Skip to main content
SpringerLink
Log in

Enrichment of human bone marrow mononuclear phagocytes and characterization of macrophage subpopulations by immunoenzymatic double staining

  • Published:
The Histochemical Journal Aims and scope Submit manuscript

Abstract

In order to isolate and enrich bone marrow mononuclear phagocytes, we performed magnetic-activated cell sorting using beads coupled to a monoclonal antibody directed against the monocyte/macrophage surface molecule CD14. Co-localization of antigens in single cells was achieved by combining an alkaline phosphatase--anti-alkaline phosphatase and an avidin--biotin complex immunoassay, avoiding the use of peroxidase. Bone marrow macrophages were first labelled by the monoclonal antibody PG-M1 (anti-CD68). Subsequently, cytoplasmic and/or surface double staining by the monoclonal antibodies against HLA-DR and Mac-2 antigen or the lectin GSA-I-B4 was carried out. Whereas HLA-DR was co-expressed by the great majority of PG-M1+ macrophages (84.9% +/- 6.9%), only a subpopulation exhibited Mac-2 (69.9% +/- 5.9%) antigen or galactoside structures detected by GSA-I-B4 (65.0% +/- 6.7%). The latter result differed only slightly from the percentage of GSA-I-B+4 macrophages determined in a previous comparative immunomorphometrical study. Therefore, using our method of isolation and enrichment by magnetic-activated cell sorting, only a negligible portion of macrophages is apparently stimulated, as shown by GSA-I-B4 staining. This methodology seems to be a valuable tool for further studies on the monocyte--macrophage system. © 1998 Chapman & Hall

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Baldus, S.E., Thiele, J., Park, Y.O., Charles, A., Mross, C., Hanisch, F.G., Zirbes, T.K., Wickenhauser, C. & Fischer, R. (1995) Carbohydrate and peptide antigens in macrophage populations derived from human bone marrow and milk: an immunomorphological and immunochemical analysis. Histochem. J. 27, 630–8.

    Google Scholar 

  • Bhatia, R., Mcglave, P.M., Dewald, G.W., Blazar, B.R. & Verfaillie, C.M. (1995) Abnormal function of the bone marrow microenvironment in chronic myelogenous leukemia: role of malignant stromal macrophages. Blood 85, 3636–45.

    Google Scholar 

  • Dilly, S.A., Jagger, C.J. & Sloane, J.P. (1993) Stromal cell populations in necropsy bone marrow sections from allogeneic marrow recipients and non-transplant patients. J. Clin. Pathol. 46, 611–16.

    Google Scholar 

  • EichmÜller, S., Stevenson, P.A. & Paus, R. (1996) A new method for double immunolabelling with primary antibodies from identical species. J. Immunol. Methods 190, 255–65.

    Google Scholar 

  • Falini, B., Abdulaziz, Z., Gerdes, J., Canino, S., Ciani, C., Cordell, J.L., Knight, P.M., Stein, H., Grignani, F., Martelli, M.F. & Mason, D.Y. (1986) Description of a sequential staining procedure for double immunoenzymatic staining of pairs of antigens using monoclonal antibodies. J. Immunol. Methods 93, 265–73.

    Google Scholar 

  • Falini, B., Flenghi, L., Pileri, S.A., Gambacorta, M., Bigerna, B., Durkop, H., Eitelbach, F., Thiele, J., Pacini, R., Cavaliere, A., Martelli, M., Cardarelli, N., Sabattini, E., Poggi, S. & Stein, H. (1993) PG-M1: a new monoclonal antibody directed against a fixative-resistant epitope on the macrophage-restricted form of the CD68 molecule. Am. J. Pathol. 142, 1359–72.

    Google Scholar 

  • Ho, M.K. & Springer, T.A. (1982) Mac-2 a novel 32 000 Mr mouse macrophage subpopulation specific antigen defined by monoclonal antibodies. J. Immunol. 128, 1221–8.

    Google Scholar 

  • Holness, C.L. & Simmons, D.L. (1993) Molecular cloning of CD68, a human macrophage marker related to lysosomal glycoproteins. Blood 81, 1607–13.

    Google Scholar 

  • Irimura, T., North, S.M. & Nicolson, G.L. (1987) Glycoprotein profiles of macrophages at different stages of activation as revealed by lectin binding after electrophoretic separation. Eur. J. Immunol. 17, 73–8.

