17 epi rvd1

Neutrophils were isolated (51 (link)) from the venous blood of healthy volunteers. The Clinical Research Committee at the Maisonneuve-Rosemont Hospital approved the experimental protocols (permit No. 99097) and each blood donor gave written informed consent. Neutrophils (5 × 10⁶ cells/mL, purity >96%, viability >98%) were cultured in RPMI medium 1640 supplemented with 10% autologous serum, IFN-β (12.5 to 50 ng/mL; Peprotech), 15-epi-LXA4 (1 μM; Cayman Chemical) or 17-epi-RvD1 (200 nM; Cayman Chemical), and then challenged with CpG DNA (E. coli strain B, 0.4 to 1.6 μg/mL). In some experiments, neutrophils were cultured with the FPR1 inhibitor cyclosporin H (1 μM, Tocris) or the ALX/FPR2 inhibitors N-t-Boc-Phe-Leu-Phe-Leu-Phe (Boc-2, 50 μM; MP Biomedicals) or Trp-Arg-Trp-Trp-Trp (WRW4, 5 μM; Tocris) (20 (link)), and then challenged with IFN-β and CpG DNA.

Neutrophils (5 × 10⁶ cells per milliliter, purity > 95%, viability > 98%, apoptotic < 2%) were resuspended in RPMI medium 1640 supplemented with 10% (vol/vol) autologous serum on a rotator with neutrophil elastase inhibitor IV (20 μM; Calbiochem), neutralizing anti-proteinase 2 Ab (5 μg/mL; Abcam), cathepsin G inhibitor (20 μM; Calbiochem), 1,10-phenantroline (4 mM; Sigma-Aldrich), and PMSF (2 mM), and then challenged with CpG DNA or mtDNA (0.4 to 1.6 μg/mL). In some experiments, PMNs were preincubated with 15-epi-LXA₄ (5S,6R,15R-trihydroxy-7E,9E,11Z,13E-eicosatetraenoic acid; 0.06 to 1 μM; Cayman Chemical) or 17-epi-RvD1 (7S,8R,17R-trihydroxy-4Z,9E,11E,13Z,15E,19Z-docosahexaenoic acid; 0.12.5 to 200 nM; Cayman Chemical) with or without the ALX/FPR2 inhibitor Boc-2 (50 μM; MP Biomedicals) or WRW4 (5 μM; Tocris) and then challenged with CpG DNA. In additional experiments, neutrophils were cultured with purified NE or PR3 (Athens Research and Technology).