FIG. 2.

Incubation of bovine PMNs with IL-1β enhances LKT binding in a β₂-integrin-dependent manner. Freshly isolated bovine PMNs (10⁶ cells/ml) were incubated with recombinant bovine IL-1β (50 ng) or medium for 15 min at 37°C. As an additional control, some PMNs were incubated with heat-inactivated (100°C for 10 min) IL-1β for 15 min at 37°C. Some of the IL-1β-stimulated and control PMNs were incubated (40 min at 4°C) with anti-β₂-integrin MAb BAT75A (50-μg/ml final concentration). PMNs were then incubated with biotinylated partially purified P. haemolytica LKT (10 to 20 U) for 10 min on ice. The cells were washed, Extra-avidin-FITC was added, and the cells were incubated for 20 min on ice. The stained cells were washed, fixed with paraformaldehyde, and analyzed by flow cytometry (10,000 cells were scored for green fluorescence). As an additional control, PMNs were incubated with partially purified culture filtrate from an LKT mutant that lacks LKT activity (prepared in the same manner as the wild-type LKT) and stained as indicated above. The data indicate the mean (± standard error of the mean) percent positive cells from five independent experiments. Asterisks indicate statistically significant differences, compared with control PMNs incubated with LKT alone (P < 0.05), determined using the Student-Newman-Keuls multiple-comparison test.