the expression of these same cytokines. IgG from individuals without known autoimmune disease fails to elicit this chemoattractant production. Furthermore, RA-IgG fails to induce IL-16 or RANTES expression in synovial fibroblasts from donors with osteoarthritis. RA-IgG-provoked IL-16 and RANTES production also appears to involve the IGF-1R because receptor-blocking Abs prevent the response. RA fibroblasts transfected with a dominant-negative mutant IGF-1R fail to respond to RA-IgG. IGF-1 and the IGF-1R-specific analog Des(1–3) also induce cytokine production in RA ...
Thyroid-associated ophthalmopathy (TAO), an autoimmune component of Graves’ disease, is associate... more Thyroid-associated ophthalmopathy (TAO), an autoimmune component of Graves’ disease, is associated with profound connective tissue remodeling and fibrosis that appear to involve the selective activation of orbital fibroblasts. Accumulation of extracellular matrix molecules is a hallmark of this process. Here we report that orbital fibroblasts treated with IL-1β express high levels of tissue inhibitor of metalloproteinase-1 (TIMP-1), an important modulator of matrix metalloproteinase activity. These high levels are associated with increased TIMP-1 activity. The induction is mediated at the pretranslational level and involves activating the TIMP-1 gene promoter. IL-1β activates the ERK 1/2 pathway in these fibroblasts and interrupting this signaling either with PD98059, a chemical inhibitor of MEK, or by transfecting cells with a dominant negative ERK 1 plasmid results in the attenuation of TIMP-1 induction. Surprisingly, treatment with IL-4 or IFN-γ could also block the IL-1β inducti...
Human fibroblasts can express numerous regulatory molecules that influence immune function. IL-16... more Human fibroblasts can express numerous regulatory molecules that influence immune function. IL-16, a ligand for CD4, is a chemoattractant molecule expressed by lymphocytes, eosinophils, mast cells, and lung epithelium. It appears that the sole target for IL-16 is the CD4-bearing cell. Here we demonstrate that fibroblasts from several tissues can express IL-16 mRNA and protein as well as IL-16-dependent chemoattractant activity. The transcript is expressed abundantly under basal culture conditions as a 2.5-kb band on Northern analysis, similar to that observed in lymphocytes. IL-16 protein and activity are undetectable in fibroblast cultures under these same control conditions. However, when treated with proinflammatory cytokines such as IL-1β, they express very high levels of IL-16 protein and chemoattractant activity, a substantial component of which can be blocked with IL-16-neutralizing Abs. The amount of IL-16 protein released into the medium is 3- to 4-fold greater, on a per ce...
Thyroid-associated ophthalmopathy and dermopathy are connective tissue manifestations of Graves’ ... more Thyroid-associated ophthalmopathy and dermopathy are connective tissue manifestations of Graves’ disease (GD). Tissue remodeling is a prominent feature of both and is apparently driven by recruited T cells. In this study, we report that IgG isolated from patients with GD (GD-IgG) up-regulates T lymphocyte chemoattractant activity in GD-derived fibroblasts from orbit, thyroid, and several regions of skin. This chemoattractant activity, absent in fibroblasts from donors without known thyroid disease, is partially susceptible to neutralization by anti-IL-16 and anti-RANTES Abs. IL-16 is a CD4+-specific chemoattractant and RANTES is a C-C-type chemokine. IL-16 and RANTES protein levels, as determined by specific ELISAs, are substantially increased by GD-IgG in GD fibroblasts. Addition of the macrolide, rapamycin, to fibroblast culture medium blocked the up-regulation by GD-IgG of IL-16, implicating the FRAP/mTOR/p70s6k pathway in the induction of IL-16 expression. These findings suggest...
