MICL modulates MSU-induced IL-8 secretion in human neutrophils. The PLB-985 cell line was differentiated with dibutyryl cyclic adenosine monophosphate (dibutyryl cAMP) for 72 h as described in Methods. The plasma membrane expression of Mac-1 (OKM1), the formyl peptide receptor-like 1 (FPRL1) and MICL were analyzed by flow cytometry on differentiated and undifferentiated PLB-985 cells. Immunoglobulin G2b (IgG2b) and IgG2a were used as control isotypes. (B) Dibutyryl cAMP-differentiated, neutrophil-like PLB-985 (20 × 106 cells/ml) were transfected using a nucleofection system with a MICL-specific small interfering RNA (siMICL) or with a negative control small interfering RNA (siCtrl). Cells were then stimulated for 3 h at 37°C with 1 mg/ml MSU in RPMI 1640 medium. Cells were centrifuged (16,000 × g for 5 min), and the supernatants were harvested and filtered. Extracellular interleukin 8 (IL-8) was quantitated using commercially available enzyme-linked immunosorbent assay (ELISA) kits. All samples were measured in duplicate. This graph is a compilation of four independent experiments. (C) MICL surface expression was monitored by flow cytometry following siRNA transfections. Cells (10 × 106 cells/ml) were incubated with 50C1 antibody (2 µg/ml) for 30 min on ice, washed and incubated with fluorescein isothiocyanate (FITC)-labeled goat antimouse Fcγ-specific IgG (diluted 1:100 in Hanks' balanced salt solution/bovine serum albumin (HBSS/BSA)) for 30 min on ice. Cells were then washed twice in HBSS/BSA and analyzed by flow cytometry using a BD FACSCanto II flow cytometer obtained from BD Biosciences. This graph is a compilation of four independent experiments. (D) Human neutrophils (20 × 106 cells/ml) were incubated with anti-MICL antibody (clone 50C1, 2 μg/ml) or IgG2a isotype for 5 min at 37ºC and then washed. Cells were then stimulated with MSU at 1 mg/ml or not for 3 h at 37°C. Extracellular IL-8 was monitored as described in (B). All samples were measured in triplicate. This result is a compilation of five independent experiments.