The Histochemical Journal

, Volume 20, Issue 3, pp 147–155 | Cite as

Differential staining of neutrophils and monocytes: Surface and cytoplasmic iron-binding proteins

  • James C. Barton
  • Richard T. Parmley
  • Thomas W. Butler
  • Sue E. Williamson
  • Michael B. Lilly
  • Richard J. Gualtieri
  • Louis W. HeckJr.


Lactoferrin, transferrin, and ferritin were systematically visualized and semiquantified in neutrophils and monocytes/macrophages using indirect immunofluorescence and functional cytochemical techniques. They localized on cell surfaces and within the cytoplasm at the light and electron microscopical levels. In normal subjects, subpopulations of blood neutrophils and monocytes had surface lactoferrin, but little surface transferrin or ferritin was observed on these cells. Most neutrophils had brilliant granular cytoplasmic positivity for lactoferrin; variable fractions of monocytes had weak to moderate diffuse cytoplasmic lactoferrin staining localized most prominently to the cytoplasmic matrix. Most neutrophils had cytoplasmic ferritin, but few had cytoplasmic transferrin, whereas larger subpopulations of monocytes had cytoplasmic staining reactions for both proteins. To analyse maturing cells, the iron nitrilotriacetate-acid ferrocyanide method was adapted for the light microscopical analaysis of neutrophils and monocytes/macrophages in soft agar culture. Further, a combined stain that visualizes iron nitrilotriacetate-acid ferrocyanide reactivity and α-naphthyl butyrate esterase activity in cells in blood and marrow smears was developed. The relative quantities and subcellular distribution of iron-binding proteins in neutrophils and monocytes/macrophages defined by the present methods can be correlated with biochemical, maturational, and functional properties of these cells.


