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Cell-surface lactoferrin as a marker for degranulation of specific granules in bovine neutrophils

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  • 1 Department of Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717.
  • | 2 Department of Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717.
  • | 3 Department of Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717.

Abstract

Objective—To develop a rapid and accurate flow cytometric method for measuring degranulation of specific granules in bovine neutrophils.

Sample Population—Blood samples obtained from four 6- to 18-month-old Holstein cattle.

Procedure—A monoclonal antibody (BL97) was generated against bovine lactoferrin and tested for applicability in ELISA, immunoprecipitation tests, immunofluorescence microscopy, and flow cytometric analyses. Using this antibody, cell-surface lactoferrin was measured concurrent with amount of secreted lactoferrin from bovine neutrophils activated with phorbol myristate acetate (PMA). Cell-surface lactoferrin also was measured on neutrophils in bovine whole blood stimulated with PMA, platelet-activating factor (PAF), N-formyl-methionyl-leucyl-phenylalanine (fMLF), and interleukin 8 (IL-8).

Results—Antibody BL97 recognized bovine lactoferrin in ELISA and western immunoblots and was useful for immunoprecipitation testing, immunofluorescence microscopy, and flow cytometric analyses of bovine leukocytes. Neutrophils activated with PMA had parallel increases in content of secreted lactoferrin (measured by ELISA) and cell-surface lactoferrin (measured by flow cytometry) with increasing PMA concentrations. In addition, fluorescein-conjugated BL97 antibody detected increases in cell-surface lactoferrin on neutrophils in bovine whole blood after activation with PMA, PAF, and IL-8. In contrast, increases in cell-surface lactoferrin were not detected on bovine neutrophils treated with fMLF.

Conclusion and Clinical Relevance—Measurement of cell-surface lactoferrin on bovine neutrophils by flow cytometry is a valid and rapid method for assessment of release of lactoferrin from specific granules in these cells and represents a means to rapidly measure neutrophil activation. This technique allows for investigation of mechanisms of neutrophil modification in isolated cells as well as in whole blood. (Am J Vet Res 2000;61:29–37)

Abstract

Objective—To develop a rapid and accurate flow cytometric method for measuring degranulation of specific granules in bovine neutrophils.

Sample Population—Blood samples obtained from four 6- to 18-month-old Holstein cattle.

Procedure—A monoclonal antibody (BL97) was generated against bovine lactoferrin and tested for applicability in ELISA, immunoprecipitation tests, immunofluorescence microscopy, and flow cytometric analyses. Using this antibody, cell-surface lactoferrin was measured concurrent with amount of secreted lactoferrin from bovine neutrophils activated with phorbol myristate acetate (PMA). Cell-surface lactoferrin also was measured on neutrophils in bovine whole blood stimulated with PMA, platelet-activating factor (PAF), N-formyl-methionyl-leucyl-phenylalanine (fMLF), and interleukin 8 (IL-8).

Results—Antibody BL97 recognized bovine lactoferrin in ELISA and western immunoblots and was useful for immunoprecipitation testing, immunofluorescence microscopy, and flow cytometric analyses of bovine leukocytes. Neutrophils activated with PMA had parallel increases in content of secreted lactoferrin (measured by ELISA) and cell-surface lactoferrin (measured by flow cytometry) with increasing PMA concentrations. In addition, fluorescein-conjugated BL97 antibody detected increases in cell-surface lactoferrin on neutrophils in bovine whole blood after activation with PMA, PAF, and IL-8. In contrast, increases in cell-surface lactoferrin were not detected on bovine neutrophils treated with fMLF.

Conclusion and Clinical Relevance—Measurement of cell-surface lactoferrin on bovine neutrophils by flow cytometry is a valid and rapid method for assessment of release of lactoferrin from specific granules in these cells and represents a means to rapidly measure neutrophil activation. This technique allows for investigation of mechanisms of neutrophil modification in isolated cells as well as in whole blood. (Am J Vet Res 2000;61:29–37)