ze~t~h,,I t~r ~le g~amle
Bltit
Blut 39, 375-381 (1979)
9 Springer-Verlag 1979
Leitartikel / Leading Article
Myeloid Differentiation in Cultures of Human Hemopoietie Precursor Cells Catherine Nissen-Druey H/imatologische Laboratorien, Kantonsspital Basel CH-4031 Basel, Switzerland
Granulocyte-macrophage colony-forming cells (GM-CFC) from human bone marrow and peripheral blood differentiate along the neutrophil, macrophage, and eosinophil pathway in culture. They proliferate in the presence of specific glycoproteins, which have been termed colony-stimulating factors (CSF), and form colonies containing recognizable end stage cells [5,8,10,11,13,21,29]. Culture results can be expressed in total colony counts. Preferably, neutrophil, macrophage, and eosinophil colonies are counted separately. Information on myeloid differentiation can then be drawn from relative colony counts at varying culture conditions: Pathway-specific colony-stimulating factors can be recognized by their capacity to stimulate proliferation of single type colonies. In vivo mechanisms of differentiation are to some extent reflected by the relative frequency of neutrophil, macrophage, and eosinophil colonies in culture. The proportion is remarkably constant in normals and varies in disease, indicating that myeloid precursors have been primed in vivo to mature preferentially along one of the pathways in vitro. Recognition of the various colony types depends on the culture medium used. They are easily distinguished in methylcellulose cultures after 2 weeks of incubation [8,10,13,21]. In agar cultures recognition is difficult without previous staining [16a]. Bone Marrow Cultures
1. Neutrophil Colony Formation Large neutrophil colonies in human cultures are visible by eye. In the inverted microscope, the colonies appear tightly packed in the center and loosely dispersed at the periphery. Single cells are transparent, i.e., they do not display cytoplasmic structures in culture. Peripheral cells develop motility by the formation of pseudopods, hence, variation of cell shapes is observed. On Giemsa stain, they are recognized as neutrophil myelocytes, metamyelocytes, and granulocytes. The stage of segmented granulocytes is rarely achieved in culture, and the development of specific granules remains incomplete [20, 23, 34]. Neutrophil colonies vary in size. 0006-5242/79/0039/0375/$1.40
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Aggregates containing less than 50 cells in agar cultures, and less than 20 cells in methylcellulose cultures, respectively, have been termed "clusters". Since a high ratio of "clusters" in relation to larger colonies is seen in cultures from patients with acute myelogenous leukemia (AML), the term "cluster" has frequently been associated with leukemia. Small colonies, however, are a normal phenomenon. They may even outweigh large colonies in cultures from patients with diseases other than leukemia, e.g., aplastic anemia. These small colonies contain neutrophils of normal appearance and do not resemble leukemic "clusters" in methylcellulose cultures. The latter are composed of visibly abnormal cells. It is misleading to use the term "cluster" for both phenomena since they are of such different significance. Preferably, small colonies of normal morphology should be distinguished from "clusters" of abnormal cells. In normal human bone marrow cultures, small neutrophil colonies appear early, reach their maximal size after 7 days, and then desintegrate. Large colonies appear later and are fewer in numbers [1]. In the mouse, precursors giving rise to small and large colonies could be separated by velocity sedimentation and by their response to CSF: More rapidly sedimenting, presumably more mature cells, formed small colonies and were highly sensitive to purified CSF. The more slowly sedimenting, probably more immature cells formed larger colonies and required another factor in addition to CSF. This non-CSF-activity was found in nonpurifled conditioned medium [17]. It is likely to be identical with the factor inducing CSF-responsiveness of immature precursors, which has been isolated from human leukocyte conditioned medimn [32]. About 60 ~ of all myeloid colonies in bone marrow cultures are neutrophil [10, 21]. One third contain macrophages in varying proportions [10]. An absolute and relative increase of neutrophil colonies occurs after successful chemotherapy of acute myelogenous leukemia and in agranulocytosis. Neutrophil precursor cells have thus been programmed in vivo-the signal probably being neutropenia-to meet the increased requirement for end stage cells. On the other hand, we did not observe an increase of neutrophil colonies in patients with neutrophil leukocytosis due to bacterial infection. This suggests that mature neutrophils can be recruited in vivo from a pool of more mature cells without primary involvement of myeloid precursors.
