Abstract
Regulation of haemopoiesis is closely mediated by a number of growth factors in the marrow microenvironment. The identification of the cell type secreting these regulatory polypeptides is difficult due to the heterogeneity of bone marrow cells. To analyse the release of haemopoietic growth factors by normal human bone marrow cells at the single cell level, we employed the reverse haemolytic plaque assay (RHPA). Freshly isolated human marrow cells were examined for the release of interleukin-1α (IL-1α), IL-3, IL-6 and granulocyte-monocyte colony stimulating factor (GM-CSF). In order to identify various cytokine-secreting cell types, the RHPA was combined with immunocytochemical or enzymatic staining. The total of secreting marrow cells as well as the amount of several secretory haemopoietic subpopulations could be determined with this technique under various conditions. Following incubation with pure serum-free medium without addition of any mediator, only few cells secreting either IL-1α, IL-3, IL-6 or GM-CSF could be observed. After 2 h incubation with recombinant human-IL-1α (rhIL-1α) (10.0 ng/ml) or rhGM-CSF (10.0 pg/ml) the number of cytokine-secreting cells significantly increased for all secretory products tested. Using cytochemical staining reactions, we were able to identify 55% of all cells secreting a specific cytokine. Glycophorin C-positive erythropoietic cells turned out to be the largest fraction (up to 89%) of cytokine-releasing haemopoietic cells, followed by neutrophil granulocytes (between 6 and 48%), and monocytes/macrophages (between 4 and 23%). Only few CD 61-positive cytokinesecreting megakaryocytes could be detected. Dose- and time-dependent kinetics after stimulation with rhGM-CSF revealed that the bulk of secretory activity originates from haemopoietic or rather from erythropoietic cells following low level stimulation and after short stimulation time. Thus, our data are in keeping with the assumption, that especially erythropoietic cells are producing a repertoire of cytokines that is thought to exhibit regulatory functions within marrow microenvironment. In the present study the RHPA is presented as an appropriate tool for measuring cytokine release not only of cells of the haematopoietic system but also of other tissues, for example solid tumours or malignant lymphomas.
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References
Brach MA, Herrmann F (1991) Hematopoietic growth factors: interactions and regulation of production. Acta Haematol 86: 128–137
Briddell RA, Hoffman R (1990) Cytokine regulation of the human burst-forming unit-megakaryocyte. Blood 76: 516–522
Briddell RA, Brandt JE, Leemhuis TB, Hoffman R (1992) Role of cytokines in sustaining long-term human megakaryocytopoiesis in vitro. Blood 79: 332–337
Broxmeyer HE (1986) Biomolecule-cell interactions and the regulation of myelopoiesis. Int J Cell Cloning 4: 378–405
Carcacciolo D, Clark SC, Ronera G (1989) Human IL-6 supports granulocytic differentiation of hematopoietic progenitor cells and acts synergistically with GM-CSF. Blood 73: 666–670
Cicco NA, Lindemann A, Content J, Vandenbussche P, Lübbert M, Gauss J, Mertelsmann R, Herrmann F (1990) Inducible production of interleukin-6 by human polymorphonuclear neutrophils: role of granulocyte-macrophage colony-stimulating factor and tumor necrosis factor-alpha. Blood 75: 2049–2052
Cordell JL, Falini B, Erber WN, Gosh A, Abulaziz Z, Mac Donald S, Pulford KAF, Stein H, Mason DY (1984) Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes). J Histochem Cytochem 32: 219–229
Czerkinsky CC, Nilsson LA, Nygren H, Ouchterlony O, Tarkowski A (1983) A solid-phase enzyme-linked immunospot (ELISPOT) assay for enumeration of specific antibody-secreting cells. J Immunolog Methods 65: 109–121
De Groote D, Zangerle PF, Gevaert Y, Fassotte MF, Beguin Y, Noizat-Pirenne F, Pirenne J, Gathy R, Lopez M, Dehart I, Igot D, Baudrihaye M, Delacroix D, Franchimont P (1992) Direct stimulation of cytokines (IL-α, TNF-α, IL-6, IFN-τ and GM-CSF) in whole blood. I. Comparison with isolated PBMC stimulation. Cytokine 4: 239–248
Dorshkind K (1990) Regulation of hemopoiesis by bone marrow stromal cells and their products. Annu Rev Immunol 8: 111–137
Eaves CJ, Cashman JD, Kay RJ, Dougherty GJ, Otsuka T, Gaboury LA, Hogge DE, Landsdorp PE, Eaves AC, Humphries RK (1991) Mechanisms that regulate the cell cycle status of very primitive hematopoietic cells in long-term human marrow cultures. II. Analysis of positive and negative regulators produced by stromal cells within the adherent layer. Blood 78: 110–117