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  1. ANDREESEN, R., OSTERHOLZ, J., BODEMANN, H., BROSS, K. J., COSTABEL, U. & LÖHR, G. W. (1984) Expression of transferrin receptors and intracellular ferritin during terminal differentiation of human monocytes.Blut 49, 195–202.Google Scholar
  2. ARNOLD, R. R., COLE, M. F. & MCGHEE, J. R. (1977) A bactericidal effect for human lactoferrin.Science 197, 263–5.Google Scholar
  3. BAGBY, G. C., RIGAS, V. D., BENNETT, R. M., BANDENBARK, A. A. & GAREWAL, H. S. (1981) Interaction of lactoferrin, monocytes, and T-lymphocyte subsets in the regulation of steady state granulopoiesisin vitro.J. Clin. invest. 68, 56–63.Google Scholar
  4. BARTON, J. C. & PARMLEY, R. T. (1986) Light microscopic, non-immunologic demonstration of iron-binding proteins in hematopoietic cells.J. Histochem. Cytochem. 34, 299–305.Google Scholar
  5. BENNETT, R. M. & KOKOCINSKI, T. (1978) Lactoferrin content of peripheral blood cells.Br. J. Haematol 39, 509–21.Google Scholar
  6. BENNETT, R. M. & DAVIS, J. (1981) Lactoferrin binding to human peripheral blood cells: an interaction with a B-enriched population of lymphocytes and a subpopulation of adherent mononuclear cells.J. Immunol. 127, 1211–16.Google Scholar
  7. BENNETT, R. M., DAVIS, J., CAMPBELL, S. M. & PORT-NOFF, S. (1983) Lactoferrin binds to cell membrane DNA: an association of surface DNA with an enriched population of B cells and monocytes.J. Clin. Invest. 71, 611–18.Google Scholar
  8. BENNETT, R. M., MERRITT, M. M. & GABOR, G. (1986) Lactoferrin binds to neutrophils membrane DNA.Br. J. Haematol. 63, 105–17.Google Scholar
  9. BERKOW, R. L., TZENG, D. Y., WILLIAMS, L. V. & BAEHNER, R. L. (1983) The comparative responses of human polymorphonuclear leukocytes obtained by counterflow centrifugal elutriation and Ficoll—Hypaque density centrifugation. I. Resting volume, stimulus-induced superoxide production, and primary and specific granule release.J. Lab. Clin. Med. 102, 732–42.Google Scholar
  10. BIRGENS, H. S., HANSEN, N. E., KARLE, H. & KRISTENSEN, L. O. (1983) Receptor binding of lactoferrin by human monocytes.Br. J. Haematol. 54, 383–91.Google Scholar
  11. BOXER, L. A., BJORKSTEIN, B., BJORK, J., YANG, H. H., ALLEN, J. M. & BAEHNER, R. L. (1982a) Neutropenia induced by systematic infusions of lactoferrin.J. Lab. Clin. Med. 99, 866–71.Google Scholar
  12. BOXER, L. A., HAAK, R. A., YANG, H. H., WOLACK, J. B., WHITCOMB, J. A., BUTTERICK, C. J. & BAEHNER, R. L. (1982b) Membrane bound lactoferrin alters surface properties of polymorphonuclear leukocytes.J. Clin. Invest. 70, 1049–57.Google Scholar
  13. BROXMEYER, H. E. (1984) Negative regulators of hematopoiesis,Long-Term Bone Marrow Culture (edited by WRIGHT, D. H. & GREENBERGER, J. S.), p. 36. New York: Alan R. Liss.Google Scholar
  14. BROXMEYER, H. E. & PLATZER, E. (1984) Lactoferrin acts on I-A and I-E/C antigen subpopulations of mouse peritoneal macrophages in the absence of T lymphocytes and other cell types to inhibit production of granulocyte macrophage colony stimulatory factorsin vitro.J. Immunol. 133, 306–14.Google Scholar
  15. BROXMEYER, H. E., RALPH, P., BOGNACKI, J., KINCADE, P. W. & DE SOUSA, M. (1980) A subpopulation of human polymorphonuclear neutrophils contains an active form of lactoferrin capable of binding to human monocytes and inhibiting production of granulocyte-macrophage colony stimulating activity.J. Immunol. 125, 903–9.Google Scholar
  16. BROXMEYER, H. E., BOGNACKI, J., RALPH, P., DORNER, M. H., LU, L. & CASTRO-MALASPINA, H. (1982) Monocytemacrophage derived acidic isoferritins: normal feedback regulators of granulocyte-macrophage progenitor cells.Blood 60, 595–607.Google Scholar
  17. BROXMEYER, H. E., JULIANO, L., LU, L., PLATZER, E. & DUPONT, B. (1984) HLA-DR human histocompatibility leukocyte antigen-restricted lymphocyte—monocyte interactions in the release from monocytes of acidic isoferritins that suppress hematopoietic progenitor cells.J. Clin. Invest. 73, 939–53.Google Scholar
  18. CAMPBELL, E. J. (1982) Human leukocyte elastase, cathepsin G, and lactoferrin: family of neutrophil granule glycoproteins that bind to an alveolar macrophage receptor.Proc. Natl. Acad. Sci. USA 79, 6941–5.Google Scholar
  19. CRAMER, E., PRYZWANSKY, K. B., VILLEVAL, J.-L., TESTA, U. & BRETON-GORIUS, J. (1985) Ultrastructural localization of lactoferrin and myeloperoxidase in human neutrophils by immunogold.Blood 65, 423–32Google Scholar
  20. DUNCAN, R. L. & MCARTHUR, W. P. (1981) Lactoferrin-mediated modulation of mononuclear cell activities. I. Suppression murinein vitro primary antibody response.Cell Immunol. 63, 308–20.Google Scholar
  21. HAURANI, F. I., MEYER, A. & O'BRIEN, R. (1973) Production of transferrin by the macrophage.J. Reticuloendothelial. Soc. 14, 309–16.Google Scholar
  22. HEMMAPLARDH, D. & MORGAN, E. H. (1974) Transferrin and iron uptake by human cells in culture.Exp. Cell. Res. 87, 207–12.Google Scholar