2. Macrophage Colony Formation Monocyte macrophages develop a characteristic morphology in culture [9,21]: They are larger than neutrophils, and appear greyish by cytoplasmic structures, which are recognized as vacuoles on Giemsa stain. They develop functional characteristics of macrophages in culture: Fc-receptors [16 d, 22] are detectable on their surface and they are able to ingest antibody-coated erythrocytes [22]. Macrophage colonies are loose. They frequently contain neutrophils in varying proportion [10,16c, 19]. Human macrophage precursors could be separated from neutrophil colony forming cells by velocity sedimentation [12]: Macrophage colony forming cells sediment more slowly and thus appear to be smaller than those committed strictly to the neutrophil pathway. They respond to a subtype of CSF, which was found in human serum and urine rather than in conditioned media [16 f].
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The relative frequency of macrophage colonies in bone marrow cultures is approximately 3 0 ~ [10,21]. One third contain neutrophils, the other two thirds are pure macrophage colonies [10]. The proportion increases at suboptimal culture conditions [16e] and with ageing of the cultures [16b]. In cultures from patients with untreated Hodgkin's disease, myeloma, benign monoclonal paraproteinemia, lymphoma, and cancer, a higher percentage of macrophage colonies is seen [21]. Most of these patients do not have monocytosis in vivo. Macrophage maturation appears to be more favored by in vitro than by in vivo conditions. Therefore, increased commitment toward the macrophage pathway as a sign of disease is recognized in vitro, but not necessarily in vivo.
3. Eosinophil Colony Formation Eosinophil colonies in methylcellulose cultures are distinguished from neutrophil colonies by their conspicuous dark appearance at small magnification. They are relatively small and consist of tightly packed cells containing cytoplasmic granules which can be recognized at high magnification in the inverted microscope. Single eosinophils are larger than neutrophils and do not vary in shape and size. This morphology is typical of human eosinophil colonies and differs from the appearance described for mouse eosinophil colonies [18]. On Giemsa stain single cells from eosinophil colonies are immature: They have a large, nonsegmented nucleus and strongly eosinophilic cytoplasmic granules. Vacuolization of the cytoplasma is frequently observed. Development of granules remains incomplete when examined by electron microscopy and cytochemistry [23, 34]. In vitro conditions apparently do not support eosinophil maturation beyond the stage of myelocytes [21]. Neutrophil differentiation in vitro is comparably more complete. There is evidence that the eosinophil pathway separates from the granulocytemacrophage pathway at a very primitive level: Eosinophil colonies are always pure, suggesting that they are derived from an eosinophil precursor which restricted differentiation capacity. In the mouse, heterogeneity of eosinophil and neutrophil precursors has been demonstrated by velocity sedimentation: Eosinophil colonyforming cells sediment more slowly and respond to a specific eosinophil colony stimulating activity derived from stimulated lymphocytes [18]. Further evidence for the specificity of eosinophil-CSF was obtained from experiments in animals with suppressed neutrophil-CSF: 1. Antibodies raised against neutrophil-CSF do not affect eosinophil-CSF [16i]. 2. Mice with a congenital defect of endotoxin mediated CSF-production have a normal eosinophil-CSF-response [2]. Human eosinophil-CSF could so far not be separated from neutrophil-CSF. Human conditioned media of all sources contain both activities (16k]. Factors, which promote maturation of eosinophils in liquid culture [2, 3, 26] or induce eosinophilia in animals [15] and in humans [33] have been described. None of them has been found to stimulate colony formation in vitro, i.e., it is not clear if they are identical with eosinophil-CSF. They could represent a maturation hormone acting at a more mature level of eosinophilopoiesis.
