PMNs from term and preterm neonates respond to LPS and PAF, excluding developmental deficiency in these signaling systems as a mechanism of defective NET formation. (A) Expression of mRNA for TLR4, the PAF receptor, and GAPDH in PMNs isolated from each subject group was determined by semiquantitative RT-PCR. (B) FITC-labeled LPS uptake was determined in each subject group by immunocytochemistry and confocal microscopy. (C) Intracellular Ca⁺⁺ release was determined by fluorometry following preincubation with Fluo4, a Ca⁺⁺-dependent fluorochrome, and stimulation with the indicated concentrations of PAF. The data bars indicate mean plus or minus SEM fluorescence in 3 separate experiments with PMNs from adult, term, and preterm subjects. (D) Secretion of IL-8 at baseline and at 240 minutes was determined by ELISA following stimulation of PMNs with PAF (10 nM) or LPS (100 ng/mL). The data indicate mean plus or minus SEM supernatant IL-8 concentrations in 3 separate experiments with PMNs from each subject group. A similar pattern was seen after a 120-minute incubation time (not shown).