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. 2001 Jan;65(1):38–44.

Phagocytic response of bovine polymorphonuclear leukocytes to different incubation conditions and following exposure to some effectors of phagocytosis and different anticoagulants in vitro.

R J Ducusin 1, T Sarashina 1, Y Uzuka 1, S Tanabe 1, M Ohtani 1
PMCID: PMC1189640  PMID: 11227193

Abstract

The ability of bovine polymorphonuclear leukocytes (PMN) to phagocytose fluorescent beads in vitro was studied using flow cytometry. The effects of varying laboratory conditions (bead:PMN ratio, length of incubation, and temperature) were first determined, then the effects of lipopolysaccharide (LPS), phorbol myristate acetate (PMA), cytochalasin B, and formyl-met-leu-phe (fMLP) on phagocytosis were evaluated. The recommended bead:PMN ratio, incubation period, and incubation temperature are 20:1, 30 min, and 38.5 degrees C, respectively. Lipopolysaccharide increased phagocytosis at a relatively high minimum dose; PMA increased phagocytosis even at low doses; cytochalasin B increased and decreased phagocytosis at low and high doses, respectively; and fMLP had no significant effect on phagocytosis. Also, the effects of ethylene diamine tetraacetic acid (EDTA) and acid citrate dextrose (ACD) as anticoagulants were compared with heparin-treated blood PMNs. Both EDTA and ACD decreased phagocytosis. Although there are reports that demonstrated that heparin reduced PMN phagocytosis, at least among the 3 anticoagulants used, heparin remains to be the standard anticoagulant for the study of PMN phagocytosis.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Branum E., Cummins L., Bartilson M., Hopper M., Pruett S., O'Brien J. F. Effect of two anticoagulants on leukocyte yield and function, and on lysosomal enzyme activity. Clin Chem. 1988 Jan;34(1):110–113. [PubMed] [Google Scholar]
  2. Böhmer R. H., Trinkle L. S., Staneck J. L. Dose effects of LPS on neutrophils in a whole blood flow cytometric assay of phagocytosis and oxidative burst. Cytometry. 1992;13(5):525–531. doi: 10.1002/cyto.990130512. [DOI] [PubMed] [Google Scholar]
  3. Carroll E. J., Mueller R., Panico L. Chemotactic factors for bovine leukocytes. Am J Vet Res. 1982 Sep;43(9):1661–1664. [PubMed] [Google Scholar]
  4. Carulli G. Applications of flow cytometry in the study of human neutrophil biology and pathology. Hematopathol Mol Hematol. 1996;10(1-2):39–61. [PubMed] [Google Scholar]
  5. Confer A. W., Simons K. R. Effects of Pasteurella haemolytica lipopolysaccharide on selected functions of bovine leukocytes. Am J Vet Res. 1986 Jan;47(1):154–157. [PubMed] [Google Scholar]
  6. De Guise S., Bernier J., Lapierre P., Dufresne M. M., Dubreuil P., Fournier M. Immune function of bovine leukocytes after in vitro exposure to selected heavy metals. Am J Vet Res. 2000 Mar;61(3):339–344. doi: 10.2460/ajvr.2000.61.339. [DOI] [PubMed] [Google Scholar]
  7. Elvinger F., Hansen P. J., Natzke R. P. Modulation of function of bovine polymorphonuclear leukocytes and lymphocytes by high temperature in vitro and in vivo. Am J Vet Res. 1991 Oct;52(10):1692–1698. [PubMed] [Google Scholar]
  8. Fattorossi A., Nisini R., Pizzolo J. G., D'Amelio R. New, simple flow cytometry technique to discriminate between internalized and membrane-bound particles in phagocytosis. Cytometry. 1989 May;10(3):320–325. doi: 10.1002/cyto.990100311. [DOI] [PubMed] [Google Scholar]
  9. Gómez-Estrada H., Ramos-Damián E., Vazquez-Escobosa C. Phagocytic activity of human polymorphonuclear leukocytes at different temperatures. Arch Invest Med (Mex) 1980;11(1):65–71. [PubMed] [Google Scholar]
  10. Hampton M. B., Winterbourn C. C. Methods for quantifying phagocytosis and bacterial killing by human neutrophils. J Immunol Methods. 1999 Dec 17;232(1-2):15–22. doi: 10.1016/s0022-1759(99)00147-7. [DOI] [PubMed] [Google Scholar]
  11. Haslett C., Guthrie L. A., Kopaniak M. M., Johnston R. B., Jr, Henson P. M. Modulation of multiple neutrophil functions by preparative methods or trace concentrations of bacterial lipopolysaccharide. Am J Pathol. 1985 Apr;119(1):101–110. [PMC free article] [PubMed] [Google Scholar]
  12. Hoeben D., Dosogne H., Heyneman R., Burvenich C. Effect of antibiotics on the phagocytotic and respiratory burst activity of bovine granulocytes. Eur J Pharmacol. 1997 Aug 13;332(3):289–297. doi: 10.1016/s0014-2999(97)01107-2. [DOI] [PubMed] [Google Scholar]
  13. Johansen K. S., Berger E. M., Repine J. E. Effect of temperature on polymorphonuclear leukocyte function. Acta Pathol Microbiol Immunol Scand C. 1983 Dec;91(6):355–359. [PubMed] [Google Scholar]