  23. KINKADE, J. M. JR, KELLAR, K. L. & WINTON, E. F. (1979) Immunochemical quantification ofin vitro neutrophilic granulocyte differentiation.Nature 277, 225–7.Google Scholar
  24. KUBOTA, K., MIZOGUCHI, H., MIURA, Y., SUDA, T. & TAKAKU, F. (1980) A new technique for the cytochemical examination of human hemopoietic cells grown in agar gel.Exp. Hematol. 8, 339–44.Google Scholar
  25. LI, C. Y., LAM, K. W. & YAM, L. T. (1973) Esterase in human leukocytes.J. Histochem. Cytochem. 21, 1–12.Google Scholar
  26. MANEVA, A. I., SIRAKOV, L. M. & MANEV, V. V. (1983) Lactoferrin binding to neutrophilic polymorphonuclear leucocytes.Int. J. Biochem. 15, 981–4.Google Scholar
  27. MASON, D. Y. & TAYLOR, C. R. (1978) Distribution of transferrin, ferritin, and lactoferrin in human tissues.J. Clin. Pathol. 31, 316–27.Google Scholar
  28. MASSON, P. L., HEREMANS, J. F. & SCHONNE, E. (1969) Lactoferrin, an iron-binding protein in neutrophilic leukocytes.J. Exp. Med. 130, 643–58.Google Scholar
  29. MIYAUCHI, J., WATANABE, Y., ENOMOTO, Y. & TAKEUCHI, K. (1983) Lactoferrin-deficient neutrophil polymorphonuclear leucocytes in leukaemias: a semiquantitative and ultrastructural cytochemical study.J. Clin. Pathol. 36, 1397–405.Google Scholar
  30. NELSON, D. A., & DAVEY, F. R. (1983) Leukocyte esterases. InHematology (edited by WILLIAMS, W. J., BEUTLER, E. ERSLEV, A. J. & LICHTMAN, M. A., p. 1651. New York, McGraw-Hill.Google Scholar
  31. NISHIYA, K. & HOROWITZ, D. A. (1982) Contrasting effects of lactoferrin on human lymphocyte and monocyte natural killer activity and antibody-dependent cell-mediated cytotoxicity.J. Immunol. 129, 2519–23.Google Scholar
  32. NISHIYA, K., CHIAO, J. W. & DE SOUSA, M. (1980) Iron binding proteins in selected human peripheral blood cell sets: immunofluorescence.Br. J. Haematol. 46, 235–45.Google Scholar
  33. OSEAS, R., YANG, H.-H., BAEHNER, R. L. & BOXER, L. A. (1981) Lactoferrin: a promoter of polymorphonuclear leukocyte adhesiveness.Blood 57, 939–45.Google Scholar
  34. PARMLEY, R. T., TAKAGI, M., BARTON, J. C., BOXER, L. A. & AUSTIN, R. L. (1982) Ultrastructural localization of lactoferrin and iron-binding protein in human neutrophils and rabbit heterophils.Am. J. Pathol. 109, 343–58.Google Scholar
  35. PHILLIPS, P. G., CHIKKAPPA, G. & BRINSON, P. S. (1983) A triple stain technique to evaluate monocyte, neutrophil, and eosinophil proliferation in soft agar cultures.Exp. Hematol. 11, 10–17.Google Scholar
  36. POLLACK, M. S., DA SILVA, B. M., MOSHIEF, R. D., GORSHEN, S., BOGNACKI, J., DUPONT, B. & DE SOUSA, M. (1983) Ferritin secretion by human mononuclear cells: association with HLA phenotype.Clin. Immunol. Immunopathol. 27, 124–34.Google Scholar
  37. RADO, T. A., BOLLOKENS, J., ST LAURENT, G., PARKER, L. & BENZ, E. J. JR (1984) Lactoferrin biosynthesis during granulocytopoiesis.Blood 64, 1103–9.Google Scholar
  38. SEGARS, F. M. & KINKADE, J. M. JR (1977) Radioimmunoassay for murine lactoferrin, a protein marker of myeloid and mammary epithelial secretory cell differentiation.J. Immunol. Methods 14, 1–14.Google Scholar
  39. STEINMAN, G., BROXMEYER, H. E., DE HARVEN, E. & MOORE, M. A. S. (1982) Immunoelectron microscopic tracing of lactoferrin, regulator of myelopoiesis, into a subpopulation of human peripheral blood monocytes.Br. J. Haematol. 50, 75–84.Google Scholar
  40. SUMMERS, M., WORWOOD, W. & JACOBS, A. (1974) Ferritin in normal erythrocytes, polymorphs, lymphocytes, and monocytes.Br. J. Haematol. 28, 19–26.Google Scholar
  41. SUMMERS, M., WHITE, G. & JACOBS, A. (1975) Ferritin synthesis in lymphocytes, polymorphs and monocytes.Br. J. Haematol. 30, 425–34.Google Scholar
  42. VAN SNICK, J. L., MASSON, P. L. & HEREMANS, J. F. (1974) The involvement of lactoferrin in the hyposideremia of acute inflammation.J. Exp. Med. 140, 1068–84.Google Scholar
  43. WORWOOD, M., HOURAHANE, D. & JONES, B. M. (1984) Accumulation and release of isoferritins during incubationin vitro of human peripheral blood mononuclear cells.Br. J. Haematol. 56, 31–43.Google Scholar
  44. YAM, L. T., LI, C. Y. & CROSBY, W. H. (1971) Cytochemical identification of monocytes and granulocytes.Am. J. Clin. Pathol. 55, 283–90.Google Scholar

Copyright information

© Chapman and Hall Ltd 1988

Authors and Affiliations

  • James C. Barton
    • 1
    • 2
  • Richard T. Parmley
    • 3
  • Thomas W. Butler
    • 2
  • Sue E. Williamson
    • 1
  • Michael B. Lilly
    • 2
  • Richard J. Gualtieri
    • 2
  • Louis W. HeckJr.
    • 1
    • 2
  1. 1.Veterans Administration Medical CenterBirminghamUSA
  2. 2.Department of Medicine, Comprehensive Cancer Center, and Multipurpose Arthritis CenterUniversity of Alabama at BirminghamBirminghamUSA
  3. 3.Department of PediatricsThe University of Texas Health Science Center at San AntonioSan AntonioUSA
  4. 4.Division of Hematology/OncologyUniversity StationBirminghamUSA

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