leukotoxin and 4-bromophenacyl-bromide

Pasteurella haemolytica A1 leukotoxic culture supernatant was evaluated for its ability to cause aggregation of bovine peripheral neutrophils. Neutrophils were isolated by a hypotonic lysis method and incubated with zymosan-activated plasma (ZAP), leukotoxic culture supernatant, antileukotoxin serum, calcium and magnesium-free media, p-bromophenacyl bromide and protein kinase C inhibitors. Aggregation was evaluated by changes in infrared light transmittance. Leukotoxic culture supernatant caused neutrophils to aggregate, and this effect was significantly removed by preincubation with antileukotoxin serum. Aggregation to ZAP and leukotoxin was dependent on the presence of extra-cellular calcium. Activation of protein kinase C by phorbol myristate acetate induced aggregation which was reduced by staurosporine; however, aggregation to leukotoxin did not involve protein kinase C activation. Phospholipase A2 inhibition did not alter the aggregation response to ZAP or to leukotoxin. The in vitro measurement of neutrophil aggregation induced by the leukotoxin of P. haemolytica reflects cytoskeletal and other activation events that may contribute to the intense inflammatory process which this organism induces in the lungs of cattle.

Topics: Acetophenones; Alkaloids; Animals; Bacterial Toxins; Cattle; Cell Aggregation; Cells, Cultured; Culture Media; Cytotoxins; Exotoxins; Mannheimia haemolytica; Neutrophils; Phospholipases A; Phospholipases A2; Protein Kinase C; Staurosporine; Tetradecanoylphorbol Acetate

Pasteurella haemolytica A1 culture supernatant containing leukotoxin, and modifiers of cyclic nucleotide and arachidonate metabolism, were evaluated for their ability to alter oxygen radical production by pulmonary alveolar macrophages obtained from seven Holstein calves. Calves were sedated, and underwent bronchoalveolar lavage to harvest macrophages, which were then incubated with culture supernatant and/or the drugs and toxins under study, and challenged with opsonized zymosan to induce oxygen radical generation. This was measured by a chemiluminescence technique. Pasteurella haemolytica A1 culture supernatant alone delayed the time to maximum oxygen radical production, although total production was increased. The cyclooxygenase inhibitor indomethacin and the phospholipase inhibitor p-bromophenacyl bromide significantly reduced maximum oxygen radical production, but their effects were diminished in the presence of culture supernatant. Although forskolin markedly inhibited oxygen radical generation, this effect was not altered by culture supernatant. Incubation of macrophages with pertussis toxin had no effect on oxygen radical production, while incubation with cholera toxin did inhibit production. This inhibitory effect was significantly lessened by concurrent incubation with P. haemolytica A1 culture supernatant.

Topics: Acetophenones; Animals; Bacterial Toxins; Bronchoalveolar Lavage Fluid; Cattle; Cells, Cultured; Cholera Toxin; Colforsin; Exotoxins; Free Radicals; Immunosuppressive Agents; Indomethacin; Macrophages; Male; Oxygen; Pasteurella; Pertussis Toxin; Virulence Factors, Bordetella