Graves’ disease (GD), an autoimmune process involving thyroid and orbital tissue, is associated w... more Graves’ disease (GD), an autoimmune process involving thyroid and orbital tissue, is associated with lymphocyte abnormalities including expansion of memory T cells. Insulin-like growth factor receptor-1 (IGF-1R)-bearing fibroblasts overpopulate connective tissues in GD. IGF-1R on fibroblasts, when ligated with IgGs from these patients, results in the expression of the T cell chemoattractants, IL-16 and RANTES. We now report that a disproportionately large fraction of peripheral blood T cells express IGF-1R (CD3+IGF-R+). CD3+IGF-1R+ T cells comprise 48 ± 4% (mean ± SE; n = 33) in patients with GD compared with 15 ± 3% (n = 21; p < 10−8) in controls. This increased population of IGF-1R+ T cells results, at least in part, from an expansion of CD45RO+ T cells expressing the receptor. In contrast, the fraction of CD45RA+IGF-1R+ T cells is similar in GD and controls. T cells harvested from affected orbital tissues in GD reflect similar differences in the proportion of IGF-1R+CD3+ and I...
The Journal of Clinical Endocrinology & Metabolism, 2014
Context: The IL-1 family plays important roles in normal physiology and mediates inflammation. Th... more Context: The IL-1 family plays important roles in normal physiology and mediates inflammation. The actions of IL-1 are modulated by multiple IL-1 receptor antagonists (IL-1RA), including intracellular and secreted forms. IL-1 has been implicated in autoimmunity, such as that occurring in Graves' disease (GD) and its inflammatory orbital manifestation, thyroid-associated ophthalmopathy (TAO). We have previously reported that CD34 ϩ fibrocytes, monocyte-lineage bone marrowderived cells, express functional TSH receptor, the central antigen in GD. When activated by TSH, they produce IL-6, IL-8, and TNF-␣. Moreover, they infiltrate the orbit in TAO in which they transition into CD34 ϩ fibroblasts and comprise a population of orbital fibroblasts (OFs). Little is known currently about any relationship between TSH, TSH receptor, and the IL-1 pathway. Objective: The objective of the study was to determine whether TSH regulates IL-1RA in fibrocytes and OFs. Design/Setting/Participants: Fibrocytes and OFs were collected and analyzed from healthy individuals and those with GD in an academic clinical practice. Main Outcome Measures: Real-time PCR, Western blot analysis, reporter gene assays, and cell transfections were performed. Results: TSH induces the expression of IL-1RA in fibrocytes and GD-OFs. The patterns of induction diverge quantitatively and qualitatively in the two cell types. This results from relatively small effects on gene transcription-related events but a greater influence on secreted IL-1RA and intracellular IL-1RA mRNA stabilities. These actions of TSH are dependent on the intermediate induction of IL-1␣ and IL-1. Conclusions: Our findings for the first time directly link activities of the TSH and IL-1 pathways. Furthermore, they identify novel molecular interactions that could be targeted as therapy for TAO. (J Clin Endocrinol Metab 99: E625-E633, 2014) T he IL-1 family comprises multiple members, including the isoforms of IL-1 receptor antagonist (IL-1RA), which modulates the activities of IL-1␣ and IL-1 (1-3). These related proteins are synthesized in response to a diverse set of factors that vary with cell-type (3). IL-1RA competitively binds the IL-1 receptor and attenuates downstream signaling. Four different IL-1RA forms have been identified, including the secreted isoform (sIL-1RA) and three intracellular proteins (icIL-1RA) (1). Transcripts encoding sIL-1RA and icIL-1RA differ only in the sequence extending from ϩ4 nt to ϩ63 nt and encoding a 21-amino acid peptide present in sIL-1RA. Two distinct gene promoters drive the expression of these proteins (4).
