Tgf β2

PBMCs were isolated from whole heparinized blood samples from SSc patients and healthy controls or from the buffy coats of healthy controls, by density gradient centrifugation using Ficoll-Paque Plus (GE Healthcare, Chicago, IL, USA). CD14+ monocytes were purified from PBMCs using the MACS Human Monocyte Isolation Kit II (Miltenyi Biotec, Bergisch Gladbach, Germany) on the autoMACs Pro Separator (Miltenyi Biotec) according to the manufacturer’s instructions. For subsequent analysis, only cell preparations with more than 95% purity (measured by FACS analysis) for CD14+ cells were used.
For selected experiments, CD14+ monocytes purified from buffy coats were cultured in RPMI 1640 + 10% FCS (fetal calf serum, <0.5 EU/mL, Sigma-Aldrich, St. Louis, MO, USA) + 2 mM Glutamine at a concentration of 2 × 10⁶ cells/mL. Cultured cells were left untreated (medium control) or treated with one of the following stimuli: 100 ng/mL ultra-pure E. coli lipopolysaccharide (LPS, strain O111:B4, Invivogen, San Diego, CA, USA), 5 µM R848 (Invivogen), 1000 U/mL IFNα-2a (Cell Sciences, Newburyport, MA, USA), and TGF-β2 (Bio-Techne, Minneapolis, MN, USA) according to the conditions and times indicated for each experiment in the results section.

The human cytokines Interleukin 10 (IL-10), Transforming Growth Factor beta-1 (TGFβ1), Transforming Growth Factor beta-2 (TGFβ2), Transforming Growth Factor beta-3 (TGFβ3), and Tumor Necrosis Factor Alpha (TNFα) were purchased from Sigma-Aldrich, Interleukin 4 (IL-4) and Interferon Gamma (IFNy) from R&D Systems/Bio-Techne (R&D Systems/Bio-Techne, Wiesbaden-Nordenstadt, Germany), and Interleukin 6 (IL-6) and Vascular Endothelial Growth Factor165 (VEGF) from PeproTech (PeproTech, Winterhude, Germany). Porcine TGFβ3 was purchased from Biozol (Biozol, Eching, Germany), whereas porcine TGFβ1, TGFβ2, IL-4, IL-6, IL-10, IFNy and TNFα were from R&D System/Bio-Techne. Since there was no porcine VEGF available, the above mentioned human VEGF was also used for stimulation of pRMG.
To diminish the influence of cytokines present in FCS, both confluent pRMG and MIO-M1 cells were rinsed two times with prewarmed serum-free medium, followed by starvation for 1 h at 37°C and 5% CO2 with serum-deprived medium. Afterwards, cells were treated over night with IFNy, IL-4, IL-6, IL-10, TGFβ1, TGFβ2, TGFβ3, TNFα or VEGF165, respectively, in a randomized plate design at a concentration of 5 ng/ml in 2 ml medium without FCS. Untreated cells cultured in serum-free medium served as a control. For this study, cells were treated with each cytokine separately, but not with multiple cytokines in combination.