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Delayed appearance and long survival of eosinophil colonies is characteristic [8, 11,16g]. Very few are seen before 2 weeks in culture. They desintegrate later than neutrophils during prolonged incubation. It has been suggested that late appearance could indicate that eosinophil precursors are more primitive cells than neutrophil colony-forming cells [11 ]. The relative frequency of eosinophil colonies in normal bone marrow cultures is low: Dao reports an incidence of 3 . 7 ~ [8], in our series it was 12~ [21]. However, compared to the percentage of mature eosinophils in vivo, eosinophil colony counts are relatively high, suggesting that in vitro conditions favor early eosinophil commitment, although they do not support terminal eosinophil maturation. A higher relative number of eosinophil colonies is observed in a variety of malignant diseases, in graft-versus-host disease [21] and in some patients with acute myelogenous leukemia (AML) in remission. Interestingly, the formation of eosinophil clusters is sometimes observed in A M L in relapse [16h and personal observation]. The prognostic significance of this finding is not yet established. It has been reported that eosinophils inhibit granulopoiesis in vitro [31]. They may therefore be involved in the control of leukemic growth. The increase of eosinophil colonies in disease was not, or not adequately paralleled by eosinophilia in peripheral blood. On the other hand, in cultures from two patients with massive eosinophilia which was due to allergic disease in one, and had developed in the course of acute lymphoblastic leukemia in the other, the percentage of eosinophil colonies was not increased. From these observations one would assume that there is more than one mechanism by which the eosinophil pathway can be stimulated: One seems to act at the level of eosinophil precursor cells, and thus to be recognized in vitro by increased eosinophil colony formation. It does not necessarily produce eosinophilia in vivo. Another factor seems to promote final eosinophil maturation rather than early commitment. It is not recognized in vitro but produces eosinophilia in vivo. These observations support the hypothesis that in vitro eosinophilCSF is not necessarily identical with the in vivo "eosinophilopoietin" described above. Eosinophils are known to act as effector cells in several types of immune reactions: Killing of parasites [7] and tumor cells [25, 28] by eosinophils has been observed. Increased eosinophil commitment in vitro in malignant disease therefore probably reflects an in vivo defense mechanism.
Peripheral Blood Cultures from peripheral blood differ from bone marrow cultures by a lower concentration of colony-forming cells. In addition, the relative frequency of different colony types in peripheral blood cultures shows some characteristic features: 1. Only large, late-appearing neutrophiI colonies are seen in peripheral blood cultures, more mature neutrophil precursors with restricted proliferation capacity appear to be excluded from the circulation. 2. Macrophage colonies are rare (7 ~ compared to 30 ~ in bone marrow cultures).
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3. The percentage of eosinophil precursors is high in peripheral blood, the relative frequency was over 50 % in six patients described by Chervenick [8]. We found a mean of 40% eosinophil colonies in 10 normals. In simultaneous bone marrow cultures the incidence was 12 %. Thus, there is a true prevalence of eosinophil precursors in the circulation. Eosinophil colonies in peripheral blood cultures are large and multicentered. Since they appear dark, they may be mistaken for erythroid bursts in cultures containing erythropoietin. The ability to form multicentered colonies is characteristic for erythroid burst-forming cells. It indicates a high proliferation capacity and thus immaturity. Likewise, "eosinophil burstforming cells" may be very primitive cells. These observations suggest that primitive myeloid precursors prevail in the circulation. The same phenomenon is known for the erythroid line. Only immature precursors (burst-forming cells) are detected in peripheral blood whereas the more mature precursors (CFU-E) are excluded [23]. Peripheral blood thus seems to be a pool of very immature hemopoietic precursors.
Conclusions and Summary Culture conditions support the early phase of myeloid differentiation. Mechanisms of regulation can therefore be recognized in vitro by differential counts of neutrophil, macrophage, and eosinophil colonies under varying conditions. From such studies information of theoretical and clinical interest has emerged: Neutrophils and macrophages mature along a common pathway, from which the eosinophil line separates at an early stage of differentiation. Specific regulators exist for each of the three pathways. Colony-stimulating factors acting at the level of neutrophil, eosinophil, and macrophage precursor cells are detectable in vitro. They initiate maturation which is probably completed by other factors in vivo. An increase of macrophage and eosinophil colonies without monocytosis and eosinophilia in vivo is frequently observed in malignant disease. This phenomenon is likely to reflect a tumor defense mechanism. The relative frequency of various precursor cells in the circulation does not reflect their distribution in the bone marrow. There seems to be a prevalence of immature cells in peripheral blood.
