FIGURE 3.

Effect of hypoxia on neutrophil phagocytic function. A and B, Hypoxia preserves neutrophil surface expression of CD11b, but not CD16. Neutrophils were incubated under normoxic or hypoxic conditions for 1–4 h prior to stimulation with fMLP (100 nM) or buffer. Fixed cells were stained with FITC-labeled anti-CD11b (A) or anti-CD16 (B), as described, and analyzed by flow cytometry. Data represent mean ± SEM of n = 4 independent experiments performed in triplicate. *p < 0.05. Owing to donor variability in baseline expression, results were normalized to normoxic controls. C–F, Hypoxia does not compromise the ability of neutrophils to phagocytose opsonized streptococci. Neutrophils were preincubated under normoxia or hypoxia for 1 or 3 h prior to the addition of opsonized FITC-labeled S. pneumoniae for 1 h; cells kept at 4°C were used to control for nonspecific adhesion. Representative histograms (C) illustrating phagocytosis of FITC-labeled S. pneumoniae by normoxic and hypoxic neutrophils (1 h) assessed by flow cytometry. Electron microscope image (D) of a neutrophil (osmium tetroxide stained, original magnification ×18,000) containing ingested bacteria; arrows point to S. pneumoniae within phagosomes. Phagocytosis of FITC-labeled S. pneumonia did not differ when neutrophils were maintained under normoxia or hypoxia for 1 or 3 h, assessed by % of FITC-positive neutrophils (E) or mean fluorescence intensity (F). Data represent mean ± SEM of three independent experiments performed in triplicate.