Tweak

Spleen cells were washed with phosphate-buffered saline (PBS; pH 7.2). After centrifugation at 1300 rpm and at 4 °C, the cells were incubated with CD19-coated magnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) and isolated on MACS separation columns (Miltenyi Biotec). Positively selected CD19⁺ B cells were stimulated with TWEAK (0.1 ng/ml; R&D Systems, Minneapolis, MN, USA) for 3 days. Total RNA was extracted using the TRI Reagent (Molecular Research Center, Cincinnati, OH, USA).

Proinflammatory cytokine, hyaluronan, and TWEAK levels in the culture supernatant of confluent orbital fibroblasts or in serum were determined using commercially available ELISA kits (IL-6, IL-8, MCP-1, and hyaluronan: R&D Systems; TWEAK: Bender Medsystems, Vienna, Austria) according to the manufacturers’ instructions. To investigate whether TWEAK-induced cytokine production is dependent on Fn14, cells were incubated with the Fn14-specific mAb ITEM4 (2.5 μg/ml) prior to stimulation. For comparison, GO cells were exposed to the signaling pathway inhibitors PD98059, SP600125, SB203580, and LY294002 (20 μM) and SC514 (10 μM) for 1 h prior to stimulation with TWEAK. The production of hyaluronan induced by TWEAK was analyzed in the same manner. hyaluronan concentration in the sample was determined from a standard binding curve generated with known amounts of hyaluronan. Samples were diluted 1:10 before analysis, and the mean value of triplicate samples is reported.
To analyze serum level of TWEAK, blood samples were drawn into test tubes containing 10% (v/v) sodium citrate. Platelet-free plasma was obtained by centrifugation at 3000 × g for 15 min at room temperature and stored at −80°C until analysis. Serum level of TWEAK protein (pg/ml) was measured using a commercial ELISA kit. Each sample was tested three times. All serum samples were tested in the same assay.