Recombinant human il 4

HASCs (10 × 10³ cells/cm²) were seeded into 24‐wells plates and cultured in αMEM containing 1% PSF, 10 IU/ml heparin, and 2% human PL in a humidified atmosphere containing 20% O₂ (normoxia [standard condition]) or 1% O₂ (hypoxia), and allowed to attach for 24 hr at 37°C. The next day, the medium was replaced with osteogenic medium, consisting of αMEM with 1% PSF, 10 IU/ml heparin, 2% human PL, 50 µM ascorbic acid‐2‐phosphate (vitamin C; Sigma‐Aldrich), 5 mM β‐glycerophosphate (Sigma‐Aldrich) and 10 nM 1,25‐(OH)₂vitamin D₃ (Sigma‐Aldrich). Recombinant human IL‐4 (R&D Systems, Minneapolis, MN), and/or recombinant human IL‐6 (R&D Systems) and recombinant human IL‐6Rα (R&D Systems) were added to the osteogenic medium. IL‐4 or IL‐6 were added in a final concentration of 1 and 10 ng/ml, respectively, and IL‐4 in combination with IL‐6 in a final concentration of 10 ng/ml. After addition of osteogenic medium supplemented with the cytokines, hASCs were incubated in 1% O₂ or 20% O₂ at 37°C during 3 days. Then, the medium was replaced by osteogenic medium without cytokines, and refreshed every 3 days during 11 days. hASCs were harvested after 2, 7, and 14 days of culture for analysis of proliferation, osteogenic differentiation, and VEGF expression as described below.

The acquisition of the reagents and media were as follows: Roswell Park Memorial Institute-1640 (RPMI-1640) medium with Ultraglutamine from Lonza (Verviers, Belgium); fetal bovine serum (FBS) from GE Health Care Life Sciences (GE Health Care, UT, USA), 2-mercaptoethanol from VWR International (Leuven, Belgium); Dulbecco’s Modified Eagle Medium/F-12 Nutrient Mixture (Ham) (DMEM/F-12; 1:1) from Gibco (Paisley, UK); phosphate-buffered saline (PBS) from Fisher Reagent (Geel, Belgium); dimethyl sulfoxide (DMSO) and phosphoric acid from Merk (Darmstadt, Germany); dimethylformamide (DMF) from Romil (Cambridge, UK), TripleXtractor and RNA Kit—Blood & Cultured Cells from GRiSP (Porto, Portugal), recombinant human IL-4 from R&D Systems (Minneapolis, USA). High-Capacity RNA-to-cDNA Kit and Master Mix from Applied Biosystems (Foster City, CA, USA) were used. When not specified, the reagents were from Sigma-Aldrich (ST. Louis, MO, USA).

NHBE culture was performed as previously described (40 (link)). Briefly, primary NHBEs (Lonza, Basel, Switzerland) of four genetically independent donors were grown as monolayers in 100% humidity and 5% CO₂ at 37 °C in serum-free defined growth media (BEGM, Lonza). NHBEs (passage 3) were used at ∼80% confluence in 6-well plates. To avoid gene expression changes or influences by growth factors in the BEGM medium, cells were rested in basal medium (BEBM, Lonza) for 12 h, then stimulated with HDM extract at a final concentration of 40µg/mL (CITEQ), recombinant human IL-4 at 50 ng/ml (R&D Systems, Minneapolis, MN, USA) for 6 h. When indicated, cells were pretreated for two h prior to stimulation with the AhR inhibitor CH-223191 (Sigma-Aldrich) at a concentration of 1µM/mL. For RNA analysis, harvested cells were lysed in RLT buffer (Qiagen, Hilden, Germany) containing 1% β-mercaptoethanol (Roth, Karlsruhe, Germany) directly in the cell culture well.

Buffy coats were bought from the Blood Transfusion Clinic at the Karolinska University Hospital, and monocytes were isolated from the buffy coats using commercial kits (RosetteSep Human Monocyte Enrichment Cocktail solution, Stemcell, Grenoble, France) and density gradient centrifugation on lymphoprep (Fresenius Kabi Norge AS, Oslo, Norway). To generate DC, the purified monocytes were cultured in RPMI 1640 medium containing GlutaMAX (Invitrogen Gibco, Paisly, UK) supplemented with 10% fetal bovine serum (HyClone,, Logan, UT, USA), 100 µg/mL streptomycin and 100 U/mL penicillin (Invitrogen Gibco), 250 ng/ml of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) (R&D systems or PeproTech, London, UK) and 6.5 ng/ml of recombinant human IL-4 (R&D systems). RPMI 1640 is described above, but without GM-CSF and IL-4 is referred to as complete RPMI, whereas RPMI 1640 with no supplementary agents is referred to as incomplete RPMI. On day 3 of culture, half of the medium was replaced with fresh complete RPMI containing IL-4 and GM-CSF. On day 6 or 7 the cells were harvested and washed with incomplete RPMI medium. The cell population used for the experiments described below typically contained >70% CD14− CD1a+ live cells, assessed by flow cytometry (data not shown).

Human naive B cells (>95% pure) were purified by negative selection, using the EasySep Human Naive B Cell Enrichment Kit (19254; StemCell Technologies), from healthy donor peripheral blood mononuclear cells, following the manufacturer's instructions. Naive B cells were then cultured in FBS–RPMI and stimulated with mCD154 (1, 2 or 4 U ml⁻¹), recombinant human IL-4 (20 ng ml⁻¹; R&D Systems) and recombinant human IL-21 (50 ng ml⁻¹; R&D Systems) for 24, 48, 72 and 96 h. RNA was extracted from stimulated and unstimulated B cells and used for qRT–PCR analysis.

Blood buffy coats were obtained from healthy volunteers after informed consent was obtained. The study protocol and any amendments were reviewed and approved by an independent review board (New England IRB, Newton, MA) before the start of the study. This study was conducted in accordance with the ethical principles of the Declaration of Helsinki.
PBMCs were freshly isolated from blood by Ficoll-Hypaque gradient centrifugation as previously described (39 (link)). Monocyte-derived dendritic cells were differentiated in vitro from freshly isolated human monocytes as previously described (39 (link)). Briefly, CD14⁺ monocytes were isolated from freshly purified PBMCs by negative selection and magnetic bead sorting (Miltenyi). Cells were then incubated in complete RPMI 1640 in the presence of 50 ng/ml recombinant human granulocyte-macrophage colony-stimulating factor and 20 ng/ml recombinant human IL-4 (R&D Systems) for 7 days. For all experiments using human donor cells, data were generated independently with at least two donors. A representative data set was selected for incorporation into the figure.