Thyrotropin and CD40L Stimulate Interleukin-12 Expression in Fibrocytes: Implications for Pathogenesis of Thyroid-Associated Ophthalmopathy

Abstract

Background: Increased numbers of bone marrow-derived progenitor cells, known as fibrocytes, populate the peripheral circulation, orbit, and thyroid of patients with Graves' disease (GD). These cells have been implicated in the development of thyroid-associated ophthalmopathy. They can differentiate into myofibroblasts or adipocytes, produce inflammatory cytokines, and remodel tissue. This study sought to determine whether thyrotropin (TSH) and CD40 ligand (CD40L), implicated in the pathogenesis of GD, induce interleukin-12 (IL-12) in human fibrocytes.

Materials and methods: IL-12 protein concentrations and mRNA levels were measured by Luminex and real-time polymerase chain reaction, respectively. Flow cytometry assessed intracellular IL-12 concentrations. Vector containing IL-12p40 promoter was transfected into cultured fibrocytes, and promoter activity was monitored using luciferase assay.

Results: TSH and CD40L stimulated intracellular IL-12 protein accumulation in peripheral blood fibrocytes. Inhibiting Akt and nuclear factor-κB (NF-κB) activity diminished IL-12 expression in fibrocytes, while TSH did not induce promoter activity. TSH-mediated IL-12 production required de novo synthesized proteins and augmented IL-12 mRNA stability. IL-12 production mediated by CD40L required tumor necrosis factor receptor-associated factor 6.

Conclusion: TSH and CD40L induce IL-12 expression in fibrocytes, and Akt and NF-κB mediate this activity. Given the importance of IL-12 in immune function, its production by fibrocytes may promote an inflammatory immune response and tissue remodeling in thyroid-associated ophthalmopathy.

Keywords: Graves' disease; autoimmunity; fibrocyte; interleukin-12; thyroid-associated ophthalmopathy.

FIG. 1.

Thyrotropin (TSH) and CD40L induced interleukin-12 (IL-12) production in circulating fibrocytes (A) and in cultured fibrocytes (B). (A) Freshly isolated peripheral blood mononuclear cells (PBMCs) were stimulated for 18 h. Flow cytometry showed that TSH and CD40L induced IL-12p40/p70 (the combined p40 monomer and p70 heterodimer) in circulating fibrocytes, resulting in MFI values that were 1.54- and 1.25-fold higher, respectively, than in untreated cells. The MFIs of IL-12 p70 (heterodimer) were 2.43- and 2.33-fold higher after TSH or CD40L was added (right panels). (B) The fibrocytes in cultures were treated with TSH and CD40L for 48 h. The time course of extracellular IL-12p40/p70 concentrations shows that TSH (▲) and CD40L (■) stimulated IL-12 production, whereas unstimulated cells (◯) produced negligible amounts. Peak production was reached at 24 h (p < 0.0001 for TSH and CD40L).

FIG. 2.

TSH and CD40L induced IL-12 at the pre-translational level. Steady-state IL-12p40 mRNA expression stimulated with TSH (▲) or CD40L (■) was followed by real-time polymerase chain reaction (PCR) in cultured fibrocytes for 24 h (A). IL-12p40 mRNA production increased responding to TSH, M22, and CD40L stimulation in fibrocytes from healthy controls (B, left panel) and from patients with GD (B, right panel).

FIG. 3.

IL-12 protein production was stimulated by TSH or CD40L induced through the Akt–NF-κB pathway in circulating (A) and cultured (B) fibrocytes. Intracellular IL-12p40/p70 (the combined monomer and heterodimer) and IL-12p70 (the heterodimer) were measured by flow cytometry in circulating fibrocytes 18 h after TSH or CD40L was added. Extracellular IL-12p40/p70 concentration was measured by Luminex technology in cultured fibrocytes after 24 h of stimulation. Akt inhibitor (AKTi) and NF-κB inhibitor (MG132) were added to cells 1 h before stimulation. The stimulatory effect of TSH or CD40L was markedly diminished by adding AKTi or MG132.

FIG. 4.

TSH and CD40L induced IL-12p40 mRNA expression through Akt (A) and NF-κB (B) signaling in cultured fibrocytes. Akt inhibitor (AKTi) and NF-κB inhibitor (MG132) were added 1 h before TSH or CD40L stimulation. RNA was isolated after 6 h of induction. AKTi and MG132 blocked IL-12p40 mRNA expression induced by TSH (p < 0.01 and p < 0.001, respectively) or CD40L (p < 0.01 and p < 0.0001, respectively).

FIG. 5.

CD40 signaling of IL-12 production proceeded through TRAF6 in fibrocytes. Two doses of 50 μM control peptide or TRAF6 or TRAF2 inhibitor peptide were added to cells 24 h and 1 h before CD40L stimulation. Six hours after CD40L stimulation, RNA was isolated, and IL-12p40 mRNA levels were measured by real-time PCR. TRAF6 inhibitor peptide significantly inhibited the action of CD40L, whereas the TRAF2 peptide had no effect.

FIG. 6.

TSH did not induce IL-12 promoter activity (A). The vector containing the promoter was transfected into fibrocytes and cells were incubated with TSH for 2 h. TSH stabilized IL-12p40 mRNA (B, left panel), whereas CD40L did not affect mRNA stability (B, right panel). IL-12 mRNA production was stimulated for 12 h with TSH. Cells were then washed and treated with 50 μM of DRB alone, DRB and TSH, or DRB and CD40L (time zero). Degradation of existing mRNA was slower in the presence of TSH and DRB (●) than in the presence of DRB alone (◯), whereas mRNA degradation proceeded at similar rate in the absence (□) and presence (■) of CD40L. TSH stimulation of IL-12p40 mRNA depended on de novo protein production (C, left panel). Adding cycloheximide (10 μg/mL) 1 h before TSH stimulation significantly lowered the TSH-induced IL-12p40 mRNA expression. Adding cycloheximide did not affect CD40L stimulation of IL-12p40 mRNA (C, right panel), indicating that CD40L does not depend on de novo protein synthesis.