Background Macrophages and fibroblasts are key effector cell types present in scleroderma tissue1. While the effect of scleroderma fibroblast conditioned medium on macrophages has been previously studied2, less is known about the effect of scleroderma macrophages on fibroblasts. Interleukin-6 (IL-6) is an important mediator of fibrosis and is overexpressed in scleroderma sera and cells such as skin fibroblasts, monocytes and endothelial cells3. Given the increase in IL-6 levels in scleroderma and presence of the IL-6 receptor on the cell surface of macrophages, we are now investigating the phenotype of macrophages exposed to IL-6. Here we present our work into the paracrine function of IL-6-treated macrophages in stimulating fibroblasts using a media transfer approach.
Objectives To investigate the effect of macrophage conditioned media on fibroblast activation.
Methods PBMC-derived macrophages from healthy control (n=3 females, mean age 50.8±21.9 years) and diffuse scleroderma (n=4 females, mean age 54.8±15.7 years, mean disease duration 73.2±90.3 months, 2 with antiScl70 and 2 with antiRNA polymerase antibodies) individuals were cultured in RPMI/10% FBS/M-CSF (4ng/ml)/P/S, quiesced in media with 1% BSA replacing the FBS, and left untreated (M0) or treated with IL-6 (50ng/ml, M (IL-6)) for 24 hours. The cultures were replaced with fresh media and collected after 24 hours. Conditioned media were applied to healthy control skin fibroblasts (24 hours) and fibroblast expression of fibrotic proteins was assessed by Western Blot, using β-tublin and TBP as loading controls. As control, fibroblasts from healthy volunteers were left untreated by culturing in non-conditioned media (fresh RPMI/ 1% BSA/M-CSF (4ng/ml)/P/S).
Results Fibroblast expression of collagen type I and connective tissue growth factor (CTGF) were not significantly different between untreated and macrophage conditioned medium treatment groups. Baseline levels of collagen type I were high in the fibroblasts cultured in non-conditioned media, and there was a trend towards increased CTGF expression in all conditioned media-treated groups compared to untreated fibroblasts in non-conditioned media. A 2.6-fold increase in α-smooth muscle actin (α-SMA) was observed in the healthy control M (IL-6)-conditioned medium-treated group compared to the group of fibroblasts cultured in non-conditioned media (one-way ANOVA with Sidak multiple comparison, p=0.048).
Conclusions After 24 hours treatment, control dermal fibroblasts treated with media of IL-6-polarised healthy control macrophages expressed higher levels of α-SMA compared to fibroblasts cultured in non-conditioned medium. A trend towards increased CTGF was also observed. These results suggest that paracrine factors in the IL-6-activated macrophage secretome may promote differentiation of fibroblasts into myofibroblasts, which is a key component of wound healing and scleroderma fibrosis.
Fuschiotti P. Current perspectives on the immunopathogenesis of systemic sclerosis. Immunotargets Ther. 2016;5:21–35.
Denton CP, Shi-Wen X, Sutton A, et al. Scleroderma fibroblasts promote migration of mononuclear leucocytes across endothelial cell monolayers. Clin Exp Immunol. 1998;114:293–300.
Khan K, Xu S, Nihtyanova S, et al. Clinical and pathological significance of interleukin 6 overexpression in systemic sclerosis. Ann Rheum Dis. 2012;71:1235–42.
Disclosure of Interest None declared