  14. Klesius P. H., Chambers W. H., Schultz R. D. Effect of bacterial lipopolysaccharide on bovine polymorphonuclear neutrophil migration in vitro. Vet Immunol Immunopathol. 1984 Oct;7(3-4):239–244. doi: 10.1016/0165-2427(84)90082-5. [DOI] [PubMed] [Google Scholar]
  15. Levin J., Poore T. E., Zauber N. P., Oser R. S. Detection of endotoxin in the blood of patients with sepsis due to gran-negative bacteria. N Engl J Med. 1970 Dec 10;283(24):1313–1316. doi: 10.1056/NEJM197012102832404. [DOI] [PubMed] [Google Scholar]
  16. Mandell G. L. Effect of temperature on phagocytosis by human polymorphonuclear neutrophils. Infect Immun. 1975 Jul;12(1):221–223. doi: 10.1128/iai.12.1.221-223.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ogle J. D., Noel J. G., Sramkoski R. M., Ogle C. K., Alexander J. W. Effects of chemotactic peptide f-Met-Leu-Phe (FMLP) on C3b receptor (CR1) expression and phagocytosis of microspheres by human neutrophils. Inflammation. 1990 Jun;14(3):337–353. doi: 10.1007/BF00915817. [DOI] [PubMed] [Google Scholar]
  18. Peterson P. K., Verhoef J., Quie P. G. Influence of temperature on opsonization and phagocytosis of staphylococci. Infect Immun. 1977 Jan;15(1):175–179. doi: 10.1128/iai.15.1.175-179.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Roth J. A., Kaeberle M. L. Evaluation of bovine polymorphonuclear leukocyte function. Vet Immunol Immunopathol. 1981 Apr;2(2):157–174. doi: 10.1016/0165-2427(81)90047-7. [DOI] [PubMed] [Google Scholar]
  20. Roth J. A., Kaeberle M. L. Isolation of neutrophils and eosinophils from the peripheral blood of cattle and comparison of their functional activities. J Immunol Methods. 1981;45(2):153–164. doi: 10.1016/0022-1759(81)90209-x. [DOI] [PubMed] [Google Scholar]
  21. Saad A. M. Flow cytometric measurement of bovine milk neutrophil phagocytosis. Acta Vet Scand. 1987;28(3-4):333–342. doi: 10.1186/BF03548601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Saad A. M., Hageltorn M. Flow cytometric characterization of bovine blood neutrophil phagocytosis of fluorescent bacteria and zymosan particles. Acta Vet Scand. 1985;26(3):289–307. doi: 10.1186/BF03546530. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Salgar S. K., Paape M. J., Alston-Mills B., Miller R. H. Flow cytometric study of oxidative burst activity in bovine neutrophils. Am J Vet Res. 1991 Aug;52(8):1201–1207. [PubMed] [Google Scholar]
  24. Salih H., Husfeld L., Adam D. Inhibitory effect of heparin on neutrophil phagocytosis and burst production using a new whole-blood cytofluorometric method for determination. Eur J Med Res. 1997 Dec 31;2(12):507–513. [PubMed] [Google Scholar]
  25. Santos J. L., Montes M. J., Gutiérrez F., Ruiz C. Evaluation of phagocytic capacity with a modified flow cytometry technique. Immunol Lett. 1995 Feb;45(1-2):1–4. doi: 10.1016/0165-2478(94)00180-y. [DOI] [PubMed] [Google Scholar]
  26. Schnitzler N., Schweizer K., Podbielski A., Haase G., Spellerberg B., Holland R., Lütticken R. Activation of granulocytes by phorbol-12-myristate-14-acetate (PMA) enhances phagocytosis of Streptococcus pyogenes. Adv Exp Med Biol. 1997;418:897–902. doi: 10.1007/978-1-4899-1825-3_210. [DOI] [PubMed] [Google Scholar]
  27. Silva I. D., Jain N. C. Effects of glycolytic and cytoskeletal inhibitors on phagocytic and nitroblue tetrazolium reductive activities of bovine neutrophils. Am J Vet Res. 1989 Jul;50(7):1175–1179. [PubMed] [Google Scholar]
  28. Smits E., Burvenich C., Heyneman R. Simultaneous flow cytometric measurement of phagocytotic and oxidative burst activity of polymorphonuclear leukocytes in whole bovine blood. Vet Immunol Immunopathol. 1997 May;56(3-4):259–269. doi: 10.1016/s0165-2427(96)05739-x. [DOI] [PubMed] [Google Scholar]
  29. Utoh J., Harasaki H. Effects of temperature on phagocytosis of human and calf polymorphonuclear leukocytes. Artif Organs. 1992 Aug;16(4):377–381. doi: 10.1111/j.1525-1594.1992.tb00535.x. [DOI] [PubMed] [Google Scholar]
  30. Victor M., Weiss J., Elsbach P. Heparin inhibits phagocytosis by polymorphonuclear leukocytes. Infect Immun. 1981 Apr;32(1):295–299. doi: 10.1128/iai.32.1.295-299.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. White-Owen C., Alexander J. W., Sramkoski R. M., Babcock G. F. Rapid whole-blood microassay using flow cytometry for measuring neutrophil phagocytosis. J Clin Microbiol. 1992 Aug;30(8):2071–2076. doi: 10.1128/jcm.30.8.2071-2076.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. van Eeden S. F., Klut M. E., Walker B. A., Hogg J. C. The use of flow cytometry to measure neutrophil function. J Immunol Methods. 1999 Dec 17;232(1-2):23–43. doi: 10.1016/s0022-1759(99)00148-9. [DOI] [PubMed] [Google Scholar]

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