References 1. Baccarani, M., Ricci, P., Santucci, A. M., Tufa, S. : Proliferazione e maturazione dei precursori granulocitari umani coltivati in metil cellulosa. In: Emolinfopoiesi, fattori di regolazione e meccanismi di crescita. Brunelli, M.A., Bagnara, G.P., Castaldini, C. (eds.), pp. 299-312. IlI incontro nazionale di ematologia sperimentale, Reggio Calabria 11-12 maggio 1979, Esculapio, Bologna. 2. Bartelmez, S.H., Dodge, W.H., Bass, D.A.: Antigen-mediated release of eosinophit growth stimulating factor (Eo-GSF) from trichinella spiralis sensitized spleen cells: A comparison of various T. spiralis antigens. Exp. Hematol. [Suppl. 3] 6, 77 (1978)
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3. Bartelmez, S.H., Dodge, W.H., Bass, D.A, : Differential regulation of spleen cell mediated eosinophil and neutrophil production. Exp. Hematol. [Suppl. 3] 6, 46 (1978) 4. Basten, A., Beeson, P.B.: Mechanism of eosinophilia. II. Role of the lymphocyte. J. Exp. Med. 131, 1288-1305 (1970) 5. Burgess, A.W., Wilson, E.C., Metcalf, D.: Stimulation by human placental conditioned medium of hemopoietic colony formation by human marrow cells. Blood 49, 573-583 (1977) 6. Burgess, A.W., Metcalf, D. : The effect of colony-stimulating factor on synthesis of ribonucleic acid by mouse bone marrow cells in vitro. J. Cell. Physiol. 90, 471-484 (1977) 7. Butterworth, A.E., David, J.R., Franks, D., Mahmoud, A.A.F., David, P.H., Sturrock, R. F., Houba, V. :Antibody dependent eosinophil mediated damage to 5~Cr-labeled schistosomula of schistosoma mansoni: Damage by purified eosinophils. J. Exp. Med. 145, 136 (1977) 8. Chervenick, P.A., Boggs, D.R.: In vitro growth of granulocytic and mononuclear cell colonies from blood of normal individuals. Blood 37, 131-135 (1971) 9. Cohn, Z.A., Hirsch, J.G., Fedorko, M.E.: The in vitro differentiation of mononuclear phagocytes. IV. The ultrastructure of macropbage differentiation in the peritoneal cavity and in culture. J. exp. Med. 123, 747-755 (1966) 10. Dao, Ch., Metcalf, D., Bilski-Pasquier, G. : Eosinophil and neutrophil colony-forming cells in culture. Blood 50, 833-839 (1977) 11. Dresch, C., Faille, A. : On the heterogeneity of colony growth according to the colony cytological type. Exp. Hematol. [Suppl. 2] 5, 15 (1977) 12. Inoue, S., Ottenbreit, J. : Two types of human colony-forming cells (CFC) differing in size, and in response to different stimulator preparations. Exp. Hematol. [Suppl. 2] 5, 15 (1977) 13. Iscove, N.N., Senn, J.S., Till, J.E., McCulloch, E.A.: Colony formation by normal and leukemic human marrow cells in culture. Blood 37, 1-5 (1971) 14. Johnson, G.R., Dresch, C., Metcalf, D. : Heterogeneity in human neutrophil, macrophage, and eosinophil progenitor cells demonstrated by velocity sedimentation separation. Blood 50, 823-831 (1977) 15, Mahmoud, A. F. A., Stone, M.K., Kellermeyer, R.W.: Eosinophilopoietin. A circulating low molecular weight peptide-like substance which stimulates the production of eosinophils in mice. J. Clin. Invest. 60, 675-682 (1977) 16, Metcalf, D. : Hemopoietic colonies. In vitro cloning of normal and leukemic cells. P. Rentchnik, Geneve (ed.), a) p. 32, b) p. 60, c) p. 65, d) p. 67, e) p. 70, f) p. 78, g) p. 144, h) p. 145, i) p. 146, k) p. 149, Berlin, Heidelberg, New York: Springer 1977 17, Metcalf, D., Mac Donald, H.R. : Heterogeneity of in vitro colony and cluster-forming cells in the mouse marrow. Segregation by velocity sedimentation. J. Cell. Physiol. 85, 643-654 (1975) 18. Metcalf, D., Parker, J., Chester, H. M., Kincade, P. W. : Formation of eosinophil-like granulocytic colonies by mouse bone marrow cells in vitro, J. Cell. Physiol. 