The Journal of Clinical Endocrinology & Metabolism, 2013
Context: Thyroid-associated ophthalmopathy (TAO) manifests as inflammation of orbital connective ... more Context: Thyroid-associated ophthalmopathy (TAO) manifests as inflammation of orbital connective tissue. Bone marrow-derived CD34 ϩ fibrocytes infiltrate the orbit in TAO where they become CD34 ϩ orbital fibroblasts. They express thyroid-specific antigens and thus may contribute to inflammation. Evidence suggests that orbital susceptibility to TAO may involve IL-1, which is modulated by IL-1 receptor antagonists, designated secreted (sIL-1RA) and intracellular (icIL-1RA). Objective: We sought to characterize the expression of sIL-1RA and icIL-1RA in TAO orbital fibroblasts compared to CD34 ϩ fibrocytes. Design/Setting/Participants: Patients with TAO and healthy donors were recruited from an academic medical center clinical practice. Main Outcome Measures: Real-time PCR, cytokine-specific ELISA, gene promoter activities, transcriptional analysis, mRNA stability, and cytometric cell sorting were performed. Results: Orbital fibroblasts treated with IL-1 exhibit greater inductions of IL-1␣, IL-1, and prostaglandin endoperoxide H synthase-2 transcripts than do fibrocytes. Fibrocytes express dramatically higher basal levels of both icIL-1RA and sIL-1RA. When treated with IL-1, icIL-1RA is induced in orbital fibroblasts but not sIL-1RA, whereas in fibrocytes, sIL-1RA is dominantly up-regulated. These inductions result from increased steady-state levels of respective mRNAs, enhanced transcript stabilities, and modestly increased gene transcription. Conclusions: Robust responses of TAO orbital fibroblasts to IL-1 are a consequence of low-level sIL-1RA expression. This results in poorly opposed actions of IL-1. In contrast, circulating fibrocytes express high levels of sIL-1RA, which are diminished as these cells transition to orbital fibroblasts. These findings identify an explanation for the inflammatory phenotype exhibited by TAO orbital fibroblasts and provide a potential target for altering disease susceptibility.
The Journal of Clinical Endocrinology & Metabolism, 2004
A distinctive histopathological feature associated with thyroid-associated ophthalmopathy is the ... more A distinctive histopathological feature associated with thyroid-associated ophthalmopathy is the disordered accumulation of the glycosaminoglycan, hyaluronan, in orbital connective tissues. This often occurs in the context of dramatic inflammation and tissue remodeling. Orbital fibroblasts exhibit a novel phenotype including exaggerated responses to cytokines. Here, we report for the first time the ability of IgG isolated from the sera of patients with Graves' disease (GD-IgG) to provoke in orbital fibroblasts the synthesis of hyaluronan. The effect of GD-IgG can be reproduced by IGF-I, appears to be mediated through the IGF-I receptor, and is abolished with glucocorticoid treatment. TSH failed to influence the synthesis of hyaluronan. In contrast to the effects in GD fibroblasts, cultures derived from donors without known thyroid disease fail to respond to GD-IgG or IGF-I. The observation that hyaluronan production is induced by GD-IgG in fibroblasts suggests that the IGF-I receptor and its activating antibodies may represent a key pathway through which important pathogenic events in thyroid-associated ophthalmopathy are mediated.
BACKGROUND Thyroid-associated ophthalmopathy, a condition commonly associated with Graves' diseas... more BACKGROUND Thyroid-associated ophthalmopathy, a condition commonly associated with Graves' disease, remains inadequately treated. Current medical therapies, which primarily consist of glucocorticoids, have limited efficacy and present safety concerns. Inhibition of the insulin-like growth factor I receptor (IGF-IR) is a new therapeutic strategy to attenuate the underlying autoimmune pathogenesis of ophthalmopathy. METHODS We conducted a multicenter, double-masked, randomized, placebo-controlled trial to determine the efficacy and safety of teprotumumab, a human monoclonal antibody inhibitor of IGF-IR, in patients with active, moderate-to-severe ophthalmopathy. A total of 88 patients were randomly assigned to receive placebo or active drug administered intravenously once every 3 weeks for a total of eight infusions. The primary end point was the response in the study eye. This response was defined as a reduction of 2 points or more in the Clinical Activity Score (scores range from 0 to 7, with a score of ≥3 indicating active thyroid-associated ophthalmopathy) and a reduction of 2 mm or more in proptosis at week 24. Secondary end points, measured as continuous variables, included proptosis, the Clinical Activity Score, and results on the Graves' ophthalmopathy-specific quality-of-life questionnaire. Adverse events were assessed. RESULTS In the intention-to-treat population, 29 of 42 patients who received teprotumumab (69%), as compared with 9 of 45 patients who received placebo (20%), had a response at week 24 (P<0.001). Therapeutic effects were rapid; at week 6, a total of 18 of 42 patients in the teprotumumab group (43%) and 2 of 45 patients in the placebo group (4%) had a response (P<0.001). Differences between the groups increased at subsequent time points. The only drug-related adverse event was hyperglycemia in patients with diabetes; this event was controlled by adjusting medication for diabetes. CONCLUSIONS In patients with active ophthalmopathy, teprotumumab was more effective than placebo in reducing proptosis and the Clinical Activity Score. (Funded by River Vision Development and others; ClinicalTrials.gov number, NCT01868997.
