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Signal transduction pathways involved in phagocytic and oxidative burst activities of cytokine-treated bovine neutrophils

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Abstract

In vitro studies were conducted to determine the relative importance of various signal transduction factors involved in the phagocytic and oxidative burst activities of cytokine-primed bovine neutrophils. These neutrophil functions were assayed in the presence of known signal transduction pathways inhibitors which included nicotinamide, staurosporine, genistein, pertussis toxin, RO 20-1724 and U-73122.

Neutrophils were isolated (purity >91%, viability >97%) from EDTA-anticoagulated jugular blood from five healthy Holstein-Friesian heifers. Freshly isolated neutrophils (6 ml, 10 x 106 cells/ml) were incubated separately for 1 h at 37 °C with equal volumes of recombinant human cytokines such as tumour necrosis factor-alpha (500 ng/ml), interleukin-l-alpha (1 ng/ml), granulocyte colony-stimulating factor (25 ng/ml), granulocyte-macrophage colony-stimulating factor (10 ng/ml) and interferon-gamma (10 ng/ml). Aliquots (1.8 ml) of various cytokine-treated neutrophils were exposed to each signal transduction pathways inhibitor for 20 min at 37 °C in the dark. Then, percentage phagocytosis and average number of intracellular bacteria per cell were evaluated microscopically using FITC-labelled opsonised bacteria (Escheria coli 0111:134). Unlabelled opsonised bacteria and dichlorofluorescin diacetate were used to evaluate H2O2 production, a measure of oxidative burst, by flow cytometry.

Phagocytic activity and H2O2 production by bovine neutrophils treated with various cytokines were increased by 52.4–86.1% and 31.3–58.2%, respectively.

These functional activities were significantly (p <0.05) reduced after exposure to different inhibitors of signal transduction pathways. The reduction in phagocytic activity of cytokine-primed neutrophils varied greatly depending on the site of action of various inhibitors, with pertussis toxin and U-73122 being the most inhibitory. In comparison, H2O2 production decreased moderately, with pertussis toxin and U-73122 being the most inhibitory and other inhibitors inducing minimal variations. It was concluded that G-inhibitory proteins (pertussis toxin-sensitive) and phospholipase C play a major role, whereas tyrosine kinase plays a minor role in the phagocytic activity and H2O2 production by cytokine-primed bovine neutrophils.

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  1. Department of Pathology, Microbiology, and Immunology, School of Veterinary Medicine, University University of California, 95616, Davis, CA, USA

    M. B. Kabbur & N. C. Jain

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Kabbur, M.B., Jain, N.C. Signal transduction pathways involved in phagocytic and oxidative burst activities of cytokine-treated bovine neutrophils. Comparative Haematology International 5, 38–46 (1995). https://doi.org/10.1007/BF00214489

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