with Graves' Ophthalmopathy Not Only Via Cyclic Adenosine. Monophosphate Signaling Pathways. Clementine J.J. van Zeijl,1 Eric Fliers,1 Chris J. van Koppen ...
THYROID Volume 21, Number 2, 2011 ª Mary Ann Liebert, Inc. DOI: 10.1089/thy.2010.0123
Thyrotropin Receptor-Stimulating Graves’ Disease Immunoglobulins Induce Hyaluronan Synthesis by Differentiated Orbital Fibroblasts from Patients with Graves’ Ophthalmopathy Not Only Via Cyclic Adenosine Monophosphate Signaling Pathways Clementine J.J. van Zeijl,1 Eric Fliers,1 Chris J. van Koppen,2 Olga V. Surovtseva,1 Marcel E. de Gooyer,2 Maarten P. Mourits,3 Wilmar M. Wiersinga,1 Andre´ M.M. Miltenburg,2 and Anita Boelen1
Background: Both expression of the thyrotropin receptor (TSHR) and the production of hyaluronan (HA) by orbital fibroblasts (OF) have been proposed to be implicated in the pathogenesis of Graves’ ophthalmopathy (GO). HA is synthesized by three types of HA synthase. We hypothesized that TSHR activation by recombinant human TSH (rhTSH) and TSHR-stimulating Graves’ disease immunoglobulins (GD-IgGs) via induced cyclic adenosine monophosphate (cAMP) signaling increases HA synthesis in differentiated OF from GO patients. Methods: Cultured human OF, obtained during decompression surgery from 17 patients with severe GO, were stimulated in vitro to differentiate into adipocytes. Differentiation was evaluated by phase-contrast microscopy. The differentiated OF were stimulated by rhTSH or by TSHR-stimulating GD-IgG. We measured cAMP using a biochemical assay, HA synthase mRNA expression by quantitative polymerase chain reaction, and HA in the supernatant by enzyme-linked immunosorbent assay. Results: All differentiated OF cultures expressed higher levels of TSHR mRNA than nondifferentiated OF cultures. Stimulation by rhTSH induced a marked cAMP response in 11 of 12 differentiated OF cultures, but no measurable HA response in all but one differentiated OF cultures. By contrast, stimulation by GD-IgG induced a moderate cAMP response in a number of differentiated OF cultures, but a marked HA response in the majority of differentiated OF cultures. Conclusion: Stimulation of differentiated OF by GD-IgG, but not by rhTSH, induces HA synthesis in the majority of patients, suggesting that in most patients TSHR-mediated cAMP signaling does not play a pivotal role in GDIgG-induced HA synthesis in differentiated OF cultures.
Introduction
O
rbital fibroblasts (OF) are assumed to play a major role in the pathogenesis of Graves’ ophthalmopathy (GO) (1). Fibroblasts may function as structural cells in the extracellular matrix, but they are also capable of initiating and participating in acute and chronic inflammatory responses (2). Various cytokines, such as interleukin-1b (IL1b), leukoregulin, and interferon-g, induce OF to produce excessive amounts of hyaluronan (HA), which is a highly hydrophilic glycosaminoglycan. HA is synthesized at the plasma membrane by three mammalian HA synthase (HAS),
HAS1, HAS2, and HAS3, which are differentially inducible by several cytokines and growth factors (3,4). Various reports have pointed to a role for HA in the pathogenesis of GO (5–7). Inflammation may drive fibroblasts to differentiate into adipocytes in various tissues (2,8). Under appropriate culture conditions, a subgroup of OF, termed preadipocytes or lipofibroblasts, has been shown to differentiate to adipocytes in vitro. This process may contribute in vivo to the accumulation of excessive adipose tissue in the orbit frequently seen in GO, and the differentiated OF may play a distinct role in the immunopathogenesis of GO (8–12).
1
Department of Endocrinology and Metabolism, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands. Schering-Plough Research Institute, Oss, The Netherlands. Department of Ophthalmology, Orbital Center, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
2 3
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170 Functional thyrotropin receptor (TSHR) expression increases in OF after adipocytic differentiation (8,10,13). The TSHR is known to be the antigen that binds Graves’ disease immunoglobulins (GD-IgGs) in thyroid follicular cells, leading to Graves’ hyperthyroidism (14,15). TSHR gene expression was shown to increase in orbital tissue specimens of patients with GO in the active stage of the disease. As a correlation between TSHR autoantibody levels and clinical activity score of GO has been reported and in view of the prognostic value of TSHR antibody levels for the severity and the outcome of GO, the TSHR is considered a major autoantigen in the immunopathogenesis of GO (16–18). Binding of the TSHR by TSH or by GD-IgG results in activation of the adenylyl cyclase/cyclic adenosine monophosphate (cAMP) signaling pathway via G-protein-coupled pathways (19). In turn, cAMP induces HA synthesis in several cell types, among which OF (20,21). We and others have recently reported that strong activation of the cAMP pathway increases HA synthesis, mainly via upregulation of HAS1, in nondifferentiated GO OF, whereas TSH-mediated cAMP induction does not induce HA synthesis (21,22). In addition, we found that TSHR-stimulating GD-IgG did not increase cAMP production or HA synthesis in nondifferentiated OF. These findings suggested that orbital cytokines, such as IL-1b, play a more prominent role than GD-IgG to increase HA synthesis in the early stages of GO. The aim of the present study was to evaluate the effect of recombinant human TSH (rhTSH) and GD-IgG on cAMP signaling and HA synthesis in differentiated OF from GO patients.
Materials and Methods GD-IgG and control IgG isolation As reported previously (22), we isolated IgG from sera of two GO patients (GD-IgG, batch I and batch II) and two healthy controls (c-IgG, batch I and batch II) using protein G Sepharose 4 Fast Flow (ProtG; Amersham Pharmacia Biotech Benelux, Roosendaal, The Netherlands), after obtaining informed consent. The GO patients had TSH-binding inhibitory IgG serum levels of 88 and 256 U/L, respectively. They had been found to have mild (clinical activity score [CAS] score 3/ 7) and moderately severe (CAS score 4/7) GO, respectively (23), and had not been treated with corticosteroids. At the time of the blood sampling, the GO was inactive in both patients. The healthy controls were TSH-binding inhibitory IgGseronegative (