The thyroid gland secretes primarily tetraiodothyronine (T4), and some triiodothyronine (T3). Und... more The thyroid gland secretes primarily tetraiodothyronine (T4), and some triiodothyronine (T3). Under normal physiological circumstances, only one-fifth of circulating T3 is directly released by the thyroid, but in states of hyperactivation of thyroid-stimulating-hormone receptors (TSHRs), patients develop a syndrome of relative T3-toxicosis. Thyroidal T4 production results from iodination of thyroglobulin (TG) at residues Tyr5 and Tyr130, whereas thyroidal T3 production may originate in several different ways. In this study, the data demonstrate that within the carboxyl-terminal portion of mouse TG, T3 is formed de novo independently of deiodination from T4. We found that upon iodination in vitro, de novo T3 formation in TG was decreased in mice lacking TSHRs. Conversely, de novo T3 that can be formed upon iodination of TG secreted from PCCL3 (rat thyrocyte) cells was augmented from cells previously exposed to increased TSH, a TSHR agonist, a cAMP analog, or a TSHR-stimulating antibo...
Thyroid-associated ophthalmopathy, a condition commonly associated with Graves' disease, rema... more Thyroid-associated ophthalmopathy, a condition commonly associated with Graves' disease, remains inadequately treated. Current medical therapies, which primarily consist of glucocorticoids, have limited efficacy and present safety concerns. Inhibition of the insulin-like growth factor I receptor (IGF-IR) is a new therapeutic strategy to attenuate the underlying autoimmune pathogenesis of ophthalmopathy. We conducted a multicenter, double-masked, randomized, placebo-controlled trial to determine the efficacy and safety of teprotumumab, a human monoclonal antibody inhibitor of IGF-IR, in patients with active, moderate-to-severe ophthalmopathy. A total of 88 patients were randomly assigned to receive placebo or active drug administered intravenously once every 3 weeks for a total of eight infusions. The primary end point was the response in the study eye. This response was defined as a reduction of 2 points or more in the Clinical Activity Score (scores range from 0 to 7, with a sc...
The thyroid gland is a butterfly-shaped endocrine gland that is located in the lower front of the... more The thyroid gland is a butterfly-shaped endocrine gland that is located in the lower front of the neck. The thyroid makes thyroid hormones, which are secreted into the blood and then carried to every tissue in the body. Thyroid hormones help the body use energy, stay warm and keep the brain, heart, muscles, and other organs working appropriately. WHAT IS GRAVES' DISEASE? Graves' disease is an autoimmune disease that leads to a generalized overactivity of the entire thyroid gland (hyperthyroidism). It is the most common cause of hyperthyroidism in the United States. It is named after Robert Graves, an Irish physician, who described this form of hyperthyroidism about 150 years ago. It is 7-8 times more common in women than men.
A hallmark of regulatory B cells is IL-10 production, hence their designation as IL-10+ B cells. ... more A hallmark of regulatory B cells is IL-10 production, hence their designation as IL-10+ B cells. Little is known about the ability of self-antigens to induce IL-10+ B cells in Graves' disease (GD), Hashimoto's thyroiditis (HT), or other autoimmune disease. Here we pulsed purified B cells from 12 HT patients, 12 GD patients, and 12 healthy donors with the thyroid self-antigen, thyroglobulin (TG) and added the B cells back to the remaining peripheral blood mononuclear cells (PBMCs). This procedure induced IL-10+ B-cell differentiation in GD. A similar tendency was observed in healthy donors, but not in cells from patients with HT. In GD, B cells primed with TG induced IL-10-producing CD4+ T cells. To assess the maximal frequency of inducible IL-10+ B cells in the three donor groups PBMCs were stimulated with PMA/ionomycin. The resulting IL-10+ B-cell frequency was similar in the three groups and correlated with free T3 levels in GD patients. IL-10+ B cells from both patient gr...
