Interleukin-6 production in CD40-engaged fibrocytes in thyroid-associated ophthalmopathy: involvement of Akt and NF-κB

Abstract

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.

Figure 1.

Molecular density of CD40 on Graves' orbital fibroblasts (GOFB) and fibrocytes. (A) Constitutive CD40 display on fibrocytes is exhibited by immunofluorescence (inset: negative isotype control). Level of CD40 surface expression, as determined by FACS analysis, is higher on fibrocytes than that on GOFB (MFI fibrocytes, 4.5 ± 0.4 versus GOFB, 2.2 ± 0.2, P < 0.005). (B) CD40 expression is upregulated by IFN-γ (1000 units) after 24 hours of treatment in both fibrocytes and orbital fibroblasts, as shown by flow cytometry. (C) Real-time PCR demonstrates that CD40 mRNA is upregulated by IFN-γ in fibrocytes from healthy donors (n = 3, *P < 0.05; ***P < 0.0001 versus control unstimulated cultures).

Figure 2.

CD40 ligation induces several cytokines in cultivated fibrocytes. Cells were treated with control membrane or CD40L recombinant membrane (100 ng/mL) for 24 hours. Conditioned media were then subjected to Luminex assessment of the cytokines indicated. Results are combined from two experiments (*P < 0.05; **P < 0.001; ***P < 0.0001 versus control membrane-treated cultures).

Figure 3.

CD40 ligation induces IL-6 expression through Akt. (A) IL-6 mRNA expressions in cultivated fibrocytes were followed by real-time PCR after CD40L treatment for 24 hours (n = 3, *P < 0.05; **P < 0.001 versus control unstimulated cultures) AKT signaling is critical to CD40-initiated activation of IL-6 expression in fibrocytes. (B) CD40L-provoked upregulation of IL-6 in fibrocytes is blocked by Akt inhibition. Cells were pretreated with AKT inhibitor IV (AktI) and then CD40L-containing membranes were added to culture medium for 1 hour. Significant difference was found between cultures treated with control membranes versus CD40L (P < 0.0001) and CD40L versus CD40L plus AktI (P < 0.001). (C) Akt phosphorylation at Ser473 in normal fibrocytes is increased by CD40 ligation after 30 minutes of incubation, whereas AktI suppresses Akt phosphorylation as shown with Western blot analysis.

Figure 4.

CD40 signaling in fibrocytes involves activation of NF-κB and p65 nuclear translocation. (A) CD40L-mediated upregulation of IL-6 in normal fibrocytes is blocked by NF-κB inhibition. Cells were pretreated with either MG132 or PDTC for 1 hour and then treated with either control membranes or those containing CD40L. Result shows significant difference between cultures treated with control membrane versus CD40L (P < 0.0001); CD40L versus CD40L plus MG132 (P < 0.0001); and CD40L versus CD40L plus PDTC (P < 0.0001). (B) CD40 ligation increases nuclear NF-κB p65 fibrocytes. Fibrocytes were treated with control membranes or with recombinant CD40L membrane for 1 hour; cytosolic and nuclear proteins were extracted and subjected to Western blot analysis of NF-κB p65. β-Actin and c-jun served as the loading control for cytosolic and nuclear protein, respectively. (C) Immunostaining of cultured fibrocytes shows the translocation of NF-κB p65 from the cytosol to nucleus as a result of CD40L induction. Yellow arrows indicate NF-κB p65 in cytosol of untreated cells and in nucleus of CD40-stimulated fibrocytes.

Figure 5.

Circulating fibrocytes are more abundant in TAO in situ. (A) Fibrocytes were identified by FACS analysis of the peripheral blood as CD45⁺Col1⁺CD34⁺ monocytes. These cells were abundant in the PBMC from patients with TAO (n = 23) yet rare in healthy controls (n = 19) (34.8 ± 5.0% vs. 6.5 ± 2.7%, respectively; P < 0.0001). (B) The phenotype of these cells was confirmed to include expression of CD45, Col1, CD34, CD14, CD86, and CXCR4 by flow cytometry.

Figure 6.

Circulating CD40⁺ fibrocytes are more frequent in TAO in situ. The fraction of CD45⁺Col1⁺CD40⁺ fibrocytes is substantially greater in those from donors with TAO (n = 19) compared with healthy controls (n = 19, 10 ± 2% vs. 0.6 ± 0.2%, respectively; P < 0.0001).

Figure 7.

Schematic view of CD40 signaling pathway of IL-6 production in fibrocytes. CD40–CD40L interaction activates a downstream signaling pathway, resulting in the production of proinflammatory cytokine, IL-6. CD40 signaling for IL-6 is mediated by Akt phosphorylation and subsequent NF-κB translocation.