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. 2016 Aug 31;71(11):1030–1038. doi: 10.1136/thoraxjnl-2015-207604

Figure 4.

Figure 4

Hypoxic upregulation of neutrophil degranulation is not hypoxia-inducible factor (HIF)-dependent. (A and B) Hypoxia mimetics do not recapitulate the hypoxic upregulation of neutrophil degranulation. Neutrophils were incubated under normoxia for 4 h±DMOG (dimethyloxalyl glycine) (1 mM, A) or desferrioxamine (DFO) (1 mM, B) or were incubated under hypoxia (positive control), prior to priming with GM-CSF (GM) (10 ng/mL, 30 min) and activation with formylated peptide (fMLP) (100 nM, 10 min). Supernatants were assayed for active elastase. Results represent mean±SEM (n=5–6, each performed in triplicate). (C–E) Hypoxia does not increase granule protein transcription, translation or total protein. Neutrophils were incubated under normoxia or hypoxia for 4 h before RNA isolation or protein quantification. (C) Granule protein (NE, neutrophil elastase and MMP-9, matrix metalloproteinase-9) and BNIP (BCL2/adenovirus E1B 19 kd-interacting protein-2, positive control) gene expression as measured by quantitative PCR (qPCR); results represent mean±SEM (n=3, each performed in triplicate). (D) Gelatinase granule content measured by western blot analysis of MMP-9; results represent mean±SEM (n=3). (E) Hypoxic elastase release was not prevented when translation was inhibited by cycloheximide, present for the entire incubation, prior to stimulation with GM-CSF (GM) and fMLP. Results represent mean±SEM (n=5, each performed in triplicate). (F) Reoxygenation (15 min) of neutrophils before priming and activation does not significantly reduce degranulation as measured by the release of active elastase. Results represent mean±SEM (n=5, each performed in triplicate). *p<0.05, **p<0.01, ***p<0.005.