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Purpose: Orbital fibroblasts from patients with Graves' disease (GD-OF) express many different cytokines when treated with bovine thyrotropin (bTSH). The present study aimed to determine why TNF-α cannot be induced by bTSH in GD-OF., Methods: Fibrocytes and GD-OFs were cultivated from donors who were patients in a busy academic medical center practice. Real-time PCR, Western blot analysis, reporter gene assays, cell transfections, mRNA stability assays, ELISA, and flow cytometry were performed., Results: We found that bTSH induces TNF-α dramatically in fibrocytes but is undetectable in GD-OF. The induction in fibrocytes is a consequence of increased TNF-α gene promoter activity and is independent of ongoing protein synthesis. It could be attenuated by dexamethasone and the IGF-1 receptor inhibiting antibody, teprotumumab. When separated into pure CD34+ OF and CD34- OF subsets, TNF-α mRNA became highly inducible by bTSH in CD34+ OF but remained undetectable in CD34- OF. Conditioned medium from CD34- OF inhibited induction of TNF-α in fibrocytes., Conclusions: Our data indicate that CD34- OF appear to release a soluble(s) factor that downregulates expression and induction by bTSH of TNF-α in fibrocytes and their derivative CD34+ OF. We proffer that CD34- OF produce an unidentified modulatory factor that attenuates TNF-α expression in GD-OF and may do so in the TAO orbit.

Fibrocytes are monocyte progenitor cells that have been implicated in normal and pathological tissue remodeling. Among the prominent chemokine receptors expressed by these cells is CXC motif receptor 4 (CXCR4), which, with its cognate ligand CXCL motif ligand 12 (CXCL-12), directs fibrocytes to sites of fibrosis. Fibrocytes have been implicated in the pathogenesis of thyroid-associated ophthalmopathy, the ocular manifestation of Graves' disease (GD), by virtue of their unique accumulation as CD34 + orbital fibroblasts (OFs). Fibrocytes also express high levels of functional TSH receptor (TSHR). Here, we determined CXCL-12 and CXCR4 expression in fibrocytes and GD-OF and whether that pathway interacts with TSHR. CXCL-12 is highly expressed in GD-OF, whereas CXCR4 levels are dramatically higher in fibrocytes. Levels of these proteins are differentially regulated by TSH in a cell type-specific manner. Further, CXCL-12 enhances the induction by TSH of IL-6 in fibrocytes but attenuates this induction in GD-OF. In contrast, in pure CD34 + OF, the interplay between TSH and CXCL-12 reverts to that observed in fibrocytes. Our results indicate that CXCL-12 enhances TSH actions in fibrocytes but inhibits them in GD-OF, a dichotomy imposed by factors emanating from CD34 - OF. They also suggest a potentially important modulatory role for CD34 - OF in determining the factors that influence pathological TSHR signaling in the TAO orbit.

Context: Rituximab depletes CD20(+) B cells and has shown potential benefit in thyroid-associated ophthalmopathy (TAO). The impact of rituximab on T cell phenotype in TAO is unexplored., Objective: The objective of the study was to quantify the abundance of IGF-I receptor-positive (IGF-1R(+)) CD4 and CD8 T cells in active TAO before and after treatment with rituximab., Design: This was a retrospective case series assessing IGF-1R(+) T cells before and after treatment with rituximab with an 18-month follow-up., Setting: The study was conducted at a tertiary care medical center., Patients: Study participants included eight patients with severe TAO., Interventions: Two infusions of rituximab (1 g or 500 mg each) were administered 2 weeks apart., Main Outcome Measures: Quantification of IGF-1R(+) T cells using flow cytometry was measured., Results: Eight patients with moderate to severe TAO [mean pretreatment clinical activity score (CAS) 5.1 ± 0.2 (SEM)] were treated. Four to 6 weeks after treatment, CAS improved to 1.5 ± 0.3, whereas the proportion of IGF-1R(+) CD3(+) T cells declined from 41.9% to 28.3% (P = .004). The proportion of IGF-1R(+) CD4(+) and IGF-1R(+) CD8(+) T cells declined 4-6 weeks after treatment (from 45.6% to 21.5% and from 32.0% to 15.8%, P = .003 and P = .001, respectively). In two patients, IGF-1R(+) CD4(+) and IGF-1R(+) CD8(+) subsets approximated pretreatment levels after 16 weeks., Conclusions: Frequency of IGF-1R(+) T cells in patients with TAO declines within 4-6 weeks after rituximab treatment. This phenotypic shift coincides with clinical improvement. Thus, assessment of the abundance of IGF-1R(+) T cells in response to rituximab may provide a biomarker of clinical response. Our current findings further implicate the IGF-1R pathway in the pathogenesis of TAO.

Purpose: CD40-CD40 ligand (CD40L) interactions appear to play pathogenic roles in autoimmune disease. Here we quantify CD40 expression on fibrocytes, circulating, and bone marrow-derived progenitor cells. The functional consequences of CD40 ligation are determined since these may promote tissue remodeling linked with thyroid-associated ophthalmopathy (TAO)., Methods: CD40 levels on cultivated fibrocytes and orbital fibroblasts (GOFB) from patients with Graves' disease (GD), as well as fibrocyte abundance, were determined by flow cytometry. CD40 mRNA expression was evaluated by real-time PCR, whereas response to CD40 ligation was measured by Luminex and RT-PCR. Protein kinase B (Akt) and nuclear factor (NF)-kappa B (NF-κB) signaling were determined by Western blot and immunofluorescence., Results: Basal CD40 expression on fibrocytes is greater than that on GOFB. IFN-γ upregulates CD40 in both cell types and its actions are mediated at the pretranslational level. Fibrocytes produce high levels of cytokines, including interleukin-6 (IL-6), TNF-α, IL-8, MCP-1, and RANTES (Regulated on Activation, Normal T Cell Expressed and Secreted) in response to CD40L. IL-6 induction results from an increase in steady state IL-6 mRNA, and is mediated through Akt and NF-κB activation. Circulating CD40(+)CD45(+)Col1(+) fibrocytes are far more frequent in vivo in donors with TAO compared with healthy subjects., Conclusions: Particularly high levels of functional CD40 are displayed by fibrocytes. CD40L-provoked signaling results in the production of several cytokines. Among these, IL-6 expression is mediated through Akt and NF-κB pathways. The frequency of circulating CD40(+) fibrocytes is markedly increased in patients with TAO, suggesting that this receptor might represent a therapeutic target for TAO.