84, 275-290 (1974) 19. Moore, M.A.S., Williams, N., Metcalf, D. : Purification and characterisation of the in vitro colony-forming cell in monkey hemopoietic tissue. J. Cell. Physiol. "/9, 283-292 (1972) 20. Moore, M.A.S., Williams, N., Metcalf, D. : In vitro colony formation by normal and leukemic human hemopoietic cells. J. Nat. Cancer Inst. 50, 60%623 (1973) 21. Nissen-Druey, C., Speck, B. : Differential counts of neutrophil, eosinophil, and macrophage colonies in cultures from human bone marrow and peripheral blood. Blut 37, 241-248 (1978) 22. Nissen, C., Elliott, B., Groff, P., Cornu, P., Gudat, F., Mfiller, H. J., Hartmann, D., Speck, B. : Pathological cell aggregates in bone marrow cultures from patients with various hematological disorders. Blut 38, 457-4.65 (1979) 23. Ogawa, M.: Circulating erythropoietic precursors assessed in culture: Characterization in normal men and patients with hemoglobinopathies. Blood 50, 1081-1092 (1977) 24. Parmly, R.T., Ogawa, M., Spicer, S.G., Wright, N, J. : Ultrastructure and cytochemistry of bone marrow granulocytes in culture. Exp. Hematol. 4, 75-89 (1976) 25. Parrillo, J.E,, Fancis, A. S. : Human eosinophils. Purification and cytotoxic capabilities of eosinophils from patients with the hypereosinophilic syndrome. Blood 51, 457 (1978) 26. Ruscetti, F.S., Cypress, R.H., Chervenick, P.A. : Specific release of neutrophilic and eosinophilic stimulating factors from sensitized lymphocytes. Blood 47, 757-765 (1976) 27. Sanderson, C. J., Lopez, A.F., Bunn Moreno, M. M. : Eosinophils and not lymphoid K cells kill trypanosoma cruzi epimastigotes. Nature (Lond~) 268, 340 (1977)
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28. Sanderson, C.J., Thomas, J.A.: A comparison of the cytotoxic activity of eosinophils and other cells by 51chronium release and time lapse micro cinematography. Immunology 34, 771 (1978) 29. Shoham, D., Ben David, E., Rosenszajn, L.A. : Cytochemical and morphological identification of macrophages and eosinophils in tissue cultures of normal human bone marrow. Blood 44, 221-223 (1974) 30. Spry, C. J. F. : Eosinophils as effector cells in disease. Schweiz. Med. Wochenschr. 108, 1572-1576 (1978) 31. Tebbi, K., Mahmoud, A., Gross, S. : The role of eosinophils in granulopoiesis. Exp. Hematol. [Suppl. 3] 6, 50 (1978) 32. Wagemaker, G., Brouwer, A., Bol, S. J.L., Visser, T.P. : Analysis of factors in vitro inducing erythropoietin and CSF-responsiveness in primitive hemopoietic progenitor cells of the mouse. Exp. Hematol. [Suppl. 3] 6, 31 (1978) 33. Walls, R.S., Basten, A., Leuchars, E., Davies, A. J. S. : Mechanisms for eosinophilic and neutrophilic leukocytoses. Br. Med. J. 3, 1957 (1971) 34. Zucker-Franklin, D., Grusky, G.: Ultrastructural analysis of hemopoietic colonies derived from human peripheral blood. J. Cell Biol. 63, 855-863 (1974)
Received July 23, 1979
Bltit
Blut 39, 375-381 (1979)
9 Springer-Verlag 1979
Leitartikel / Leading Article
Myeloid Differentiation in Cultures of Human Hemopoietie Precursor Cells Catherine Nissen-Druey H/imatologische Laboratorien, Kantonsspital Basel CH-4031 Basel, Switzerland
Granulocyte-macrophage colony-forming cells (GM-CFC) from human bone marrow and peripheral blood differentiate along the neutrophil, macrophage, and eosinophil pathway in culture. They proliferate in the presence of specific glycoproteins, which have been termed colony-stimulating factors (CSF), and form colonies containing recognizable end stage cells [5,8,10,11,13,21,29]. Culture results can be expressed in total colony counts. Preferably, neutrophil, macrophage, and eosinophil colonies are counted separately. Information on myeloid differentiation can then be drawn from relative colony counts at varying culture conditions: Pathway-specific colony-stimulating factors can be recognized by their capacity to stimulate proliferation of single type colonies. In vivo mechanisms of differentiation are to some extent reflected by the relative frequency of neutrophil, macrophage, and eosinophil colonies in culture. The proportion is remarkably constant in normals and varies in disease, indicating that myeloid precursors have been primed in vivo to mature preferentially along one of the pathways in vitro. Recognition of the various colony types depends on the culture medium used. They are easily distinguished in methylcellulose cultures after 2 weeks of incubation [8,10,13,21]. In agar cultures recognition is difficult without previous staining [16a]. Bone Marrow Cultures
