Abstract
Background: SSc is a complex autoimmune fibrotic disease, characterized by elevated deposition of extracellular matrix (ECM) proteins, in particular collagen type I. The disease is heterogeneous; organs commonly affected by fibrosis are the skin, kidney, lung and heart. Vascular complications include pulmonary arterial hypertension (PAH), which occurs in 12–40% of patients. CD14þmonocytes are afunctionally heterogeneous cell type. They have been noted to differentiate into a number of cell phenotypes including macrophages and collagen-producing fibrocytes. In culture fibrocytes adopt a spindle shape, co-express haematopoietic CD45RO and 25F9, along with mesenchymal markers including aSMA and collagen type I. Fibrocytes amplify the inflammatory/immune response through distinct mechanisms, including antigen presentation, cytokine and chemokine secretion, and the production of MMPs. We and others have shown fibrocyte differentiation is enhanced by fibrogenic cytokines including PDGF. Here we seek to understand the mechanism by which SSc fibrocytes influence the local microenvironment of the tissue. Methods: CD14þ peripheral blood mononuclear cells (PBMCs) were isolated from SSc patient and healthy control blood. PBMCs were cultured in the presence of macrophage colony stimulating factor (MCSF; n > 10) and/or endothelin-1 (ET-1; n > 10); after 14 days of culture number of fibrocytes was assessed. The effect of pharmacological inhibitors including ETRA and ETRB antagonism on fibrocyte differentiation (n ¼ 6 SSc and control) was investigated. Secreted factors in culture media from SSc and control fibrocytes were assessed by ELISA (n ¼ 6), and the effects of conditioned media explored in 3D-collagen gel. Results: Both MCSF and ET-1 significantly induced fibrocyte differentiation, in combination differentiation was significantly augmented (P < 0.05) in comparison with mono-treatment. SSc fibrocytes more readily differentiated from CD14þ PBMCs than healthy control donors in response to MCSF (P < 0.05), ET-1 (P < 0.05) as well as MCSF with ET-1 in combination (P < 0.01). ETRA and ETRB antagonists, BQ123 and BQ788 (respectively), and Bosentan (a dual ETR antagonist) inhibited MCSF induced fibrocyte differentiation in a concentration dependant manner. Furthermore SSc fibrocytes secreted significantly more connective tissue growth factor (CTGF) than control fibrocytes (P < 0.05) cultured with MCSF. Consistent with fibrocytes acting in a paracrine manner, conditioned media from SSc fibrocytes promoted fibroblast gel contraction by control cells (P < 0.05). Conclusion: Here we show CD14þ SSc PBMCs more readily differentiate into fibrocytes and that activation via the ETRA and ETRB is essential for ET-1 and MCSF induced fibrocyte differentiation. Suggesting MCSF acts indirectly via ET-1 release; possibly resulting in a positive feedback loop. Our data suggest fibrocytes may contribute to the development of a pro-fibrotic environment through influencing tissue resident fibroblasts in a paracrine manner