MODULATION OF PHAGOCYTIC AND OXIDATIVE BURST ACTIVITIES OF BOVINE NEUTROPHILS BY HUMAN RECOMBINANT TNF-ALPHA, IL-1-ALPHA, IFN-GAMMA, G-CSF, GM-CSF

In vitro experiments were conducted to determine the effects of recombinant cytokines on phagocytic and oxidative burst activities of bovine neutrophils. Neutrophils were isolated (purity >91%, viability >97%) from EDTA-anticoagulated blood from healthy Holstein-Friesian heifers. Aliquots of neutrophils (10 x 10(6) cells/ml) were incubated for 1 h at 37 degrees C with equal volumes of recombinant human cytokines, namely, tumour necrosis factor-alpha (rhTNF-alpha, 0.5-1000 ng/ ml), interleukin-1-alpha (rhIL-1-alpha, 0.001-10 ng/ml), interferon-gamma (rhIFN-gamma, 0.01-100 ng/ml), granulocyte colony-stimulating factor (rhG-CSF, 25 ng/ml), and granulocyte-macrophage colony-stimulating factor (rhGM-CSF, 10 ng/ml). Then, the percentage phagocytosis and average number of intracellular bacteria per cell were evaluated by flow cytometry and/or fluorescent microscopy using FITC-labelled opsonised bacteria (Escherichia coli 0111:B4). Unlabelled opsonised bacteria and dichlorofluorescin diacetate were used to evaluate H2O2 production, a measure of oxidative burst, by flow cytometry. The results showed that all five cytokines significantly (p <0.05) increased percentage phagocytosis (52.4-86.1%), number of intracellular bacteria per cell (24.9-47.9%), and H2O2 production (31.3-58.2%) when compared to untreated neutrophils. A gradual increase in mean channel fluorescence but not in percentage phagocytosis was consistently seen with increasing concentrations of rhTNF-alpha, rhIL-1-alpha, and rhIFN-gamma, thereby indicating a concentration-dependent stimulation of phagocytic capacity by these three cytokines.