Murine neutrophils exhibit enhanced ICAM-1 expression in a stimulus-specific manner in vitro. Whole blood from WT or ICAM-1 KO mice was incubated (4 hours at 37°C) with IL-1β (50 ng/mL), LTB4 (30 nM), KC (30 nM), fMLP (1 µM), Zymosan (10 µg/mL), LPS (300 ng/mL), or TNF (100 ng/mL). After the red blood cell (RBC) lysis step, leukocytes were labeled with DAPI and fluorescent antibodies against CD45, Ly6G, and ICAM-1. Samples were analyzed by flow cytometry. (A) Percentage of ICAM-1–positive (ICAM-1pos) neutrophils in WT and ICAM-1 KO samples. (B) RFI compared with isotype control of unstimulated and LPS-stimulated WT neutrophils. (C) Representative histogram of ICAM-1 expression on saline or LPS-stimulated WT neutrophils compared with binding of an isotype control. (D) Time course of cell-surface ICAM-1 expression. (E) Naive or LPS-stimulated WT neutrophils labeled with fluorescent antibodies against Ly6G and ICAM-1 and the nuclear marker Draq5. Some samples were permeabilized before ICAM-1 labeling to visualize intracellular stores in addition to surface expression. Cells were imaged by confocal microscopy and analyzed using Imaris imaging software. (F) Quantitative polymerase chain reaction analysis of ICAM-1 mRNA in saline or LPS-stimulated neutrophils. Unstimulated expression levels were normalized to 1; LPS-stimulated data are shown as fold change compared with unstimulated samples. (G) Western blot of ICAM-1 and β-actin loading control in naive or LPS-stimulated purified neutrophils treated with protease inhibitor at the time of collection. Data are expressed as mean ± SEM of n = 3-27 animals/group. Statistically significant (t test) differences between treatment groups: *P < .05 and ***P < .001; differences between WT and ICAM-1 KO: #P < .05, ##P < .01, and ###P < .001.