1. Neutrophil Colony Formation Large neutrophil colonies in human cultures are visible by eye. In the inverted microscope, the colonies appear tightly packed in the center and loosely dispersed at the periphery. Single cells are transparent, i.e., they do not display cytoplasmic structures in culture. Peripheral cells develop motility by the formation of pseudopods, hence, variation of cell shapes is observed. On Giemsa stain, they are recognized as neutrophil myelocytes, metamyelocytes, and granulocytes. The stage of segmented granulocytes is rarely achieved in culture, and the development of specific granules remains incomplete [20, 23, 34]. Neutrophil colonies vary in size. 0006-5242/79/0039/0375/$1.40
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Aggregates containing less than 50 cells in agar cultures, and less than 20 cells in methylcellulose cultures, respectively, have been termed "clusters". Since a high ratio of "clusters" in relation to larger colonies is seen in cultures from patients with acute myelogenous leukemia (AML), the term "cluster" has frequently been associated with leukemia. Small colonies, however, are a normal phenomenon. They may even outweigh large colonies in cultures from patients with diseases other than leukemia, e.g., aplastic anemia. These small colonies contain neutrophils of normal appearance and do not resemble leukemic "clusters" in methylcellulose cultures. The latter are composed of visibly abnormal cells. It is misleading to use the term "cluster" for both phenomena since they are of such different significance. Preferably, small colonies of normal morphology should be distinguished from "clusters" of abnormal cells. In normal human bone marrow cultures, small neutrophil colonies appear early, reach their maximal size after 7 days, and then desintegrate. Large colonies appear later and are fewer in numbers [1]. In the mouse, precursors giving rise to small and large colonies could be separated by velocity sedimentation and by their response to CSF: More rapidly sedimenting, presumably more mature cells, formed small colonies and were highly sensitive to purified CSF. The more slowly sedimenting, probably more immature cells formed larger colonies and required another factor in addition to CSF. This non-CSF-activity was found in nonpurifled conditioned medium [17]. It is likely to be identical with the factor inducing CSF-responsiveness of immature precursors, which has been isolated from human leukocyte conditioned medimn [32]. About 60 ~ of all myeloid colonies in bone marrow cultures are neutrophil [10, 21]. One third contain macrophages in varying proportions [10]. An absolute and relative increase of neutrophil colonies occurs after successful chemotherapy of acute myelogenous leukemia and in agranulocytosis. Neutrophil precursor cells have thus been programmed in vivo-the signal probably being neutropenia-to meet the increased requirement for end stage cells. On the other hand, we did not observe an increase of neutrophil colonies in patients with neutrophil leukocytosis due to bacterial infection. This suggests that mature neutrophils can be recruited in vivo from a pool of more mature cells without primary involvement of myeloid precursors.