    Google Scholar 

  • Keller, R., Keist, R. & Joller, P. (1995) Macrophage response to microbial pathogens: Modulation of the expression of adhesion, CD14, and MHC class II molecules by viruses, bacteria, protozoa and fungi. Scand. J. Immunol. 42, 337–44.

    Google Scholar 

  • KÖller, M., Willheim, M., Krugluger, W., Kurz, M., HÖcker, P., FÖrster, O. & Boltz-Nitulescu, G. (1996) Immunophenotyping of human bone marrow-derived macrophages. Scand. J. Immunol. 43, 626–32.

    Google Scholar 

  • Lee, S.H. (1991) Phenotypic analysis of human bone marrow macrophages. Blood Cells 17, 45–58.

    Google Scholar 

  • Lee, S.H., Crocker, P.R., Westaby, S., Key, N., Mason, D.Y., Gordon, S., Weatherhall, D. J. (1988) Isolation and immunocytochemical characterization of human bone marrow stromal macrophages in hemopoietic clusters.J. Exp. Med. 168, 1193–8.

    Google Scholar 

  • Maddox, D.E., Shibata, S. & Goldstein, I.J. (1982) Stimulated macrophages express a new glycoprotein receptor reactive with Griffonia simplicifolia I-B4 isolectin. Proc. Natl. Acad. Sci. USA 79, 166–70.

    Google Scholar 

  • Petryniak, J., Huard, T.K., Nordblom, G.D. & Goldstein, I. J. (1986) Lectin binding studies on murine peritoneal cells: physicochemical characterization of the binding of lectins from Datura stramonium, Evonymus europaea, and Griffonia simplicifolia to murine peritoneal cells. Arch. Biochem. Biophys. 244, 57–66.

    Google Scholar 

  • Plenat, F., Martinet, Y., Martinet, N. & Vignaud, J.M. (1994) Immunohistochemical methods for studying mononuclear phagocytes in tissue sections. J. Immunol. Methods 174, 133–54.

    Google Scholar 

  • Pryzwansky, K.B. (1982) Applications of double-label immunofluorescence. In Techniques in Immunohistochemistry, Vol. 1 (edited by Bullock, G.R. & Petrusz, P. ), pp. 77–89. London: Academic Press.

    Google Scholar 

  • Saito, N., PUlford, K.A.F., Breton-Gorius, J., Mason, D.Y. & Cramer, E.M. (1991) Ultrastructural localisation of the CD68 macrophage-associated antigen in human blood neutrophils and monocytes. Am. J. Pathol. 139, 1053–9.

    Google Scholar 

  • Sato, S. & Hughes, R.C. (1994a) Regulation of secretion and surface expression of Mac-2, a galactosidebinding protein of macrophages. J. Biol. Chem. 269, 4424–30.

    Google Scholar 

  • Sato, S. & Hughes, R.C. (1994b) Control of Mac-2 surface expression on murine macrophage cell lines. Eur. J. Immunol. 24, 216–21.

    Google Scholar 

  • Schmitz, B., Radbruch, A., Kü mmel, T., Wickenhauser, C., Korb, H., Hansmann, M.L., Thiele, J., Fischer, R. (1994) Magnetic activated cell sorting (MACS)-a new immunomagnetic method for megakaryocytic cell isolation: comparison of different separation techniques. Eur. J. Haematol. 52, 267–75.

    Google Scholar 

  • Stanciu, L.A., Shute, J., Holgate, S.T. & Djukanovic, R. (1996) Production of IL-8 and IL-4 positively and negatively selected CD4+ and CD8+ human T cells following four-step cell separation method including magnetic cell sorting (MACS). J. Immunol. Methods 189, 107–15.

    Google Scholar 

  • Strobl, H., Scheinecker, C., Csmarits, B., Majdic, O. & Knapp, W. (1995) Flow cytometric analysis of intracellular CD68 molecule expression in normal and malignant haemopoiesis. Br. J. Haematol. 90, 774–82.

    Google Scholar 

  • Tabor, D.R., Larry, C.H. & Jacobs, R.F. (1989) Differential induction of macrophage GSIB4-activity. J. Leukoc. Biol. 45, 452–7.

