Fig. 6.

HMGB-1 blockade but not MNase treatment reduced the cytokine stimulatory capacity of NET. CXCL8/IL-8 (a) and IL-6 (b) secretion by A549 cells cultured for 18 h at 37°C with medium (Basal), SN unPMN, SN NET-MSU, or SN NET-MSU obtained in the presence of MNase (10 U/mL). The effect of MNase on A549 cells was evaluated as a control. c CXCL8/IL-8 secretion by A549 cells stimulated with SN NET-MSU, neutrophil DNA (3 µg/mL), or thymus DNA (3 µg/mL). d The effect of heparin (Hep) on the ability of SN NET-MSU to stimulate IL-6 secretion by A549 cells. CXCL8/IL-8 (e, g) and IL-6 (f) secretion by A549 cells cultured for 18 h at 37°C in the presence or absence of SN NET-MSU (Basal) or heat-treated SN NET-MSU (SN NET-MSU Ø; 30 min at 70°C) (e, f) or SN NET together with EI (g). CXCL8/IL-8 (h) and IL-6 (i) secretion by A549 cells cultured for 18 h at 37°C with or without SN NET-MSU previously incubated for 30 min with an anti-HMGB-1 (aHMGB-1) blocking antibody (2G7) or the respective IgG2b isotype control. The effect of aHMGB-1 and its isotype control on the epithelial cells was assessed as a control. Data are depicted as the mean ± SEM of 3 (a, b), 4 (d, f, h, i), 5 (c, g), and 9 (e) independent experiments performed in triplicate. * p < 0.05 and nonsignificant (ns) (two-way ANOVA with repeated measures, followed by Bonferroni's multiple-comparisons test).