Sfasl

Levels of sTRAIL, sFasL, MCP1, IP10 and Eotaxin3 were assessed in duplicates in cell culture supernatants and uterine flushing samples using commercial enzyme-linked immunosorbent assays (ELISAs) (TRAIL, MCP1, IP10, Eotaxin3: R&D Systems, Amersham, United Kingdom; sFasL: Sigma–Aldrich, St. Louis, MO, United States).

Human siRNAs for TNFRSF10A (SASI_Hs01_00139573), TNFRSF10B (SASI_Hs01_00040567), TNFRSF1A (SASI_Hs01_00033456), TNFRSF12A (SASI_Hs01_00129286), FAS (SASI_Hs02_00301734), RELA (SASI_Hs01_00171091), CUHK (SASI_Hs01_00206921), and IKBKB (SASI_Hs01_00156170), and the MISSION siRNA universal negative control were purchased from Sigma-Aldrich (St. Louis, MO). Human synovial cells were transfected with siRNA by electroporation using the Neon Transfection System kit (Thermo Fisher Scientific), in accordance with the manufacturer’s instructions. After 24 hr of transfection, cells were stimulated with 400 ng/mL sFasL (R and D Systems).

1 × 10⁵ CD27⁺ or CD27⁻ B-cells were incubated with agonistic anti-CD40 mAb (1 μg/ml, CP-870,893; kindly provided by Pfizer, New London, CT), the dsRNA complex polyinosinic:polycytidylic acid (10 μg/ml, poly(I:C); Sigma), lipopolysaccharide (10 μg/ml, LPS; Sigma), resiquimod (10 μg/ml, R848; Sigma) or CpG oligodeoxynucleotide (ODN) 2006 (1 μg/ml, InvivoGen, San Diego, CA). After 48 hours, cells were washed in complete medium and were then cultured in complete medium containing agonistic anti-CD95 mAb (2 μg/ml, CH11; MBL International, Woburn, MA) or human rTRAIL (1 μg/ml; R&D Systems, Minneapolis, MN) for an additional 18 hours. In confirmatory experiments, an alternative anti-CD95 mAb (SM1/1, eBioscience, San Diego, CA) or a sFasL (R&D Systems, Minneapolis, MN) incubated with anti-His Tag (R&D Systems, Minneapolis, MN) to allow cross-linking of Fas receptors. Cells were then washed in PBS and resuspended in Annexin-V binding buffer with Annexin-V-FITC and propidium iodide (PI; BioLegend, San Diego, CA). The apoptosis induced by adding agonistic anti-CD95 mAb or rTRAIL was defined as the change in % Annexin-V⁺ and calculated by subtracting the value for percentage of Annexin-V-positive cells in culture medium alone (background apoptosis) from the value for percentage of apoptosis in a replicate culture containing agonist anti-Fas mAb or rTRAIL.