Modulation of phagocytic and oxidative burst activities of bovine neutrophils by human recombinant TNF-α, IL-1-α, IFN-γ, G-CSF, GM-CSF

M. B. Kabbur, N. C. Jain, Thomas B Farver

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Abstract

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 × 106 cells/ml) were incubated for 1 h at 37 °C with equal volumes of recombinant human cytokines, namely, tumour necrosis factor-alpha (rhTNF-α, 0.5-1000 ng/ml), interleukin-1-alpha (rhIL-1-α, 0.001-10 ng/ml), interferon-gamma (rhIFN-γ, 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-α, rhIL-1-α, and rhIFN-γ, thereby indicating a concentration-dependent stimulation of phagocytic capacity by these three cytokines.

Original languageEnglish (US)
Pages (from-to)47-55
Number of pages9
JournalComparative Haematology International
Volume5
Issue number1
DOIs
StatePublished - Mar 1995

Fingerprint

Respiratory Burst
Granulocyte Colony-Stimulating Factor
Granulocyte-Macrophage Colony-Stimulating Factor
Interleukin-1
Neutrophils
Phagocytosis
Cytokines
Bacteria
Flow Cytometry
Interleukin-1alpha
Fluorescein-5-isothiocyanate
Edetic Acid
Interferon-gamma
Microscopy
Tumor Necrosis Factor-alpha
Fluorescence
Escherichia coli

Keywords

  • Bovine
  • Cytokines
  • Granulocyte colonystimulating factor
  • Granulocyte-macrophage colonystimulating factor
  • Hydrogen peroxide production
  • Interferon-gamma
  • Interleukin-1 alpha
  • Neutrophils
  • Phagocytosis
  • Tumour necrosis factor-alpha

ASJC Scopus subject areas

  • Hematology

Cite this

@article{3b126ac330c149509db31e662030e6ee,
title = "Modulation of phagocytic and oxidative burst activities of bovine neutrophils by human recombinant TNF-α, IL-1-α, IFN-γ, G-CSF, GM-CSF",
abstract = "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 × 106 cells/ml) were incubated for 1 h at 37 °C with equal volumes of recombinant human cytokines, namely, tumour necrosis factor-alpha (rhTNF-α, 0.5-1000 ng/ml), interleukin-1-alpha (rhIL-1-α, 0.001-10 ng/ml), interferon-gamma (rhIFN-γ, 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-α, rhIL-1-α, and rhIFN-γ, thereby indicating a concentration-dependent stimulation of phagocytic capacity by these three cytokines.",
keywords = "Bovine, Cytokines, Granulocyte colonystimulating factor, Granulocyte-macrophage colonystimulating factor, Hydrogen peroxide production, Interferon-gamma, Interleukin-1 alpha, Neutrophils, Phagocytosis, Tumour necrosis factor-alpha",
author = "Kabbur, {M. B.} and Jain, {N. C.} and Farver, {Thomas B}",
year = "1995",
month = "3",
doi = "10.1007/BF00214490",
language = "English (US)",
volume = "5",
pages = "47--55",
journal = "Comparative Clinical Pathology",
issn = "1618-5641",
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TY - JOUR

T1 - Modulation of phagocytic and oxidative burst activities of bovine neutrophils by human recombinant TNF-α, IL-1-α, IFN-γ, G-CSF, GM-CSF

AU - Kabbur, M. B.

AU - Jain, N. C.

AU - Farver, Thomas B

PY - 1995/3

Y1 - 1995/3

N2 - 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 × 106 cells/ml) were incubated for 1 h at 37 °C with equal volumes of recombinant human cytokines, namely, tumour necrosis factor-alpha (rhTNF-α, 0.5-1000 ng/ml), interleukin-1-alpha (rhIL-1-α, 0.001-10 ng/ml), interferon-gamma (rhIFN-γ, 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-α, rhIL-1-α, and rhIFN-γ, thereby indicating a concentration-dependent stimulation of phagocytic capacity by these three cytokines.

AB - 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 × 106 cells/ml) were incubated for 1 h at 37 °C with equal volumes of recombinant human cytokines, namely, tumour necrosis factor-alpha (rhTNF-α, 0.5-1000 ng/ml), interleukin-1-alpha (rhIL-1-α, 0.001-10 ng/ml), interferon-gamma (rhIFN-γ, 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-α, rhIL-1-α, and rhIFN-γ, thereby indicating a concentration-dependent stimulation of phagocytic capacity by these three cytokines.

KW - Bovine

KW - Cytokines

KW - Granulocyte colonystimulating factor

KW - Granulocyte-macrophage colonystimulating factor

KW - Hydrogen peroxide production

KW - Interferon-gamma

KW - Interleukin-1 alpha

KW - Neutrophils

KW - Phagocytosis

KW - Tumour necrosis factor-alpha

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