The IGF-1R is required for optimal growth in vivo and in vitro; it plays a crucial role in the es... more The IGF-1R is required for optimal growth in vivo and in vitro; it plays a crucial role in the establishment and maintenance of the transformed phenotype and it protects tumor cells from apoptosis both in vivo and in vitro.
Graves' disease, an autoimmune process associated with thyroid dysfunction, can also manifest as ... more Graves' disease, an autoimmune process associated with thyroid dysfunction, can also manifest as remodeling of orbital connective tissue. Affected tissues exhibit immune responses that appear to be orchestrated by resident cells and those recruited from the bone marrow through their expression and release of cytokines and surface display of cytokine receptors. Cytokines are small molecules produced by many types of cells, including those of the ''professional'' immune system. Aberrant cytokine expression appears to play an important role in the pathogenesis of many human diseases, including thyroid autoimmunity. The skewed pattern of cytokine expression in the thyroid, including the T helper cell bias, may condition the response to apoptotic signals and determine the characteristics of an autoimmune reaction. Furthermore, chemoattractant cytokines, including IL16, RANTES, and CXCL10, elaborated by resident cells in the thyroid and orbit may provoke mononuclear cell infiltration. Other cytokines may drive cell activation and tissue remodeling. Thus cytokines and the signaling pathways they activate represent attractive therapeutic targets. Interruption of these might alter the natural course of Graves' disease and its orbital manifestations.
Purpose-To study the effectiveness of anti-CD20 (Rituximab, RTX, Rituxan®, Genentech Inc. USA) th... more Purpose-To study the effectiveness of anti-CD20 (Rituximab, RTX, Rituxan®, Genentech Inc. USA) therapy in patients with severe, corticosteroid (CS)-resistant thyroid-associated ophthalmopathy (TAO). Design-Retrospective interventional case series Participants-Six consecutive subjects with severe, progressive TAO unresponsive to CS. Methods-Electronic medical record review of consecutive patients receiving RTX during the previous 18 months. Responses to therapy were graded using standard clinical assessment and flowcytometric analysis of peripheral lymphocytes. Main outcome measures-Clinical activity score (CAS), proptosis, strabismus, treatment sideeffects, and quantification of regulatory T cells. Results-Six patients were studied. Systemic CS failed to alter clinical activity in all patients (CAS= 5.3 + 1.0 (mean + standard deviation) before vs 5.5 + 0.8 during therapy for 7.5+ 6.4 months,
The thyroid target Ag for disease-inducing autoantibodies in Graves’ disease is the receptor for ... more 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 ...
Human orbital fibroblasts exhibit a unique inflammatory phenotype. In the present study, we repor... more Human orbital fibroblasts exhibit a unique inflammatory phenotype. In the present study, we report that these fibroblasts, when treated with IL-1β, express high levels of IL-6, a cytokine involved in B cell activation and the regulation of adipocyte metabolism. The magnitude of this induction is considerably greater than that in dermal fibroblasts and involves up-regulation of IL-6 mRNA levels. IL-1β activates both p38 and ERK 1/2 components of the MAPK pathways. Disrupting these could attenuate the IL-6 induction. The up-regulation involves enhanced IL-6 gene promoter activity and retardation of IL-6 mRNA decay by IL-1β. Dexamethasone completely blocked the effect of IL-1β on IL-6 expression. Orbital fibroblasts also express higher levels of IL-6R than do skin-derived cells. When treated with rIL-6 (10 ng/ml), STAT3 is transiently phosphorylated. Thus, the exaggerated capacity of orbital fibroblasts to express high levels of both IL-6 and its receptor in an anatomic site-selective ...
