FIGURE 6.

Effect of Ca²⁺ chelator and anti-IL-8, -TNF, or -FasL Ab on AGE-mediated cell death. A, U-937 cells were preincubated with 1 μg/ml anti-IL-8 or -TNF Ab for 2 h and then stimulated with AGE-HSA (100 μg/ml) for 24 h or with IL-8 (100 ng/ml) or TNF (100 pm) for 2 h. Cells were washed, and intracellular Ca²⁺ was measured using Fura-2AM as fluorescence probe in a fluorometer fixing emission at 510 nm and excitation from 300 to 400 nm. B, U-937 cells were preincubated with CsA (2.5 μm) and BAPTA-AM (5 μm) or with anti-TNF or -IL-8 Ab for 2 h and then stimulated with AGE-HSA for different times. Nuclear extracts were used to assay NF-κB DNA binding by gel shift assay. Cells were preincubated with CsA and anti-IL-8 Ab for 2 h and then stimulated with AGE-HSA for 24 h. C, calcineurin activity was measured from whole cell extracts and indicated as -fold of activation considering value of unstimulated cells as 1-fold. U-937 cells were transfected with Qiagen SuperFect reagent for 3 h with plasmids for FasL promoter DNA that had been linked to luciferase (FasL-luciferase) and GFP. After washing, cells were cultured for 12 h. Cells, incubated with anti-IL-8 Ab for 2 h, were stimulated with AGE-HSA (100 μg/ml) for 48 h. D, the luciferase activity was measured from whole cell extracts and indicated as -fold of activation. E, cells were preincubated with CsA, BAPTA-AM, anti-TNF Ab, or anti-IL-8 Ab for 2 h and then stimulated with AGE-HSA for 48 h. FasL was detected by RT-PCR from total RNA isolated from cells. F, U-937 cells, treated with cystamine (500 μm), brefeldin A (5 μg/ml), or diltiazem (100 μm) for 2 h, were stimulated with AGE-HSA for 24 h. Culture supernatant was concentrated 10 times and used to detect IL-8 and proteinase 3 by Western blot. Cells, treated with diltiazem, brefeldin A, or cystamine for 2 h in triplicate, were stimulated with AGE-HSA for 48 h. G, the MTT assay was done and indicated as inhibition of cell death in percentage. U-937 cells, incubated with anti-IL-8 Ab, anti-TNF Ab, CsA, or BAPTA-AM for 2 h, were stimulated with AGE-HSA for 48 h. Error bars in C, D, and G indicate ± S.D. of triplicate samples. H, after these treatments, cell death was detected by annexin V-phycoerythrin and analyzed in FACS.