    Google Scholar 

  • Thiele, J., Braeckel, C., Wagner, S., Falini, B., Dienemann, D., Stein, H. & Fischer, R. (1992a) Macrophages in normal bone marrow and in chronic myeloproliferative disorders: an immunohistochemical and morphometric study by a new monoclonal antibody (PG-M1) on trephine biopsies. Virchows Arch. A Pathol. Anat. Histol. 421, 33–9.

    Google Scholar 

  • Thiele, J., Romatowski, C., Wagner, S., Dienemann, D., Stein, H. & Fischer, R. (1992b) Macrophages (phagocytic-histiocytic reticular cells) in reactive-inflammatory lesions of the bone marrow and in myelodysplastic syndromes (MDS). An immunohistochemical and morphometric study by use of a new monoclonal antibody (PG-M1). Pathol. Res. Pract. 188, 995–1001.

    Google Scholar 

  • Thiele, J., Meuter, R.B., Titius, R.B., Zankovich, R. & Fischer, R. (1993) Proliferating cell nuclear antigen expression by erythroid precursors in normal bone marrow, in reactive lesions and in polycythaemia rubra vera. Histopathology 22, 429–35.

    Google Scholar 

  • Thiele, J., Wickenhauser, C., Baldus, S.E., Kuemmel, T., Zirbes, T.K., Drebber, U., Wirtz, R., Thiel, A., Hansmann, M.L. & Fischer, R. (1995) Characterization of CD34+ human hemopoietic progenitor cells from the peripheral blood: enzyme-, carbohydrateand immunocytochemistry, morphometry, and ultrastructure. Leukoc. Lymph. 16, 483–91.

    Google Scholar 

  • Titius, B.R., Thiele, J., Schaefer, H., Kreipe, H. & Fischer, R. (1994) KI-S1 and proliferating cell nuclear antigen expression of bone marrow macrophages. Acta Haematol. 91, 144–9.

    Google Scholar 

  • Van der loos, C.M., Van den oord, J. J., Das, P.K. & Houthoff, H. J. (1988) Use of commercially available monoclonal antibodies for immunoenzyme double staining. Histochem. J. 20, 409–13.

    Google Scholar 

  • Van der loos, C.M., Becker, A.E. & Van den oord, J.J. (1993) Practical suggestions for successful immunoenzyme double-staining experiments. Histochem. J. 25, 1–13.

    Google Scholar 

  • Warfel, A.H. & Zucker-Franklin, D. (1992) Specific ligation of surface á-D-galactosyl epitopes markedly affects the quantity of four major proteins secreted by macrophages. J. Leukoc. Biol. 52, 80–4.

    Google Scholar 

  • Warfel, A.H., Zucker-Franklin, D. & Zheng, Z.Y. (1991) Macrophage membrane glycoproteins that bind Griffonia simplicifolia I-B4: effect on cytotoxicity and protein secretion. J. Cell. Physiol. 147, 265–73.

    Google Scholar 

  • Wickenhauser, C., Thiele, J., Drebber, U., Kvasnicka, H.M., Thiel, A., Schmitz, B., Hansmann, M.L. & Fischer, R. (1995) CD34á human hemopoietic progenitor cells of the bone marrow differ from those of the peripheral blood: an immunocytochemical and morphometric study. Acta Haematol. 93, 83–90.

    Google Scholar 

  • WÜrden, S. & Homberg, U. (1993) A simple method for immunofluorescent double staining with primary antisera from the same species. J. Histochem. Cytochem. 41, 627–30.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Pathology, University of Cologne, Cologne, Germany

    STEPHAN E. BALDUS, CLAUDIA WICKENHAUSER, ALEXANDRA STEFANOVIC, BEATE SCHMITZ, JUERGEN THIELE & ROBERT FISCHER

Authors
  1. STEPHAN E. BALDUS
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. CLAUDIA WICKENHAUSER
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. ALEXANDRA STEFANOVIC
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. BEATE SCHMITZ
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. JUERGEN THIELE
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. ROBERT FISCHER
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

BALDUS, S.E., WICKENHAUSER, C., STEFANOVIC, A. et al. Enrichment of human bone marrow mononuclear phagocytes and characterization of macrophage subpopulations by immunoenzymatic double staining. Histochem J 30, 285–291 (1998). https://doi.org/10.1023/A:1003268008228

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1003268008228

Keywords

Search

Navigation