Graves’ disease (GD) is associated with T cell infiltration, but the mechanism for lymphocyte tra... more Graves’ disease (GD) is associated with T cell infiltration, but the mechanism for lymphocyte trafficking has remained uncertain. We reported previously that fibroblasts from patients with GD express IL-16, a CD4-specific chemoattractant, and RANTES, a C-C chemokine, in response to GD-specific IgG (GD-IgG). We unexpectedly found that these responses result from a functional interaction between GD-IgG and the insulin-like growth factor (IGF)-I receptor (IGF-IR). IGF-I and the IGF-IR-specific IGF-I analog, des(1–3), mimic the effects of GD-IgG. Neither GD-IgG nor IGF-I activates chemoattractant expression in control fibroblasts from donors without GD. Interrupting IGF-IR function with specific receptor-blocking Abs or by transiently transfecting fibroblasts with a dominant negative mutant IGF-IR completely attenuates signaling provoked by GD-IgG. Moreover, GD-IgG displaces specific 125I-labeled IGF-I binding to fibroblasts and attenuates IGF-IR detection by flow cytometry. These findi...
We have reported recently that IgG from patients with Graves’ disease (GD) can induce the express... more We have reported recently that IgG from patients with Graves’ disease (GD) can induce the expression of the CD4-specific T lymphocyte chemoattractant, IL-16, and RANTES, a C-C chemokine, in their fibroblasts. This induction is mediated through the insulin-like growth factor-1 receptor (IGF-1R) pathway. We now report that Abs from individuals with active rheumatoid arthritis (RA-IgG) stimulate in their synovial fibroblasts the expression of these same cytokines. IgG from individuals without known autoimmune disease fails to elicit this chemoattractant production. Furthermore, RA-IgG fails to induce IL-16 or RANTES expression in synovial fibroblasts from donors with osteoarthritis. RA-IgG-provoked IL-16 and RANTES production also appears to involve the IGF-1R because receptor-blocking Abs prevent the response. RA fibroblasts transfected with a dominant-negative mutant IGF-1R fail to respond to RA-IgG. IGF-1 and the IGF-1R-specific analog Des(1–3) also induce cytokine production in RA ...
Thyroid-associated ophthalmopathy (TAO), an autoimmune component of Graves’ disease, is associate... more Thyroid-associated ophthalmopathy (TAO), an autoimmune component of Graves’ disease, is associated with profound connective tissue remodeling and fibrosis that appear to involve the selective activation of orbital fibroblasts. Accumulation of extracellular matrix molecules is a hallmark of this process. Here we report that orbital fibroblasts treated with IL-1β express high levels of tissue inhibitor of metalloproteinase-1 (TIMP-1), an important modulator of matrix metalloproteinase activity. These high levels are associated with increased TIMP-1 activity. The induction is mediated at the pretranslational level and involves activating the TIMP-1 gene promoter. IL-1β activates the ERK 1/2 pathway in these fibroblasts and interrupting this signaling either with PD98059, a chemical inhibitor of MEK, or by transfecting cells with a dominant negative ERK 1 plasmid results in the attenuation of TIMP-1 induction. Surprisingly, treatment with IL-4 or IFN-γ could also block the IL-1β inducti...
Human fibroblasts can express numerous regulatory molecules that influence immune function. IL-16... more Human fibroblasts can express numerous regulatory molecules that influence immune function. IL-16, a ligand for CD4, is a chemoattractant molecule expressed by lymphocytes, eosinophils, mast cells, and lung epithelium. It appears that the sole target for IL-16 is the CD4-bearing cell. Here we demonstrate that fibroblasts from several tissues can express IL-16 mRNA and protein as well as IL-16-dependent chemoattractant activity. The transcript is expressed abundantly under basal culture conditions as a 2.5-kb band on Northern analysis, similar to that observed in lymphocytes. IL-16 protein and activity are undetectable in fibroblast cultures under these same control conditions. However, when treated with proinflammatory cytokines such as IL-1β, they express very high levels of IL-16 protein and chemoattractant activity, a substantial component of which can be blocked with IL-16-neutralizing Abs. The amount of IL-16 protein released into the medium is 3- to 4-fold greater, on a per ce...
