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

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.

Increased expression of TSH receptor by fibrocytes in thyroid-associated ophthalmopathy leads to chemokine production.

CONTEXT: The molecular basis for anatomically dispersed clinical manifestations in Graves' disease (GD) eludes our understanding. Bone marrow-derived, pluripotent fibrocytes represent a subset of peripheral blood mononuclear cells and infiltrate the orbital and thyroid tissues in GD. These cells may be involved in the pathogenesis of thyroid-associated ophthalmopathy (TAO). OBJECTIVE: The objective of the study was to quantify fibrocyte display of functional cell surface TSH receptor (TSHR), identify the profile of chemokines they express after TSHR activation, and determine whether circulating TSHR(+) peripheral blood fibrocytes are more frequent in situ in patients with TAO. DESIGN/SETTING/PARTICIPANTS: Using a newly developed technique, fibrocytes were directly identified in peripheral blood from 31 patients with TAO and 19 healthy subjects receiving care at a multidisciplinary academic center. MAIN OUTCOME MEASURES: The frequency in situ of fibrocytes (collagen 1(+), CD45(+), CD34(+), CD14(+), CD86(+) peripheral blood mononuclear cells) was assessed by multiparameter flow cytometry and correlated to clinical disease activity and smoking status. Levels of TSHR-displaying fibrocytes and their response to TSH and TSHR-activating antibody, M22, were measured by flow cytometry, Luminex, and real-time PCR. RESULTS: The levels of TSHR expression by fibrocytes are substantially higher than those found in orbital fibroblasts. Moreover, the frequency of TSHR(+) fibrocytes in patients with TAO was greater than that in healthy subjects in situ. Their abundance is not influenced by disease activity or smoking history. These cells produce high levels of several cytokines and chemokines including IL-8, regulated upon activation, normal T cell expressed and secreted, and monocyte chemoattractant protein-1 when treated with TSH or M22. TSH induces IL-8 production at the pretranslational level. This induced cytokine can be detected in intact fibrocytes ex vivo. CONCLUSIONS: Frequency of circulating TSHR(+) fibrocytes is markedly increased in patients with TAO, and they express proinflammatory chemokines in response to TSH. Because they infiltrate both orbit and thyroid in GD, they may represent the link between systemic immunoreactivity and organ-specific autoimmunity.