Inflammatory cytokines enhance the interaction of Mannheimia haemolytica leukotoxin with bovine peripheral blood neutrophils in vitro

Abstract

Mannheimia (Pasteurella) haemolytica A1 produces several virulence factors that play an important role in the pathogenesis of bovine pneumonic pasteurellosis. Foremost among these is a leukotoxin (LKT) that specifically kills ruminant leukocytes. Recent evidence suggests that M. haemolytica LKT binding to bovine leukocytes is mediated by the beta(2)-integrin CD11a/CD18 (lymphocyte function-associated antigen 1 [LFA-1]), which subsequently induces activation and cytolysis of these cells. Inflammatory cytokines, which are released during viral and bacterial infection, are reported to increase LFA-1 expression and conformational activation. We investigated the effects of the inflammatory cytokines interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNF-alpha), and gamma interferon (IFN-gamma) on the interaction of M. haemolytica LKT with bovine peripheral blood neutrophils (PMNs). In this study we demonstrated, by flow cytometry, that bovine PMNs increased their binding to an anti-bovine LFA-1 monoclonal antibody (BAT75A) following in vitro incubation with IL-1beta, TNF-alpha, or IFN-gamma. Incubation with cytokines also increased CD18 expression, as assessed by real-time PCR and by Western blotting. Increased LFA-1 expression by PMNs exposed to cytokines was associated with increased LKT binding and cytotoxicity. The latter represented, at least in part, enhanced PMN apoptosis, as assessed by propidium iodine staining and caspase-3 activation. The results of this study suggest that inflammatory cytokines may play an important role in enhancing the biological response of bovine PMNs to M. haemolytica LKT.

FIG. 1.

Incubation with inflammatory cytokines increases the staining of bovine PMNs with the anti-LFA-1 MAb BAT75A. Freshly isolated bovine PMNs (1 × 10⁶ cells/ml) were incubated with recombinant bovine IL-1β (50 ng), recombinant human TNF-α (50 ng), recombinant bovine IFN-γ (50 ng), or medium (control) for 15 min (A) or 60 min (B) at 37°C. The cells were then washed and incubated (40 min at 4°C) with anti-LFA-1 MAb BAT75A (final concentration, 50 μg/ml). The cells were washed, incubated with an FITC-labeled second antibody, and analyzed by flow cytometry (10,000 cells were scored for green fluorescence). The data are the means ± standard errors of the means for four independent experiments. An asterisk indicates that the data for stimulated cells was statistically significantly different from the data for control cells (P < 0. 05).

FIG. 2.

Incubation with inflammatory cytokines increases the expression of CD18 mRNA in bovine PMNs. Freshly isolated bovine PMNs (1 × 10⁶ cells/ml) were incubated with recombinant bovine IL-1β (50 ng), recombinant human TNF-α (50 ng), recombinant bovine IFN-γ (50 ng), or medium (control) for 60 min at 37°C. RNA was extracted and transcribed to cDNA, and then CD18 cDNA expression was assessed by real-time PCR. The results were expressed as fold increases relative to the data obtained with unstimulated (control) PMNs. The data are the means ± standard errors of the means for three independent experiments. An asterisk indicates that the data for cytokine-stimulated cells was statistically significantly different from the data for unstimulated cells (P < 0.01).

FIG. 3.

Western blot analysis of LFA-1 expression by bovine PMNs incubated with inflammatory cytokines. Freshly isolated bovine PMNs (1 × 10⁶ cells/ml) were incubated with recombinant bovine IL-1β (50 ng), recombinant human TNF-α (50 ng), recombinant bovine IFN-γ (50 ng), or medium (control) for 60 min at 37°C. Total cell lysates were prepared, and equal amounts of total protein were loaded onto Tris-HCl-4 to 20% polyacrylamide gradient gels, electrophoresed, and transferred to nitrocellulose membranes. The membranes were blocked, washed, and probed with anti-LFA-1 MAb BAT75A or HI111 at 37°C for 1 h. The blots were washed and probed with horseradish peroxidase-conjugated anti-mouse IgG at 37°C for 1 h. Immunoreactive proteins were visualized with a SuperSignal West Pico chemiluminescence kit, and relative band intensities were determined by using ImageQuaNT software. The numbers below the bands are the mean fold increases in LFA-1 expression by bovine PMNs incubated with the cytokines compared with the expression by unstimulated PMNs (three separate experiments).

FIG. 4.

Incubation of bovine PMNs with inflammatory cytokines enhances LKT binding. Freshly isolated bovine PMNs (1 × 10⁶ cells/ml) were incubated with recombinant bovine IL-1β (50 ng), recombinant human TNF-α (50 ng), recombinant bovine IFN-γ (50 ng), or medium (control) for 15 min (A) or 60 min (B) at 37°C. The cells were then incubated for 10 min in an ice bath with biotinylated LKT (1 U) and washed. Extra-avidin-FITC was added and 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). The data are the means ± standard errors of the means for four independent experiments. An asterisk indicates that the data for stimulated cells was statistically significantly different from the data for control cells (P < 0.05).

FIG. 5.

Incubation of bovine PMNs with inflammatory cytokines enhances LKT cytotoxicity. Freshly isolated bovine PMNs (1 × 10⁶ cells/ml) were incubated with recombinant bovine IL-1β (50 ng), recombinant human TNF-α (50 ng), recombinant bovine IFN-γ (50 ng), or medium (control) for 15 min (A) or 1 h (B) at 37°C. In the latter experiments (B) some of the cells were incubated with an anti-LFA-1 MAb (BAT75A) (final concentration, 50 μg/ml) for 40 min before addition of LKT. Control and treated PMNs were then plated in 96-well plates and incubated with partially purified M. haemolytica LKT (1 U) for 1 h at 37°C. Cell viability was assessed by XTT reduction. The data are the means ± standard errors of the means for four independent experiments. One asterisk indicates that the value for cytokine-stimulated cells was statistically significantly greater than the value for control cells (P < 0.05). Two asterisks indicate that the value for anti-LFA-treated cytokine-stimulated PMNs was significantly less than the value for untreated cytokine-stimulated PMNs.

FIG. 6.

Incubation of bovine PMNs with inflammatory cytokines enhances LKT-mediated apoptosis. Freshly isolated bovine PMNs (1 × 10⁶ cells/ml) were incubated with recombinant bovine IL-1β (50 ng), recombinant human TNF-α (50 ng), recombinant bovine IFN-γ (50 ng), or medium (control) for 1 h at 37°C. Partially purified M. haemolytica LKT (0.1 U) was then added to the PMNs and incubated for 6 h at 37°C. Apoptosis was assessed by PI staining by flow cytometry (A) and by caspase-3 activation by a colorimetric assay (B). The data are the means ± standard errors of the means for three independent experiments. An asterisk indicates that the data for cytokine-stimulated cells was statistically significantly different from the data for control cells (P < 0.05).

FIG. 7.

Correlation between LKT biological activities and LFA-1 staining of bovine PMNs as determined by linear regression. (A) LKT binding versus LFA-1 staining (r = 0.76; y = 10.7 + 0.78x; P = 0.0006). (B) LKT cytotoxicity versus LFA-1 staining (r = 0.60; y = 15.5 + 0.73x; P = 0.0126). (C) LKT-mediated apoptosis assessed by PI staining versus LFA-1 staining (r = 0.68; y = 26.9 + 1.2x; P = 0.0138). (D) LKT-mediated caspase-3 activation versus LFA-1 staining (r = 0.89; y = 15.5 + 25.3x; P < 0.0001).