Thyroid-associated ophthalmopathy and dermopathy are connective tissue manifestations of Graves’ ... more Thyroid-associated ophthalmopathy and dermopathy are connective tissue manifestations of Graves’ disease (GD). Tissue remodeling is a prominent feature of both and is apparently driven by recruited T cells. In this study, we report that IgG isolated from patients with GD (GD-IgG) up-regulates T lymphocyte chemoattractant activity in GD-derived fibroblasts from orbit, thyroid, and several regions of skin. This chemoattractant activity, absent in fibroblasts from donors without known thyroid disease, is partially susceptible to neutralization by anti-IL-16 and anti-RANTES Abs. IL-16 is a CD4+-specific chemoattractant and RANTES is a C-C-type chemokine. IL-16 and RANTES protein levels, as determined by specific ELISAs, are substantially increased by GD-IgG in GD fibroblasts. Addition of the macrolide, rapamycin, to fibroblast culture medium blocked the up-regulation by GD-IgG of IL-16, implicating the FRAP/mTOR/p70s6k pathway in the induction of IL-16 expression. These findings suggest...
Graves’ disease (GD), an autoimmune process involving thyroid and orbital tissue, is associated w... more Graves’ disease (GD), an autoimmune process involving thyroid and orbital tissue, is associated with lymphocyte abnormalities including expansion of memory T cells. Insulin-like growth factor receptor-1 (IGF-1R)-bearing fibroblasts overpopulate connective tissues in GD. IGF-1R on fibroblasts, when ligated with IgGs from these patients, results in the expression of the T cell chemoattractants, IL-16 and RANTES. We now report that a disproportionately large fraction of peripheral blood T cells express IGF-1R (CD3+IGF-R+). CD3+IGF-1R+ T cells comprise 48 ± 4% (mean ± SE; n = 33) in patients with GD compared with 15 ± 3% (n = 21; p < 10−8) in controls. This increased population of IGF-1R+ T cells results, at least in part, from an expansion of CD45RO+ T cells expressing the receptor. In contrast, the fraction of CD45RA+IGF-1R+ T cells is similar in GD and controls. T cells harvested from affected orbital tissues in GD reflect similar differences in the proportion of IGF-1R+CD3+ and I...
The Journal of Clinical Endocrinology & Metabolism, 2014
Context: The IL-1 family plays important roles in normal physiology and mediates inflammation. Th... more Context: The IL-1 family plays important roles in normal physiology and mediates inflammation. The actions of IL-1 are modulated by multiple IL-1 receptor antagonists (IL-1RA), including intracellular and secreted forms. IL-1 has been implicated in autoimmunity, such as that occurring in Graves' disease (GD) and its inflammatory orbital manifestation, thyroid-associated ophthalmopathy (TAO). We have previously reported that CD34 ϩ fibrocytes, monocyte-lineage bone marrowderived cells, express functional TSH receptor, the central antigen in GD. When activated by TSH, they produce IL-6, IL-8, and TNF-␣. Moreover, they infiltrate the orbit in TAO in which they transition into CD34 ϩ fibroblasts and comprise a population of orbital fibroblasts (OFs). Little is known currently about any relationship between TSH, TSH receptor, and the IL-1 pathway. Objective: The objective of the study was to determine whether TSH regulates IL-1RA in fibrocytes and OFs. Design/Setting/Participants: Fibrocytes and OFs were collected and analyzed from healthy individuals and those with GD in an academic clinical practice. Main Outcome Measures: Real-time PCR, Western blot analysis, reporter gene assays, and cell transfections were performed. Results: TSH induces the expression of IL-1RA in fibrocytes and GD-OFs. The patterns of induction diverge quantitatively and qualitatively in the two cell types. This results from relatively small effects on gene transcription-related events but a greater influence on secreted IL-1RA and intracellular IL-1RA mRNA stabilities. These actions of TSH are dependent on the intermediate induction of IL-1␣ and IL-1. Conclusions: Our findings for the first time directly link activities of the TSH and IL-1 pathways. Furthermore, they identify novel molecular interactions that could be targeted as therapy for TAO. (J Clin Endocrinol Metab 99: E625-E633, 2014) T he IL-1 family comprises multiple members, including the isoforms of IL-1 receptor antagonist (IL-1RA), which modulates the activities of IL-1␣ and IL-1 (1-3). These related proteins are synthesized in response to a diverse set of factors that vary with cell-type (3). IL-1RA competitively binds the IL-1 receptor and attenuates downstream signaling. Four different IL-1RA forms have been identified, including the secreted isoform (sIL-1RA) and three intracellular proteins (icIL-1RA) (1). Transcripts encoding sIL-1RA and icIL-1RA differ only in the sequence extending from ϩ4 nt to ϩ63 nt and encoding a 21-amino acid peptide present in sIL-1RA. Two distinct gene promoters drive the expression of these proteins (4).
