Healthy monocytes can differentiate into α-SMA and collagen expressing cells upon GM-CSF stimulation.

<p>A) CD14⁺ monocytes from healthy donors were cultured for 14 days with GM-CSF, IL-4 or ET-1. Western blot analysis showed expression of type-1 collagen and α-SMA under distinct conditions. Fibroblasts were used as a positive control, GAPDH was used as loading control. B) Monocytes were treated with GM-CSF and 0–500 ng/ml of ET-1 over 14 days. Western Blot analysis for type-1 collagen and α-SMA was conducted. GAPDH was used as loading control. C) and D) Quantification of the western blots from 3 independent experiments are shown. E) Expression of CD14, HLA-DR and CD11c was assessed by flow cytometry.</p

Morphological analysis of monocytes from SSc patients and healthy controls.

<p>A) Immunofluorescence staining of α-SMA (red) in GM-CSF treated monocytes from healthy control or a SSc patient. Fibroblasts were used as positive control. Nuclei were stained with DAPI (blue). For quantification, spindle shaped cells per 5×5 cm field were counted and normalized to cell numbers (B). ** Statistically highly significant (P-value<0.01).</p

Patient monocytes need less stringent stimulation to upregulate α-SMA.

<p>A) Monocytes from healthy controls or SSc patients were cultured for 14 days with GM-CSF, IL-4 or ET-1. α-SMA levels were detected by Western blot analysis. GAPDH was used as loading control. B) Quantification of Western blots from 8 patients and 8 healthy controls showed stronger expression of α-SMA in SSc monocytes compared to healthy controls despite no significant difference could be detected. C) qPCR analysis of the procollagen expression in GM-CSF treated monocytes from healthy controls (HC) (n = 4) or SSc patients (n = 8). Data were normalized to expression of 18S. (p = 0.481). D) and E) Monocytes from HC (n = 4) and SSc patients (n = 3) were treated with various combinations of GM-CSF, IL-4 and ET-1. Procollagen expression was measured by qPCR. Data were normalized to expression of 18S. The concentration of ET-1 in all experiments was 100 ng/ml. * Statistically significant (P-value<0.05).</p

Patient characteristics.

<p>Abbreviations: f:female, m:male, ANA: anti-nuclear antibodies; ACA: anti-centromere antibodies; nd: no data, AZA, azathioprine, Cyc: cyclophosphamide, MMF: mycophenolate mofetil, MTX: methotrexate.</p