Stimulated PMNs from term and preterm infants extrude less DNA and release less neutrophil elastase, a surrogate marker of NET formation, than do PMNs from adults. (A) Supernatant DNA content was determined via fluorometry by quantitation of a non–cell-permeable DNA dye (Sytox Orange) in samples from PMNs stimulated with LPS (100 ng/mL) for 1 hour. The data are expressed as mean plus or minus SEM values for extracellular DNA. The asterisk indicates a significant difference (P < .001) between values from adult PMNs and those from term and preterm neutrophils. (B) NET-associated neutrophil elastase was examined by immunocytochemical analysis of PAF-stimulated and control PMNs from healthy adults and term infants. Stimulated PMNs from adults (left) extruded NETs that stained robustly for NE (magenta fluorescence; white arrows). Stimulated PMNs isolated from term infants (right) failed to form NET lattices containing extracellular NE. NE was detected in primary granules in PMNs from both term neonates and adults (yellow arrows). Extracellular and intracellular DNA counterstaining as in Figure 1 demonstrated both nuclear and, depending on the source of PMNs (adult vs neonate), extracellular NET-associated DNA. (C) NET-associated NE activity was determined in DNase-treated incubations of control PMNs and neutrophils stimulated with LPS (100 ng/mL) for 120 minutes. Mean plus or minus SEM NE concentrations are shown. The asterisk indicates a significant difference (P < .05) in NE concentration in DNase-treated samples from neonatal PMNs compared with the DNase-treated samples from adult PMNs. The images and data in Figure 3 are from a minimum of 3 separate experiments in each case.