Figure 1.

Neonatal neutrophils form CR3-mediated clusters and rapid neutrophil extracellular traps (NETs) in response to fibronectin (Fn) plus β-glucan, which is independent of respiratory burst. Neonatal neutrophils on fibronectin plus β-glucan form aggregates and NETs. A, Micrographs show adult and neonatal polymorphonuclear leukocytes (PMNs) that were adhered to wells precoated with Fn alone (left columns) or Fn supplemented with β-glucan (right columns). PMNs were pretreated with 10⁻⁹ M fMLF and 1 mM Mn⁺² immediately before adding cells to the coated wells and incubating for 30 minutes at 37°C. Data represent at least 5 independent experiments done using neutrophils from different individual donors. All images were obtained at 20× original magnification (bar, 100 µm). B, Adult and neonatal PMNs prepared as described above were pretreated with CR3-blocking monoclonal antibody (clone 44abc; left columns) or the respiratory burst inhibitor DPI (right columns) before they were adhered to wells coated with Fn plus β-glucan. Images were taken at 20× original magnification (bar, 100 µm). These results represent at least 4 independent experiments, using neutrophils from different individual donors. C, Quantification of NET formation as the percentage of the total imaged field. Data represent 4–20 wells per condition. Error bars represent standard errors of the mean (SEM). *P < .01 vs cells treated with Fn alone and those treated with CR3-blocking monoclonal antibody. D, Scanning electron microscopy images of neonatal PMNs obtained at 40× original magnification, demonstrating NET elaboration. Neonatal neutrophils were prepared as described above and were then fixed and prepared for scanning electron microscopy. Abbreviation: NS, nonsignificant.