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Host-pathogen interactions: leukocyte phagocytosis and associated sequelae

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Methods in Cell Science

Abstract

Polymorphonuclear leukocytes (PMNs) are a critical component of the human innate immune response and are the first line of defense against invading microorganisms. Phagocytosis of invading microbes induces production of reactive oxygen species (ROS) by PMNs, which facilitates bactericidal activity. In addition to eliminating microorganisms, phagocytosis also accelerates PMN apoptosis, a process critical for resolution of inflammation. Inasmuch as leukocyte phagocytosis and ROS production are key components of the innate immune response, we developed flow cytometric methods to evaluate these processes in human PMNs. In contrast to traditional microscopy-based analyses, the methods described herein provide objective and high throughput measures of host cell-pathogen interactions. Importantly, they can be adapted for use with a number of fluorometric probes, and bacterium and host cell of choice, and each is based upon a common phagocytosis assay system. We also describe methods to measure phagocytosis-induced PMN apoptosis with this assay system. These methods entail detecting surface-exposed phosphatidylserine (early apoptosis), and measuring PMN chromatin condensation and DNA fragmentation (late apoptosis). Taken together, these assays provide rapid and accurate assessment of critical PMN processes.

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References

  1. Ahmed HJ, Johansson C, Svensson LA, Ahlman K, Verdrengh M, Lagergard T (2002). In vitroand in vivointeractions of haemophilus ducreyi with host phagocytes. Infect Immun 70: 899-908.

    Google Scholar 

  2. Allen LA, DeLeo FR, Gallois A, Toyoshima S, Suzuki K, Nauseef WM (1999). Transient association of the nicotinamide adenine dinucleotide phosphate oxidase subunits p47phox and p67phox with phagosomes in neutrophils from patients with X-linked chronic granulomatous disease. Blood 93: 3521-3530.

    Google Scholar 

  3. Babior BM, Kipnes RS, Curnutte JT (1973). Biological defense mechanisms. The production by leukocytes of superoxide, a potential bactericidal agent. J Clin Invest 52: 741-744

    Google Scholar 

  4. Bassoe CF, Smith I, Sornes S, Halstensen A, Lehmann AK (2000). Concurrent measurement of antigen-and antibody-dependent oxidative burst and phagocytosis in monocytes and neutrophils. J Immunol Methods 21: 203-220.

    Google Scholar 

  5. Boyum A (1968). Isolation of mononuclear cells and granulocytes from human blood. Isolation of mononuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl 97: 77-89.

    Google Scholar 

  6. Cunningham MW (2000). Pathogenesis of group a streptococcus infections. Clin Microbiol Rev 3: 470-511.

    Google Scholar 

  7. DeLeo FD, Allen LA, Apicella MA, Nauseef WM (1999). NADPH oxidase activation and assembly during phagocytosis. J Immunol 163: 6733-6740.

    Google Scholar 

  8. Ernst JD (2000). Bacterial inhibition of phagocytosis. Cell Microbiol 2: 379-386.

    Google Scholar 

  9. Gao LY, Kwaik YA (2000). The modulation of host cell apoptosis by intracellular bacterial pathogens. Trends Microbiol 8: 306-313.

    Google Scholar 

  10. Gao LY, Kwaik YA (2000). Hijacking of apoptotic pathways by bacterial pathogens. Microbes Infect 2: 1705-1719.

    Google Scholar 

  11. Holland SM, Gallin JI (1998). Evaluation of the patient with recurrent bacterial infections. Annu Rev Med 49: 185-199.

    Google Scholar 

  12. Jansen WTM, Gootjes J, Zelle M, Madore DV, Verhoef J, Snippe H, Verheul AFM (1998). Use of highly encapsulated streptococcus pneumoniae strains in a flow-cytometric assay for assessment of the phagocytic capacity of serotype-specific antibodies. Clin Diagn Lab Immunol 5: 703-710.

    Google Scholar 

  13. Kerr JF, Wyllie AH, Currie AR (1972). Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br J Cancer 26: 239-257.

    Google Scholar 

  14. Kobayashi SD, Voyich JM, Buhl CL, Stahl RM, DeLeo FR (2002). Global changes in gene expression by human polymorphonuclear leukocytes during receptor-mediated phagocytosis: cell fate is regulated at the level of gene expression. Proc Natl Acad Sci USA 99: 601-6906.

    Google Scholar 

  15. Koopman G, Reutelingsperger CP, Kuijten GA, Keehnen RM, Pals ST, van Oers MH (1994). Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. Blood 84: 1415-1420.

    Google Scholar 

  16. Lehmann AK, Halstensen A, Bassoe CF (1998). Flow cytometric quantitation of human opsonindependent phagocytosis and oxidative burst responses to meningococcal antigens. Cytometry 33: 406-413.

    Google Scholar 

  17. Lehmann AK, Sornes S, Halstensen A (2000). Phagocytosis: measurement by flow cytometry. J Immunol Methods 243: 229-242.

    Google Scholar 

  18. Lekstrom-Himes JA, Gallin JI (2000). Immunodeficiency diseases caused by defects in phagocytes. Adv Immunol 343: 1703-1714.

    Google Scholar 

  19. Li X, Darzynkiewicz Z (1995). Labeling dna strand breaks with brd utp. Detection of apoptosis and cell proliferation. Cell Prolif 28: 571-579.

