|
Neutrophil treatment with exogenous SEMA3F blocks chemotactic responses while preserving phagocytic capacity and respiratory burst functions.(A–F) Isolated peripheral blood neutrophils from healthy volunteers were incubated with recombinant SEMA3F (0–100 nM), and functional assays were performed. (A and B) Chemotactic behavior of neutrophils to fMLF (0–100 nM) and LTB4 were measured by Boyden chamber, CC chemokinesis control (A), and microfluidic chip assay (B). (C) Neutrophils were incubated with Alex 488 E. coli and phagocytic uptake was determined by flow cytometry, with adhesion excluded by 4°C control. (D) Phagocytic indices were calculated by cytospin following neutrophil culture with opsonized Zymosan particles for 30 minutes. (E) ROS generation was determined following a 1 hour preincubation with SEMA3F and treatment with fMLF for 30 minutes. (F) Neutrophil release of elastase was measured by fluorimetric assay following pretreatment with 1 hour of SEMA3F, 30 minutes of GM-CSF (10 ng/mL), and 10 minutes of fMLF (100 nM). All data are mean ± SEM with individual data points from independent experiments (n = 3–6). Statistical analysis was by 1-way ANOVA and Sidak’s post hoc test (A, B, E, and F) or paired t test (C–E). *P < 0.05; **P < 0.01; ****P < 0.0001.
|
