Fig. 4. Cell-surface expression of NE on mouse blood neutrophils in vitro and in vivo occurs via PECAM-1-independent mechanisms. (A) Blood neutrophils from WT or PECAM KO mice were incubated for 30 minutes in 96-well plates coated with BSA or the combination of PECAM-1, ICAM-1 and IL-8 (total protein being 10 µg ml^–1) before being permeabilized and immunostained for expressions of NE (red; Alexa Fluor 633) or 6 integrins (green; Alexa Fluor 488), and analysed at RT using a Zeiss LSM 5 Pascal confocal laser-scanning microscope (using a 100x oil A-plan objective with a numerical aperture of 1.25) equipped with HeNe (excitation wavelength of 633 nm) and argon (excitation wavelength of 488 nm) lasers. Images are shown of mid-level sections of representative cells from three independent experiments. Bar, 10 µm. (B,C) Cell-surface expression of NE was quantified on blood or IL-1ß-elicited peritoneal neutrophils using a fluorogenic substrate. Briefly, mice (WT or PECAM-1 KO) were injected i.p. with saline (1 ml) or IL-1ß (10 ng) and, 4 hours later, killed by CO₂ asphyxiation. Purified blood neutrophils or peritoneal leukocytes were added to 96-well plates such that each well contained a total of 4x10⁵ neutrophils. After centrifugation, the samples were fixed and assayed for NE activity. (B) A comparison between NE activity on the cell surface of WT blood and peritoneal neutrophils (significant statistical difference between samples is indicated by asterisks: ***P<0.001). (C) A comparison between NE activity on the cell surface of IL-1ß-induced transmigrated neutrophils in WT and PECAM-1 KO mice. Results represent the means±s.e.m. of samples obtained from seven to ten mice in each group.