Chemokine receptor CCR2 expression by systemic sclerosis fibroblasts - Evidence for autocrine regulation of myofibroblast differentiation

Objective. To investigate expression of the chemokine receptor CCR2 on key cell types involved in the pathogenesis of systemic sclerosis (SSc) and to assess the potential for autocrine activation of SSc dermal fibroblasts via CCL2/CCR2. Methods. Chemokine receptor expression in skin biopsy tissues and explanted dermal fibroblasts from a well-characterized cohort of SSc patients was examined using immunohistochemistry and flow cytometry techniques. Autocrine regulation of the expression of fibrotic markers in CCR2+ SSc fibroblast cell lines was assessed using specific ligand or receptor antagonists. Results. We identified strong CCR2 expression in skin biopsy samples of early-stage diffuse cutaneous SSc (dcSSc), but not late-stage dcSSc or limited cutaneous SSc. Double labeling confirmed up-regulation of CCL2/ CCR2 on myofibroblasts, pericytes, lymphocytes, macrophages, and endothelial cells. Explanted dermal fibroblasts from early dcSSc tissues expressed CCR2 and CXCR2 in 55% and 66% of cell strains, respectively. There was no expression in control fibroblasts. CCR2+ fibroblasts demonstrated a profibrotic phenotype, with overexpression of alpha-smooth muscle actin (alpha-SMA), connective tissue growth factor (CTGF), and CCL2. Flow cytometric analysis identified a subset of CCR2+ SSc fibroblasts expressing the myofibroblast marker alpha-SMA. In these cultures, specific inhibition of CCL2 or CCR2 attenuated the overexpression of alpha-SMA, but not CTGF or plasminogen activator inhibitor 1. Conclusion. Our results show that CCR2 is up-regulated in early dcSSc on cell types known to be activated in the disease, which is consistent with a key role in SSc pathogenesis. CCR2 expression on SSc fibroblasts appears to regulate the expression of CCL2 and alpha-SMA. Our findings suggest potential autocrine regulation of key profibrotic properties via a CCL2/ CCR2 loop in early-stage dcSSc.