(A) Neutrophil chemotaxis toward different chemoattractants. n = 3; *P < 0.05. (B) Neutrophil chemotaxis toward chemoattractants (control) or chemoattractants that were incubated with lactoferrin (10 μg/ml) followed by the addition of isotype or anti-lactoferrin monoclonal antibody (10 μg/ml). Antibodies were removed using magnetic IgG beads. n = 3; *P < 0.05, NS compared with chemoattractant control. (C) Immunoblot analysis of lysates of neutrophils incubated with or without biotinylated lactoferrin (10 μg/ml) at 37°C for 1 hour. (D) Neutrophil chemotaxis toward lactoferrin (10 μg/ml) purified from human neutrophils or human milk. **P < 0.001 vs. fMLP. C5a-induced monocyte (E) or macrophage (F) chemotaxis. (G) Neutrophil migration in the presence of lactoferrin (10 μg/ml) in the top or bottom compartment of the Transwell insert (n = 3; NS vs. corresponding +LTF controls). (H) Chemotaxis assay to determine neutrophil migration toward purified recombinant iron-depleted (Apo-), partially iron-saturated, and fully iron-saturated (Holo-) recombinant lactoferrin (10 μg/ml). Milk-purified lactoferrin and partially iron-saturated transferrin (TF; 10 μg/ml) were added as control. n = 4; ^†P < 0.001 compared with fMLP control. Error bars indicate SEM.