Figure 2.

NETs released from particle-treated neutrophils induce secretion of TNF-α by macrophages. (A) Human neutrophils were treated with mineralo-organic particles for 30 min, followed by incubation with PMA-primed macrophages for 8 hours. TNF-α secretion was measured using ELISA. Data are shown as means ± SEM and the results of three independent experiments. *p < 0.05, as compared to untreated neutrophils. (B) Experimental design used to investigate the neutrophil-macrophage interaction. THP-1 cells were either directly co-cultured with neutrophils (top panel) or grown in the upper chamber of the Transwell system, separated from the neutrophils in the lower chamber by a semi-permeable membrane, which prevented cell-to-cell contact but allowed diffusion of soluble material (bottom panel). (C) Co-culture of THP-1 cells with particle-treated neutrophils enhanced TNF-α secretion. Data are shown as means ± SEM and the results of three independent experiments. *p < 0.05 and **p < 0.005, vs. untreated neutrophils. (D) Indirect co-culture of THP-1 cells and particle-treated neutrophils in the Transwell system failed to induce TNF-α secretion, but treatment of NETs with DNase enhanced TNF-α secretion. Data are shown as means ± SEM and the results of three independent experiments. **p < 0.005, vs. untreated neutrophils. (E) THP-1 cells or (F) neutrophils in the Transwell system were subjected to immunoblotting to detect intracellular TNF-α. Data are representative of three independent experiments.