Gatter KC, Cordell JL, Turley H, Heryet A, Kiefer N, Anstee DJ, Mason DY (1988) The immunohistological detection of plate-lets, megakaryocytes and thrombi in routinely processed specimens. Histopathology 13: 257–267
Greenberger JS (1991) The hematopoietic microenvironment. Crit Rev Oncol/Hematol 11: 65–84
Hermine O, Beru N, Pech Nancy, Goldwasser E (1991) An autocrine role for erythropoietin in mouse hematopoietic cell differentiation. Blood 78: 2253–2260
Heyworth CM, Ponting ILO, Dexter TM (1988) The response of haemopoietic cells to growth factors: developmental implications of synergistic interactions. J Cell Sci 91: 239–247
Heyworth CM, Vallance SJ, Whetton AD, Dexter TM (1990) The biochemistry and biology of the myeloid haemopoietic cell growth factors. J Cell Sci Suppl 13: 57–74
Ikebuchi K, Clark SC, Ihle JN, Souza LM, Ogawa M (1988) GM-CSF enhances IL-3-dependent proliferation of multipotential haemopoietic progenitores. Proc Natl Acad Sci USA 85: 3445–3449
Jerne NK, Henry AA, Nordin H, Fuji T, Koros AMC, Lefkovits S (1974) Plaque-forming cells: methodology and theory. Transplant Rev 18: 130–191
Kittler ELW, McGrath H, Temeles D, Crittenden RB, Kister VK, Quesenberry PJ (1992) Biologic significance of constitutive and subliminal growth factor production by bone marrow stroma. Blood 79: 3168–3178
Lewis CE (1991) Detecting cytokine production at the single-cell level. Cytokine 3: 184–188
Lewis CE, McCarthy SP, Lorenzen J, McGee JO'D (1989) Heterogeneity among human mononuclear cells in their secretion of lysozyme, interleukin 1 and type-b transforming growth factor: a quantitative analysis at the single-cell level. Eur J Immunol 19: 2037–2043
Lewis CE, McCarthy SP, Richards PS, Lorenzen J, Horak E, McGee JO'D (1990) Measurement of cytokine release by human cells. A quantitative analysis at the single cell level using the reverse haemolytic plaque assay. J Immunol Meth 127: 51–59
Metcalf D (1984) Haemopoietic colony stimulating factors. Elsevier, Amsterdam
Molinaro GA, Dray S (1974) Antibody-coated erythrocytes as a manifold probe for antigens. Nature 248: 515–520
Moore MAS (1991) Clinical implications of positive and negative hematopoietic stem cell regulators. Blood 78: 1–19
Otsuka T, Thacker JD, Hogge DE (1991) The effects of interleukin 6 and interleukin 3 on early hematopoietic events in long-term cultures of human marrow. Exp Hematol 19: 1042–1048
Quesenberry PJ (1989) Stromal cells in long-term bone marrow cultures. In: Tavassoli M (ed) Handbook of the hemopoietic microenvironment. Humana, Clifton NJ, pp 253–276
Radzun HJ, Hansmann ML, Heidebrecht HJ, Boedewad-Radzun S, Wacker HH, Kreipe H, Lumbeck H, Hernandez C, Kuhn C, Parwaresch MR (1991) Detection of the monocyte-macrophage differentiation antigen in routinely processed paraffin embedded tissues by monoclonal antibody KiM1P. Lab Invest 65: 306–315
Rowe JM, Rapoport AP (1992) Hemopoietic growth factors: a review. J Clin Pharmacol 32: 486–501
Shirai A, Holmes K, Klinman D (1993) Detection and quantitation of cells secreting IL-6 under physiologic conditions in BALB/c mice. J Immunol 150: 793–799
Sieff CA, Emerson SG, Donahue RE, Nathan DG, Wang EA, Wong CG, Clark SC (1985) Human recombinant granulocyte-macrophage colony stimulating factor: a multilineage hematopoetin. Science 230: 1171–1173
Sytkowski AJ, O'Hara C, Vanasse G, Armstrong MJ, Kreczko S, Dainiak N (1990) Characterization of biologically active, platelet-derived growth factor-like molecules produced by murine erythroid cells in vitro and in vivo. J Clin Invest 85: 40–46
Takahashi T, Tsuyuoka R, Ueda Y, Suzuki A, Icheba S, Okuno Y, Nakamura K, Imura H (1991) Megakaryocyte potentiating activity of IL-1, IL-6 and GM-CSF as evaluated by their action on in vitro human megakaryocytic colonies. Br J Haematol 78: 480–487
Whetton AD, Dexter TM (1989) Myeloid haemopoietic growth factors. Biochim Biophys Acta 989: 111–132
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Rohde, D., Wickenhauser, C., Denecke, S. et al. Cytokine release by human bone marrow cells: analysis at the single cell level. Vichows Archiv A Pathol Anat 424, 389–395 (1994). https://doi.org/10.1007/BF00190561
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DOI: https://doi.org/10.1007/BF00190561