Changes in intracellular containment in subsets of neutrophils is associated with increased intraphagosomal pH and decreased HOCl production. (A) Healthy volunteers provided blood samples 180 minutes after injection of LPS. Neutrophils were fluorescence-activated cell sorted based on expression of FcyRIII (CD16) and L-selectin (CD62L). (B) Mean fluorescence of coculture of sorted neutrophil subsets (5 × 106/mL) and SA-GFP (3 × 106 CFUs per mL) in fibrin gels (n = 8). (C) Corresponding TUO of experiments described in panel B (n = 8). (D) Percentage of cell death over time as determined by propidium iodide staining in fibrin gels; propidium iodide was added to the coculture of 5 × 106 neutrophils per mL in fibrin gels. (E) Migration of sorted neutrophil subsets in fibrin gels toward the bacterial peptide fMLP as studied under light microscopy. (F) Phagocytosis, as defined by neutrophil AF647 positivity, was determined over time for sorted neutrophil subsets incubated in shaken suspension with dual-labeled SA bioparticles and 40% serum using flow cytometry (n = 8). (G) Quantitative fluorescence microscopy of the number of SA bioparticles per neutrophil for sorted subsets incubated in fibrin gels after 45 and 90 minutes (n = 5). (H) Neutrophil subsets were incubated with dual-labeled SA bioparticles in shaken suspension with 40% serum (n = 8). pHrodo and (I) AF647 fluorescence were determined over time by using flow cytometry. (J) Neutrophil subsets were lysed and MPO abundance was determined (n = 3). (K) Supernatants from degranulated neutrophil subsets were incubated with protease-specific fluorescent substrates, and activity was determined by using a fluorescence plate reader (n = 4). Significance for panels C, E, G, J, and K was determined by RM 1-way ANOVA followed by Tukey’s multiple comparison test. In panels F, H, and I, data points are the mean. Lines represent the predicted value after 1-phase association nonlinear regression analyses of the data, and 1-way ANOVA was applied to the predicted plateaus. Goodness of fit was assessed by calculating the correlation coefficient (r2) and visual examination of the data. Data are mean + SEM or mean ± SEM. *P < .05; **P < .01; ***P < .001; ****P < .0001.
