Interleukin il 4

Human monocytes were isolated from buffy coats using CD14 S-pluriBead antihuman beads (PluriSelect) according to manufacturer's instructions. The moDCs were generated by culturing monocytes for 5 days in RPMI1640 (Cytivia) medium supplemented with 10% (v/v) heat inactivated fetal calf serum (FCS; Sigma-Aldrich, St. Louis, MO, USA), 89 ng × mL⁻¹ GM-CSF and 22 ng × mL⁻¹ interleukin (IL)-4 (both Immunotools). Medium was exchanged on day 3.
PBMCs were isolated from buffy coats via Lymphoprep density gradient centrifugation (Stemcell Technologies, Vancouver, BC, Canada). PBMCs were stored in FCS containing 10% (v/v) DMSO at −170°C until further analyses.

Bone marrow-derived macrophages were isolated from the long bones of hTNF-α tg mice and stimulated with 5 ng/ml M-CSF (Miltenyi Biotec, Bergisch Gladbach, Germany) (d0) for 6 days with regular medium changes (Figure 1). On day 7, wells were subdivided into three groups and either stimulated with 5 ng/ml M-CSF for M0; 4 ng/ml GM-CSF (Miltenyi Biotec), 20 ng/ml interferon-γ (IFN-γ, ImmunoTools, Friesoythe, Germany), and 20 ng/ml lipopolysaccharide (LPS) for M1; and 5 ng/ml M-CSF and 20 ng/ml interleukin- (IL-) 4 (ImmunoTools) for M2 macrophages (Figure 1(a)) or stimulated with SN of FLS for 24 h, respectively (Figures 1(b) and 1(c)). Two hours after the addition of cytokines or SN, cells were irradiated (Figures 1(a) and 1(c)). Figure 1 summarizes these experimental set-ups. Cells were kept under standard conditions (37°C, 5% CO2; 95% humidity) in a medium containing Dulbecco's modified Eagle medium (DMEM, PAN Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum (FBS, Biochrom AG, Berlin, Germany), 5% horse serum (Sigma-Aldrich, Darmstadt, Germany), 1% penicillin/streptomycin (Gibco), and 50 μM 2-mercaptoethanol (Gibco).