Cell culture

Human FLS were isolated from synovial tissues obtained from RA patients, as previously described 13. Inguinal lymph nodes from mice were excised 10 days after the onset of CIA. Cells were dissociated and cultured at a density of 2 × 10⁶ cells/ml in RPMI 1640 supplemented with 10% fetal bovine serum (FBS), 50 units/ml of penicillin/streptomycin, and 50 μM 2‐mercaptoethanol. Cells were left unstimulated or were stimulated with 100 ng/ml of anti‐CD3 monoclonal antibody (145‐2C11; eBioscience), and supernatants were collected for cytokine analysis at 48 hours after stimulation.

For generation of bone marrow–derived macrophages (BMMs), bone marrow cells were flushed from the tibias and femurs of 7–12‐week‐old male DBA/1 mice. Erythrocytes were depleted using an Erythrocyte Lysing kit (R&D Systems), and the remaining cells were cultured in RPMI 1640 supplemented with 10% FBS, 50 units/ml of penicillin/streptomycin, and 50 μM 2‐mercaptoethanol containing 50 ng/ml of macrophage colony‐stimulating factor (PeproTech). The medium was changed on day 3. BMMs were harvested on day 6 using a nonenzymatic cell dissociation solution (Sigma‐Aldrich) and were plated in a 96‐well plate (5 × 10⁴ cells/well) or a 48‐well plate (3 × 10⁵ cells/well). The next day, cells were treated with 10 nM DX‐2400 and/or TNFR‐Fc, with or without lipopolysaccharide (LPS; 1 ng/ml), and at 24 hours after treatment, supernatants and total RNA, which was extracted using an RNeasy kit (Qiagen), were collected.