TSH-induced TNFα mRNA expression involves Akt and NF-κB.

<p>AKTi (A) or MG132 (B) was added 1 hour before TSH stimulation of healthy cultured fibrocytes. RNA was isolated after 6 hours of induction. AKTi treatment leads to significant reduction in TSH-induced TNFα mRNA expression (from 23-fold to 1.3-fold expression, <i>p</i> <0.0001) (A). Similarly, MG132 treatment inhibits TSH-induced TNFα mRNA expression (from 93-fold to 16-fold expression; p< 0.001) (B).</p

Steady state TNFα mRNA expression after TSH stimulation by real-time PCR in cultured healthy fibrocytes.

<p>TNFα mRNA expression is significantly increased in response to TSH stimulation in cultured fibrocytes. The steady state TNFα mRNA expression reaches a peak level at 3 hours (301-fold expression, p<0.0001).</p

TSH-induced TNFα protein production involves Akt and NF-κB.

<p>FACS analysis shows that pretreatment with either AKTi (AKT inhibitor) or MG132 (NF-κB inhibitor) reduces TSH-induced TNFα protein production in healthy circulating fibrocytes (MFI 2.92 reduced to 1.46 with addition of AKTi and 1.33 with MG132) (A). Luminex analysis shows that pretreatment with either AKTi or MG132 reduces TSH-induced TNFα production in healthy cultured fibrocytes. TSH-induced TNFα production of 1312 pg/ml is reduced by 52% to 612 pg/ml with AKTi and by 81% to 251 pg/ml with MG132 (p <0.0001) (B).</p

TSH and M22 induce TNFα production in healthy and GD fibrocytes.

<p>A representative example of FACS analysis shows that unstimulated circulating fibrocytes from healthy (A) and GD (B) patients produce negligible amount of TNFα (MFI 1.07 and 1.11, respectively). TSH stimulation is associated with significantly higher production of intracellular TNFα (MFI 1.75 and 1.82 for healthy and GD fibrocytes, respectively). M22 treatment of circulating healthy (A) and GD (B) fibrocytes is also associated with increased production of intracellular TNFα (MFI for healthy and GD fibrocytes are 1.54 and 1.52, respectively). Luminex analysis shows that TSH increases extracellular TNFα protein production in cultured healthy (C) and GD (D) fibrocytes. TNFα protein production peaks at 12 hours (1311 pg/ml, p<0.0001) (C). Cultured GD fibrocytes show increased extracellular TNFα production after TSH stimulation (from 8 to 799 pg/ml, p<0.0001) (D).</p

TSH/M22-induced TNFα protein production in fibrocytes is attenuated by TMB, a human anti-IGF-1R monoclonal antibody.

<p>Both circulating and cultured fibrocytes were pretreated with TMB, prior to TSH or M22 stimulation. TMB decreases TSH/M22-induced TNFα protein production in healthy (from MFI value of 2.92 to 1.91 for TSH; and from 1.67 to 1.12 for M22) (A, B) and GD circulating fibrocytes (from MFI value of 1.82 to 1.23 for TSH; and from 1.66 to 1.19 for M22) (C, D).</p

Treatment with a combination of TSH and CD40L stimulates greater cytokine production in fibrocytes than treatment with either CD40L or TSH alone.

<p>The titers of IL-6 (A) and TNF-α (B) were measured, using Luminex technology, in culture media collected 24 h after stimulation.</p

TSH stimulates CD40 expression at the transcriptional level in fibrocytes.

<p>The steady-state CD40 transcript level after 6 hours of TSH addition increased 30-fold over baseline.</p

TSH increases the abundance of CD40 protein on the surface of fibrocytes.

<p>(A) Results of flow cytometric analyses show that the level of cell-surface CD40 is augmented. As shown in representative histograms, 24 h TSH stimulation resulted in comparable CD40 stimulation in fibrocytes from healthy controls and patients with Graves’ diseases (1.34- and 1.50-fold MFI increase, respectively). in cells. (B) The CD40 antibodies bound per cell (ABC) test also shows a statistically significant (n = 6, <i>P</i> < 0.01) increase in CD40 density per cell in samples treated with TSH.</p

Treatment with a combination of CD40L and TSH increases the levels of IL-6 (A) and TNF-α (B) mRNA more than treatment with either CD40L or TSH alone.

<p>Cells were treated with TSH, CD40L, or a combination of these ligands for 6 h.</p

Co-localization and protein-protein interaction of TSHR and CD40 in fibrocytes were confirmed with confocal microscopy and co-immunoprecipitation (co-IP).

<p>(A) The spindle-shaped fibrocytes indicate that THSR (green, <i>left panel</i>) and CD40 (red, <i>middle panel</i>) are colocalized on the cell surface, resulting in the yellow-colored merged image (<i>right panel</i>). (B) Co-IP studies using TSHR (<i>left panel</i>) or CD40 antibodies (<i>right panel</i>) show that TSHR antibody pulls down TSHR and CD40, and CD40 antibody also pulls down both proteins, indicating physical contact between the 2 proteins.</p