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Comparative study of phagocytosis and intracellular bactericidal activity of human monocytes and polymorphonuclear cells

Application of fluorochrome and extracellular quenching technique

Abstract

Simultaneous assessment of the total number of bacteria (TNB) ingested, phagocytosis (Ph), phagocytic index (PI), and intracellular bactericidal activity (ICBA) of human monocytes was done by applying the fluorochrome acridine orange technique. Living bacteria stained orthochromatically green, whereas the dead ones were metachromatically red. The stain of extracellular bacteria was completely quenched by crystal violet counterstain. Using the Hypaque-Ficoll separation method combined with glass adherence, the yield of monocytes was 84±11%, the purity 90±8%, and the viability 99±1%. After 60 min of incubation of monocytes withStaphylococcus aureus, phagocytosis was 94±4%, PI 10.0±0.5, ICBA 76 ±5%, and TNB ingested 946±67/100 cells.E. coli B4 was equally ingested by PMNs and monocytes and killed intracellularly more efficiently by the latter type of cells. Over the ratios of bacteria to cells of 5∶1 to 20∶1, phagocytic activity of monocytes was equal or superior to that of PMNs. Phagocytic and bactericidal activities were enhanced by AB serum, more by the fresh one than by inactivated. Phagocytic activity of monocytes was markedly influenced by temperature of incubation. Room temperature (24°C) significantly suppressed phagocytosis. Contrary to the previous beliefs no significant quantitative differences were found between phagocytic and bactericidal functions of monocytes as compared to polymorphonuclear phagocytes. The acridine orange-crystal violet method is simple, reliable, reproducible, and can be used for assessment of functional capacity of human phagocytes.

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References

  1. 1.

    Ranadive, N. S., andK. S. Korgaonkar. 1961. Spectrophotometric studies on the binding of acridine orange to RNA and dioxy RNA.Biochim. Biophys. Acta 39:547–550.

  2. 2.

    Smith, D. L., andF. Rommel. 1977. A rapid micro method for the simultaneous determination of phagocytic-microbiocidal activity of human peripheral blood leukocytes in vitro.J. Immunol. Methods 17:241–247.

  3. 3.

    Pantazis, C. G., andW. T. Kniker. 1979. Assessment of blood leukocyte microbial killing by using a new fluorochrome microassay.J. Reticuloendothel. Soc. 26:155–170.

  4. 4.

    Goldner, M., H. Farkas-Himsley, A. Kormendy, andM. Skinner. 1983. Bacterial phagocytosis monitored by fluorescence and extracellular quenching; ingestion and intracellular killing.Lab. Med. 14:291–294.

  5. 5.

    Pruzanski, W., S. Saito, andD. W. Nitzan. 1983. The influence of lysostaphin on phagocytosis, intracellular bactericidal activity and chemotaxis of human polymorphonuclear cells.J. Lab. Clin. Med. 102:298–305.

  6. 6.

    Hed, J. 1977. The extinction of fluorescence by crystal violet and its use to differentiate between attached and ingested microorganisms in phagocytosis.FEMS, Microbiol. Lett. 1:357–361.

  7. 7.

    Leb, L., T. Crusberg, N. Fortier, andL. M. Snyder. 1983. Evaluation of methods using adherence to substrate and density gradient for the isolation of human monocytes.J. Immunol. Methods 58:309–321.

  8. 8.

    Pruzanski, W., andK. J. Delmage. 1977. Cytotoxic and cytolytic activity of homogeneous cold agglutinins on peripheral blood monocytes.Clin. Immunol. Immunopathol. 7:130–138.

  9. 9.

    MacKaness, G. B. 1952. Action of drugs on intracellular tubercle bacilii.J. Pathol. Bacteriol. 64:429–446.

  10. 10.

    Kaplow, L. W. 1965. Simplified myeloperoxidase stain using benzidine dihydrochloride.Blood 26:215–219.

  11. 11.

    Yam, L. T., C. Y. Li, andW. H. Crosby. 1971. Cytochemical identification of monocytes and granulocytes.Am. J. Clin. Pathol. 55:283–290.

  12. 12.

    Steigbigel, R. T., L. H. Lambert, Jr., andJ. S. Remington. 1974. Phagocytic and bacterial properties of normal human monocytes.J. Clin. Invest. 53:131–142.

  13. 13.

    Ackerman, S. K., andS. D. Douglas. 1978. Purification of human monocytes on microexudate-coated surfaces.J. Immunol. 120:1372–1374.

  14. 14.

    Brandslund, I., J. M. Rasmussen, D. Fisker, andS. E. Svehag. 1982. Separation of human peripheral blood monocytes on continuous density gradients of polyvinylpyrrolidone-coated silica gel (Percoll).J. Immunol. Methods 48:199–211.

  15. 15.

    Cline, M. J., andR. I. Lehrer. 1968. Phagocytosis by human monocytes.Blood 32:423–435.

  16. 16.

    Feige, U., B. Overwien, andC. Sorg. 1982. Purification of human blood monocytes by hypotonic density gradient centrifugation in Percoll.J. Immunol. Methods 54:309–315.

  17. 17.

    Ferrante, A., andY. H. Thong. 1982. Separation of mononuclear and polymorphonuclear leucocytes from human blood by the one-step Hypaque-Ficoll method is dependent on blood column height.J. Immunol. Methods 48:81–85.

  18. 18.

    Loos, H., B. Blok-Schut, R. van Doorn, R. Hoksbergen, A. Brutel De Al Riviere, andL. Meerhof. 1976. A method for the recognition and separation of human blood monocytes on density gradients.Blood 48:731–742.

  19. 19.

    Pertoft, H., A. Johnsson, B. Warmegard, andR. Seljelid. 1980. Separation of human monocytes on density gradients of Percoll.J. Immunol. Methods 33:221–229.

  20. 20.

    Weiner, R. S., andV. O. Shah. 1980. Purification of human monocytes: Isolation and collection of large numbers of peripheral blood monocytes.J. Immunol. Methods 36:89–97.

  21. 21.

    Ulmer, A. J., andH. D. Flad. 1979. Discontinuous density gradient separation of human mononuclear leucocytes using Percoll as gradient medium.J. Immunol. Methods 30:1–10.

  22. 22.

    Ranade, S. S., V. G. Tatake, andK. S. Korgaonkar. 1961. Effects of ultrasonic radiation inEscherichia coli B using fluorochrome acridine orange as a vital stain.Nature 189:931–932.

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Supported in part by grant-in-aid of the Medical Research Council of Canada.

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Pruzanski, W., Saito, S. Comparative study of phagocytosis and intracellular bactericidal activity of human monocytes and polymorphonuclear cells. Inflammation 12, 87–97 (1988). https://doi.org/10.1007/BF00915894

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Keywords

  • Functional Capacity
  • Crystal Violet
  • Acridine
  • Human Monocyte
  • Separation Method