Fig. 2.

17-epi-RvD1, but not IFN-β restores impaired phagocytosis-induced neutrophil apoptosis. (A–C) Human polymorphonuclear neutrophil granulocytes (PMNs (5 × 10⁶ cells/mL) were cultured for 10 min with IFN-β (50 ng/mL) and then with CpG DNA (1.6 μg/mL) for 60 min. Surface expression of CD11b (A) and complement 5a receptor (C5aR, CD88) (B) was assessed by flow cytometry; neutrophil elastase activity in cell-free culture media was measured with a colorimetric assay using N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide as a substrate (C). Results are means ± SEM (n = 5 or 6 different blood donors). *P < 0.05, **P < 0.01. (D) Neutrophils were cultured with IFN-β for 10 min and CpG DNA for 60 min and then with opsonized FITC-labeled E. coli (seven bacteria per neutrophil) for 30 min. Extracellular fluorescence was quenched with 0.2% trypan blue and intracellular fluorescence was analyzed with flow cytometry. Results are means ± SEM (n = 5 different blood donors). *P < 0.05. (E) Neutrophils were cultured with IFN-β (50 ng/mL) for 10 min CpG DNA and then mixed with E. coli at a ratio of 1:1 or 1:7 for 3 h. Bacteria levels in culture media were assessed by growth on tryptic agarose. (F and G) Neutrophils were cultured with IFN-β (50 ng/mL), 15-epi-LXA₄ (1 μM), or 17-epi-RvD1 (200 nM) for 10 min, then with CpG DNA (1.6 μg/mL) and opsonized FITC-labeled E. coli (seven bacteria per neutrophil) for 24 h. Apoptosis was assessed by annexin-V staining (F) and nuclear DNA content (G) with flow cytometry. Results are means ± SEM (n = 5 different blood donors). *P < 0.05, **P < 0.01, ***P < 0.001 (Dunn’s multiple contrast hypothesis test).