    Google Scholar 

  20. Martinez JE, Romero-Steiner S, Pilishvili T, Barnard S, Schinsky J, Goldblatt D, Carlone DM (1999). A flow cytometric opsonophagocytic assay for measurement of functional antibodies elicited after vaccination with the 23-valent pneumococcal polysaccharide vaccine. Clin Diagn Lab Immunol 6: 581-586.

    Google Scholar 

  21. McCloskey PS, Salo RJ (2000). Flow cytometric analysis of group b streptococci phagocytosis and oxidative burst in human neutrophils and monocytes. FEMS Immunol Med Microbiol 27: 59-65.

    Google Scholar 

  22. Nauseef WM, Clark RA (2000). Granulocytic phagocytes. Chapter in: Mandell GL, Bennet JE, Dolin R. (eds), Basic principles in the diagnosis and management of infectious disease, 5th ed. New York: Churchill Livingstone, pp 89-112.

    Google Scholar 

  23. Nicoletti I, Migliorati G, Paglizcci MC, Grignani F, Riccardi C (1991). A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139: 271-279.

    Google Scholar 

  24. Park MA, Lee MJ, Lee SH, Jung DK, Kwak JY (2002). Anti apoptotic role of phopholipase d in spontaneous and delayed apoptosis of human neutrophils. FEBS Lett 519: 45-49.

    Google Scholar 

  25. Perskvist N, Long M, Stendahl O, Zheng L (2002). Mycobacterium tuberculosis promotes apoptosis in human neutrophils by activating caspase-3 and altering expression of bax/bcl-xl via an oxygen dependent pathway. J Immunol 168: 6358-6365.

    Google Scholar 

  26. Plested JS, Berne FL, Couli PA, Makepeace K, Lehmann AK, Mackinnon FG, Griffiths HG, Herbert MA, Richards JC, Moxon ER (2001). Functional opsonic activity of human serum antibodies to inner core lipopolysaccharide (gale) of serogroup b meningoccoci measured by flow cytometry. Infect Immun 69: 3203-3213.

    Google Scholar 

  27. Rodriguez ME, van der Pol WL, van de Winkel JG (2001). Flow cytometry-based phagocytosis assay for sensitive detection of opsonic activity of pneumococcal capsular antibodies in human sera. J Immunol Methods 252: 33-44.

    Google Scholar 

  28. Rozenberg-Arska M, Salters M, van Strijp J, Geuze J, Verhoef J (1985). Electron microscopic study of phagocytosis of escherichia coli by human polymorphonuclear leukocytes. Infect Immun 50: 852-859.

    Google Scholar 

  29. Rothe G, Valet G (1990). Flow cytometric analysis of respiratory burst activity in phagocytes with hydroethidine and 2'7'-dichlorofluorescin. J Leuk Biol 47: 440-448.

    Google Scholar 

  30. Rothe G, Valet G (1994). Flow cytometric assays of oxidative burst activity in phagocytes. Methods Enzymol 233: 539-548.

    Google Scholar 

  31. Savill J (1997). Apoptosis in the resolution of inflammation. J Leuk Biol 61: 375-380.

    Google Scholar 

  32. Savill JS, Wyllie AH, Henson JE, Walport MJ, Henson PM, Haslett C (1989). Macrophage phagocytosis of aging neutrophils in inflammation. Programmed cell death in the neutrophil leads to its recognition by macrophages. J Clin Invest 83: 865-875.

    Google Scholar 

  33. Stendahl O, Hed J, Kihlstrom E, Magnusson KE, Tagesson C (1977). Phagocytic internalization and the requirement for membrane perturbation. FEMS Lett 81: 118-120.

    Google Scholar 

  34. Underhill DM, Ozinsky A (2002). Phagocytosis of microbes: complexity in action. Annu Rev Immunol 20: 825-852.

    Google Scholar 

  35. van Eeden S, Klut M, Walker B, Hogg J (1999). The use of flow cytometry to measure neutrophil function. J Immunol Methods 232: 23-43.

    Google Scholar 

  36. Vermes I, Haanen C, Steffens-Nakken H, Reutelingsperger C (1995). A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labeled annexin V. J Immunol Methods 184: 39-51.

    Google Scholar 

  37. Villasenor-Sierra A, McShan WM, Salmi D, Kaplan EL, Johnson DR, Stevens DL (1999). Variable susceptibility to opsonophagocytosis of group a streptococcus m-1 strains by human immune sera. J Infect Dis 180: 1921-1928.

    Google Scholar 

  38. Weinrauch Y, Zychlinsky A (1999). The induction of apoptosis by bacterial pathogens. Annu Rev Microbiol 53: 155-187.

    Google Scholar 

  39. Whyte MK, Meagher LC, MacDermot J, Haslett C (1993). Impairment of function in aging neutrophils is associated with apoptosis. J Immunol 150: 5124-5134.

    Google Scholar 

  40. Wyllie HH, Morris RG, Smith AL, Dunlop D (1984). Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macro-molecular synthesis. J Pathol 142: 67-77.

    Google Scholar 

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Authors and Affiliations

  1. Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Hamilton, MT, 59840, USA

    Jovanka M. Voyich & Frank R. DeLeo

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  1. Jovanka M. Voyich
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  2. Frank R. DeLeo
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Voyich, J.M., DeLeo, F.R. Host-pathogen interactions: leukocyte phagocytosis and associated sequelae. Methods Cell Sci 24, 79–90 (2002). https://doi.org/10.1023/A:1024154200702

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