Figure 4.

S100A8/A9 activates neutrophils. (A) Viability of neutrophils upon 2 h of incubation at 37 °C with S100A8/A9 was assessed by flow cytometry for AnnexinV and TO-PRO. Viable neutrophils were defined as negative for both markers (n = 3, mean + SEM). Indicated in the left panel is the gating strategy. (B,C) Expression of neutrophil activation markers (B) CD11b (n = 3–5, mean + SEM) and (C) CD62L (n = 2, mean + SEM) was measured by flow cytometry upon 2 h of incubation at 37 °C with indicated concentrations of S100A8/A9 or TNF-α in the absence or presence of 10% human serum. (B) Indicated in the left panel is the gating strategy. (C) FACS plots showing CD62-L shedding are displayed. (D) Neutrophils were left unstimulated or stimulated with LPS/LBP or different concentrations of S100A8/A9 for 30 min at 37 °C in the presence of 10% human serum. Percentage of adhesion is shown compared to total input (n = 3–5, mean + SEM). (E) Neutrophils were primed by LPS/LBP or different concentrations of S100A8/A9 in the presence of 10% human serum. After 30 min of incubation at 37 °C, 1 µM fMLF was added to induce ROS production. The maximal slope of H₂O₂ production was measured using Amplex Red (n = 5, mean + SEM). Statistics were performed by paired t-test (A,B,E) or mixed effects’ model with Dunnett post hoc test (D); * p < 0.05, ** p < 0.01, ns = not significant.