The Journal of Clinical Endocrinology & Metabolism, 2013
Context: Thyroid-associated ophthalmopathy (TAO) manifests as inflammation of orbital connective ... more Context: Thyroid-associated ophthalmopathy (TAO) manifests as inflammation of orbital connective tissue. Bone marrow-derived CD34 ϩ fibrocytes infiltrate the orbit in TAO where they become CD34 ϩ orbital fibroblasts. They express thyroid-specific antigens and thus may contribute to inflammation. Evidence suggests that orbital susceptibility to TAO may involve IL-1, which is modulated by IL-1 receptor antagonists, designated secreted (sIL-1RA) and intracellular (icIL-1RA). Objective: We sought to characterize the expression of sIL-1RA and icIL-1RA in TAO orbital fibroblasts compared to CD34 ϩ fibrocytes. Design/Setting/Participants: Patients with TAO and healthy donors were recruited from an academic medical center clinical practice. Main Outcome Measures: Real-time PCR, cytokine-specific ELISA, gene promoter activities, transcriptional analysis, mRNA stability, and cytometric cell sorting were performed. Results: Orbital fibroblasts treated with IL-1 exhibit greater inductions of IL-1␣, IL-1, and prostaglandin endoperoxide H synthase-2 transcripts than do fibrocytes. Fibrocytes express dramatically higher basal levels of both icIL-1RA and sIL-1RA. When treated with IL-1, icIL-1RA is induced in orbital fibroblasts but not sIL-1RA, whereas in fibrocytes, sIL-1RA is dominantly up-regulated. These inductions result from increased steady-state levels of respective mRNAs, enhanced transcript stabilities, and modestly increased gene transcription. Conclusions: Robust responses of TAO orbital fibroblasts to IL-1 are a consequence of low-level sIL-1RA expression. This results in poorly opposed actions of IL-1. In contrast, circulating fibrocytes express high levels of sIL-1RA, which are diminished as these cells transition to orbital fibroblasts. These findings identify an explanation for the inflammatory phenotype exhibited by TAO orbital fibroblasts and provide a potential target for altering disease susceptibility.
The Journal of Clinical Endocrinology & Metabolism, 2004
A distinctive histopathological feature associated with thyroid-associated ophthalmopathy is the ... more A distinctive histopathological feature associated with thyroid-associated ophthalmopathy is the disordered accumulation of the glycosaminoglycan, hyaluronan, in orbital connective tissues. This often occurs in the context of dramatic inflammation and tissue remodeling. Orbital fibroblasts exhibit a novel phenotype including exaggerated responses to cytokines. Here, we report for the first time the ability of IgG isolated from the sera of patients with Graves' disease (GD-IgG) to provoke in orbital fibroblasts the synthesis of hyaluronan. The effect of GD-IgG can be reproduced by IGF-I, appears to be mediated through the IGF-I receptor, and is abolished with glucocorticoid treatment. TSH failed to influence the synthesis of hyaluronan. In contrast to the effects in GD fibroblasts, cultures derived from donors without known thyroid disease fail to respond to GD-IgG or IGF-I. The observation that hyaluronan production is induced by GD-IgG in fibroblasts suggests that the IGF-I receptor and its activating antibodies may represent a key pathway through which important pathogenic events in thyroid-associated ophthalmopathy are mediated.
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Papers by Terry Smith