Figure 1.

S100A8 and S100A9 induce EoL-1 cell apoptosis via TLR4. (A–D) EoL-1, HL-60, K562, U937, Jurkat cells (A,B), eosinophils, neutrophils, lymphocytes, and monocytes isolated from normal subjects (C,D) were incubated for 24, 48, or 72 h in the absence (Con) or presence of indicated concentrations of S100A8 (A,C) and S100A9 (B,D) (3 < n < 5). (E) EoL-1 cells were pretreated with anti-S100A8 (A8), anti-S100A9 (A9), or rabbit control (Ro) antibodies (2 μg/mL) and subsequently incubated with S100A8 or S100A9 (10 μg/mL) (n = 4). (F) EoL-1 cells were pretreated with or without 1 μM TLR4 inhibitor (TLR4i) and polymyxin B (10 μg/mL) for 1 h, after which the cells were incubated for 72 h in the absence or presence of S100A8 and S100A9 (50 μg/mL) (3 < n < 5). Data are presented relative to the S100A8- or S100A9-treated group, which is set at 100% (E,F). Data are expressed as the means ± SD. *p < 0.05 indicates a significant difference between the S100A8- or S100A9-treated group and stimulator-treated groups. (G) EoL-1 cells were treated with indicated concentrations of LPS and MPLA for 24, 48, and 72 h (n = 3). Apoptosis was analyzed by measuring the binding of annexin V-FITC and PI. (H) Flow cytometry was applied to determine TLR4 expression in EoL-1, HL-60, K562, U937, Jurkat cells, and normal eosinophils, neutrophils, lymphocytes, and monocytes without stimulators (3 < n < 5). Baseline fluorescence was obtained by incubating normal rabbit antibodies and was set at 1. (I, J) EoL-1 cells were incubated for 24, 48, and 72 h in the absence (Con) or presence of S100A8 and S100A9 (10 μg/mL) (n = 3). TLR4 expression was detected using Western blotting (I) and flow cytometry (J). Data are expressed as the means ± SD. *p < 0.05 and **p < 0.01 indicate a significant difference between the control and stimulator-treated groups.