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

Objective-To develop a rapid and accurate flow cytometric method for measur ing degranulation of specific granules in bovine neutrophils. Sample Population-Blood samples obtained from four 6- to 18-month-old Holst ein cattle. Procedure-A monoclonal antibody (BL97) was generated against bovine lactofe rrin and tested for applicability in ELISA, immunoprecipitation tests, immu nofluorescence microscopy, and flow cytometric analyses. Using this antibod y, cell-surface lactoferrin was measured concurrent with amount of secreted lactoferrin from bovine neutrophils activated with phorbol myristate aceta te (PMA). Cell-surface lactoferrin also was measured on neutrophils in bovi ne whole blood stimulated with PMA, platelet-activating factor (PAF), N-for myl-methionyl-leucyl-phenylalanine (fMLF), and interleukin 8 (IL-8). Results-Antibody BL97 recognized bovine lactoferrin in ELISA and western im munoblots and was useful for immunoprecipitation testing, immunofluorescenc e microscopy, and flow cytometric analyses of bovine leukocytes. Neutrophil s activated with PMA had parallel increases in content of secreted lactofer rin (measured by ELISA) and cell-surface lactoferrin (measured by flow cyto metry) with increasing PMA concentrations. In addition, fluorescein-conjuga ted BL97 antibody detected increases in cell-surface lactoferrin on neutrop hils in bovine whole blood after activation with PMA, PAF, and IL-8. In con trast, increases in cell-surface lactoferrin were not detected on bovine ne utrophils treated with fMLF. Conclusion and Clinical Relevance-Measurement of cell-surface lactoferrin o n bovine neutrophils by flow cytometry is a valid and rapid method for asse ssment 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 isola ted cells as well as in whole blood.