Hydrocortisone

Primary human umbilical vein endothelial cells (HUVECs, Lonza Verviers SPRL, Verviers, Belgium) were cultured in endothelial cell basal medium (ECBM, PromoCell, Heidelberg, Germany) supplemented with 10% fetal calf serum (FCS), 4 μL/mL endothelial cell growth supplement/heparin, 0.1 ng/mL human epithelial growth factor, 1 ng/mL human basic fibroblast growth factor, 1 μg/mL hydrocortisone (C-39210, PromoCell), 2 mM glutamine, 100 U/mL penicillin and 100 μg/mL streptomycin (Life Technologies, Cergy Pontoise, France). HUVECs from 3 independent donors were used for the assays. For activation, ECs were grown to confluence in 6-well, 12-well, or 96-well plates and starved overnight in ECBM supplemented with 2% FCS before treatment. Prior addition of TNF (200 U/mL, 6h) or IFNγ (100 U/mL, 48h) cells were preincubated with NPs or controls (diluent, PDTC, ZA, CsA, glyoxal) for the indicated periods of time. Experiments were also performed without cytokines to evaluate the effect of NPs on the constitutive expression of the cell surface molecules.

Human umbilical vein endothelial cells (HUVEC) were purchased from Promocell (Heidelberg, Germany). Expansion, cell cultivation and experiments were performed using endothelial cell growth medium (ECGM) supplemented with 0.4% endothelial cell growth supplement (ECGS), 2% fetal calf serum (FCS), 0.1 ng/ml epidermal growth factor (EGF), 1 ng/ml basic fibroblast growth factor (bFGF), 90 μg/ml heparin and 1 μg/ml hydrocortisone (all from Promocell). HUVEC at passages 2 to 6 were grown in a humidified incubator at 37°C and 5% CO₂ and used for experiments. HUVEC were seeded in the respective plates 24 h prior to medium change and stimulation. A 1-hour pre-incubation was performed in co-incubation experiments using SnPPIX or bafilomycin A₁. All incubations were performed in ECGM. Most test substances were dissolved in DMSO and further diluted with ECGM yielding final DMSO concentrations in incubates of 0.02% (v/v) (for SB202190), 0.025% (v/v) (for bafilomycin A₁) or 0.05% (v/v) (for SB202474 and SB203580). Vehicle for SnPPIX in incubates was ECGM containing 0.1% (v/v) NaOH (1 M). As vehicle control ECGM containing the respective amount of DMSO and/or NaOH was used. SiRNAs were diluted in RNase-free water according to the manufacturer’s instructions.

We cultivated primary human skin fibroblast lines (HF40 and HFF-1) (n = 2 each) that were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and cultivated in Dulbecco's minimum essential medium supplemented with 10% fetal calf serum and, when confluent, medium was supplemented with or without recombinant human (rh)-IL-17 (R&D System, Minneapolis, MN) of 200 ng ml⁻¹ (same IL-17 source and concentration used in prior experiments with human keratinocytes) [10] (link), [12] (link). After 24-hour incubation, fibroblasts were harvested for further analyses.
We also cultivated NHEKs obtained from PromoCell, in the Keratinocyte Growth Medium 2 supplemented with 0.004 ml/ml BPE, 0.125 ng/ml EGF, 5 ug/ml Insulin, 0.33 ug/ml Hydrocortisone, 0.39 ug/ml Epinephrine, 10 ug/ml Transferrin, and 0.06 mM Ca++ (all items purchased from PromoCell GmbH, Heidelberg, Germany). The experiment was performed in triplicate.
Once 70–80% confluent, the medium was changed with full media containing 0.06 mM Ca++, 1.2 mM Ca++, or 1.2 mM Ca++ plus 2.0% FBS, for 24 and 48 hours before harvesting for other analyses.