Pasteurella haemolytica leukotoxin and endotoxin induced cytokine gene expression in bovine alveolar macrophages requires NF-κB activation and calcium elevation

In bovine alveolar macrophages (BAMs), exposure to leukotoxin (Lkt) and endotoxin (LPS) from Pasteurella haemolytica results in expression of inflammatory cytokine genes and intracellular calcium ([Ca²⁺](i)) elevation. Leukotoxin from P. haemolytica interacts only with leukocytes and platelets from ruminant species. Upregulation of cytokine genes in different cells by LPS involves activation of the transcription factor NF-kappa B (NF-κB), resulting in its translocation from the cytoplasm to the nucleus. Using immunocytochemical staining and confocal imaging, we studied whether NF-κB activation represents a common mechanism for the expression of multiple cytokine genes in BAMs (Lkt-susceptible cells) stimulated with Lkt and LPS. Bovine pulmonary artery endothelial cells and porcine alveolar macrophages were used as nonsusceptible cells. The role of Ca²⁺ and tyrosine kinases in NF-κB activation and inflammatory cytokine gene expression was studied, since an inhibitor of tyrosine kinases attenuates LPS-induced [Ca²⁺](i) elevation in BAMs. The results are summarized as follows: (a) Lkt induced NF-κB activation and [Ca²⁺](i) elevation only in BAMs, while LPS effects were demonstrable in all cell types; (b) chelation of [Ca²⁺](i) blocked NF-κB activation and IL-1β, TNFα and IL-8 mRNA expression; and (c) tyrosine kinase inhibitor herbimycin A blocked expression of all three cytokine genes in BAMs stimulated with Lkt, while only the expression of IL-1β was blocked in BAMs stimulated with LPS. We conclude that cytokine gene expression in BAMs requires NF-κB activation and [Ca²⁺](i) elevation, and Lkt effects exhibit cell type- and species specificity.