Recombinant human il 4

Samples of 40-50 mL citron anticoagulant cord blood were obtained from full-term healthy pregnancies during cesarean sections and used to obtain mononuclear cells via lymphocyte separation medium (TBD, Tianjin, China), according to the manufacturer's instructions. Mononuclear cells were suspended in improved minimum essential medium (IMEM; Gibco) and cultured for 6 hours at 37 °C and 5% CO₂. After incubation, 95% of suspended cells were T cells, which were collected and cultured in RPMI 1640 plus 300 U/mL recombinant human interleukin (IL)2 and 50 ng/mL purified anti-human CD3 monoclonal antibody (eBioscience, San Diego, CA, USA). Adherent cells could be differentiated into DCs through culture in IMEM containing 1000 U/mL recombinant human colony-stimulating factor 2 and 500 U/mL recombinant human IL4 (PeproTech, Rocky Hill, NJ, USA). On day 5, the DCs were loaded with 10 mg/mL recombinant MTBHsp70-exFPR1 fusion protein, a mixture of MTBHsp70 and exFPR1, MTBHsp70, exFPR1, or phosphate-buffered saline (PBS). On day 8, the DCs were collected for flow cytometry or cocultured with autologous lymphocytes for 7 days as therapeutic vaccines for further experiments.

Resveratrol (RSV), LPS, compound C (CC), and Ficoll-Paque were purchased from Sigma-Aldrich (St. Louis, MO, USA). Recombinant human IL-4, recombinant human macrophage-colony-stimulating factor (M-CSF), and interferon (INF) γ were from Pepro Tech (Rocky Hill, NJ, USA). Sirtinol was obtained from Calbiochem (La Jolla, CA, USA). Antibodies specific for AMPKα, phosphor-AMPKα, phosphor-NF-κB p65 (Lys310), acetyl-NF-κB p65 (Lys310), acetyl-CoA carboxylase (ACC), and phosphor-ACC were from Cell Signaling (Danvers, MA, USA). Antibodies specific for SIRT1, NF-κB p65, ariginase-1, and Histone H1 were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Antibodies specific for TNFα and IL-1β were from Abcam (Cambridge, UK). Antibody specific for TRANCE/TNFSF11 was from R&D Systems (Minneapolis, MN, USA).

DCs were kindly provided by Assoc. Prof. Dr. Natthanej Luplertlop, Department of Microbiology and Immunology, Faculty of Tropical Medicine, Mahidol University. DCs were isolated from human peripheral blood mononuclear cells by density centrifugation over Ficoll-Paque Plus (Sigma-Aldrich, Germany) [20 (link)]. Magnetic beads coated with antibodies (Abs; Miltenyi Biotec, France) were used to purify monocytes by negative selection. The monocytes were seeded at 10⁶ cells/mL in RPMI-1640 medium supplemented with 10% (v/v) fetal calf serum, 1% penicillin/streptomycin, 50 ng/mL recombinant human IL-4 (PeproTech, France), and 100 ng/mL recombinant human granulocyte–macrophage colony-stimulating factor (PeproTech, France) and cultured for 7 days at 37 °C with 5% CO₂ in a humidified incubator.

Human synovial sarcoma cell line SW982 is highly recommended as a suitable human synoviocyte model for the study of RA therapy such as fluvastatin-induced apoptosis signaling [17 (link)] and hence was used in this study. The SW982 cell line was cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and incubated at 37°C in humid conditions provided with 5% CO₂. SW982 cells (2 × 10⁵ cells/ml) were plated into 6-well plates overnight before treated with various arthritis-related TLR ligands including 10 ng/ml PGN (TLR2 ligand), 10 μg/ml poly I:C (PIC, TLR3 ligand), 10 ng/ml LPS (TLR4 ligand), and 3 μg/ml imiquimod (TLR7 ligand) and inflammatory cytokines including 100 ng/ml recombinant human IFN-γ (PeproTech, USA), 10 ng/ml recombinant human IL-4 (PeproTech, USA), 10 ng/ml recombinant human TNF-α (PeproTech, USA), and 10 ng/ml recombinant human IL-1β (PeproTech, USA) for various time points. The cells were then harvested for RNA or protein isolation.

The human U87 cells line and human umbilical vein endothelial cells line (HUVECs) were obtained from American Type Culture Collection (ATCC). U87 cells were grown in high glucose Dulbecco's Modified Eagle Medium (DMEM) (Gibco, Invitrogen) supplemented with 10% FBS (Gibco, Invitrogen) and 1% penicillin/streptomycin (Gibco, Invitrogen). HUVECs were maintained in RPMI 1640 (Gibco, Invitrogen) supplemented with 10% FBS and 1% penicillin/streptomycin. All the cells were grew in 5% CO₂ at 37°C.
Fresh human peripheral blood was obtained from Sun Yat-sen Memorial Hospital, according to the guidelines approved by the ethics committee at Sun Yat-sen Memorial Hospital. Monocyte-derived M₂ macrophages were generated from human PBMC as described previously[12 (link)]. Briefly, PBMC were isolated from peripheral blood by density gradient centrifugation. After being purified by using anti-CD14 microbeads (Miltenyi Biotec), monocytes were incubated in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin for 3 days. M₂ macrophage polarization were obtained by removing the median and culturing cells for another 3 days in DMEM supplemented with 40 ng/ml Recombinant Human IL-4 (PeproTech).

ML351, PD146176, 25-hydroxycholesterol, 15(S)-HETE, 13(S)-HODE, and 17(S)-HDHA were purchased from Cayman Chemical (Ann Arbor, MI, USA), Bafilomycin A1, Methyl-β-cyclodextrin from Sigma-Aldrich (Steinheim, Germany), CC chemokine receptor 4 (CCR4) antagonist from EMD Millipore Corp. (Billerica, MA, USA), and recombinant human IL-4 from PeproTech (Hamburg, Germany). Primers were purchased from http://Biomers.net GmbH (Ulm, Germany) and their sequences are available on request. All chemicals were of the highest grade of purity and commercially available.