Recombinant bovine interleukin-1beta amplifies the effects of partially purified Pasteurella haemolytica leukotoxin on bovine neutrophils in a beta(2)-integrin-dependent manner

Abstract

The influx and death of polymorphonuclear leukocytes within the infected lung are hallmarks of bovine pasteurellosis. Recent reports have shown that the Pasteurella haemolytica leukotoxin (LKT) and other RTX toxins bind beta(2)-integrins on target cells. In this study we demonstrate that exposure of bovine neutrophils to recombinant bovine interleukin-1beta upregulates beta(2)-integrins (CD11a/CD18), which in turn enhance the binding and amplify the biological effects of partially purified LKT on these cells. LKT binding and cytotoxicity were inhibited by addition of an anti-integrin antibody (CD11a/CD18). These findings help to clarify the early events that occur in bovine pasteurellosis and support the hypothesis that inflammatory mediators might increase the severity of pasteurellosis by causing upregulation of beta(2)-integrins that serve as an LKT receptor on bovine neutrophils.

FIG. 1

Incubation with recombinant bovine IL-1β upregulates β₂-integrin expression on bovine PMNs. (A and B) Freshly isolated bovine peripheral blood PMNs (10⁶ cells/ml) were incubated with IL-1β (50 ng) for 15 min at 37°C (open trace) or with medium (solid trace) before incubation (40 min at 4°C) with anti-β₂-integrin MAb BAT75A (50-μg/ml final concentration) (A) or CA1.4E9 (50-μg/ml final concentration) (B). The cells were then washed, incubated with an FITC-labeled second antibody, and analyzed by flow cytometry (10,000 cells were scored for green fluorescence). Panels A and B represent a single representative experiment. (C and D) Mean (± standard error of the mean) percent positive cells for five independent experiments (P < 0.01 compared with unstimulated cells [Con]).

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.

FIG. 3

RGD peptide inhibits the binding of partially purified LKT to resting, but not IL-1β-stimulated, bovine PMNs. Freshly isolated bovine PMNs (10⁶ cells/ml) were incubated with IL-1β (50 ng) or medium (CON) for 15 min at 37°C. The PMNs were next incubated with RDG or a control peptide (RGES) (1 mM) for 15 min at 37°C. The cells were washed and incubated with biotinylated P. haemolytica LKT (10 to 20 U) for 10 min on ice. Extra-avidin was added, and the cell suspensions were incubated on ice for an additional 20 min. The stained cells were washed and analyzed by flow cytometry (10,000 cells were scored for green fluorescence). The data represent the mean (± standard error of the mean) percent positive cells from four independent experiments. The asterisks indicate significant differences (P < 0.01) compared with control PMNs incubated in medium alone before exposure to LKT (CON), as determined by the Student-Newman-Keuls test.

FIG. 4

Incubation of bovine PMNs with IL-1β enhances LKT cytotoxicity in a β₂-integrin-dependent manner. Freshly isolated bovine PMNs (10⁶ cells/ml) were incubated with IL-1β (50 ng) or medium (CON) for 15 min at 37°C. This was followed by a 40-min incubation with the anti-β₂-integrin MAb BAT75A (50-μg/ml final concentration). Control and IL-1β-treated PMNs were then plated in 96-well plates and incubated with partially purified P. haemolytica LKT (1 to 5 U) for 1 h at 37°C. Cell viability was assessed by XTT reduction, as described previously (32). The data indicate the mean (± standard error of the mean) percent LKT-mediated PMN death from four independent experiments. Asterisks indicate statistically significant differences (P < 0.05) for BAT75A-treated PMNs versus PMNs incubated with LKT alone, using the Student-Newman-Keuls multiple-comparison test.