2. Macrophage Colony Formation Monocyte macrophages develop a characteristic morphology in culture [9,21]: They are larger than neutrophils, and appear greyish by cytoplasmic structures, which are recognized as vacuoles on Giemsa stain. They develop functional characteristics of macrophages in culture: Fc-receptors [16 d, 22] are detectable on their surface and they are able to ingest antibody-coated erythrocytes [22]. Macrophage colonies are loose. They frequently contain neutrophils in varying proportion [10,16c, 19]. Human macrophage precursors could be separated from neutrophil colony forming cells by velocity sedimentation [12]: Macrophage colony forming cells sediment more slowly and thus appear to be smaller than those committed strictly to the neutrophil pathway. They respond to a subtype of CSF, which was found in human serum and urine rather than in conditioned media [16 f].
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The relative frequency of macrophage colonies in bone marrow cultures is approximately 3 0 ~ [10,21]. One third contain neutrophils, the other two thirds are pure macrophage colonies [10]. The proportion increases at suboptimal culture conditions [16e] and with ageing of the cultures [16b]. In cultures from patients with untreated Hodgkin's disease, myeloma, benign monoclonal paraproteinemia, lymphoma, and cancer, a higher percentage of macrophage colonies is seen [21]. Most of these patients do not have monocytosis in vivo. Macrophage maturation appears to be more favored by in vitro than by in vivo conditions. Therefore, increased commitment toward the macrophage pathway as a sign of disease is recognized in vitro, but not necessarily in vivo.
3. Eosinophil Colony Formation Eosinophil colonies in methylcellulose cultures are distinguished from neutrophil colonies by their conspicuous dark appearance at small magnification. They are relatively small and consist of tightly packed cells containing cytoplasmic granules which can be recognized at high magnification in the inverted microscope. Single eosinophils are larger than neutrophils and do not vary in shape and size. This morphology is typical of human eosinophil colonies and differs from the appearance described for mouse eosinophil colonies [18]. On Giemsa stain single cells from eosinophil colonies are immature: They have a large, nonsegmented nucleus and strongly eosinophilic cytoplasmic granules. Vacuolization of the cytoplasma is frequently observed. Development of granules remains incomplete when examined by electron microscopy and cytochemistry [23, 34]. In vitro conditions apparently do not support eosinophil maturation beyond the stage of myelocytes [21]. Neutrophil differentiation in vitro is comparably more complete. There is evidence that the eosinophil pathway separates from the granulocytemacrophage pathway at a very primitive level: Eosinophil colonies are always pure, suggesting that they are derived from an eosinophil precursor which restricted differentiation capacity. In the mouse, heterogeneity of eosinophil and neutrophil precursors has been demonstrated by velocity sedimentation: Eosinophil colonyforming cells sediment more slowly and respond to a specific eosinophil colony stimulating activity derived from stimulated lymphocytes [18]. Further evidence for the specificity of eosinophil-CSF was obtained from experiments in animals with suppressed neutrophil-CSF: 1. Antibodies raised against neutrophil-CSF do not affect eosinophil-CSF [16i]. 2. Mice with a congenital defect of endotoxin mediated CSF-production have a normal eosinophil-CSF-response [2]. Human eosinophil-CSF could so far not be separated from neutrophil-CSF. Human conditioned media of all sources contain both activities (16k]. Factors, which promote maturation of eosinophils in liquid culture [2, 3, 26] or induce eosinophilia in animals [15] and in humans [33] have been described. None of them has been found to stimulate colony formation in vitro, i.e., it is not clear if they are identical with eosinophil-CSF. They could represent a maturation hormone acting at a more mature level of eosinophilopoiesis.
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Delayed appearance and long survival of eosinophil colonies is characteristic [8, 11,16g]. Very few are seen before 2 weeks in culture. They desintegrate later than neutrophils during prolonged incubation. It has been suggested that late appearance could indicate that eosinophil precursors are more primitive cells than neutrophil colony-forming cells [11 ]. The relative frequency of eosinophil colonies in normal bone marrow cultures is low: Dao reports an incidence of 3 . 7 ~ [8], in our series it was 12~ [21]. However, compared to the percentage of mature eosinophils in vivo, eosinophil colony counts are relatively high, suggesting that in vitro conditions favor early eosinophil commitment, although they do not support terminal eosinophil maturation. A higher relative number of eosinophil colonies is observed in a variety of malignant diseases, in graft-versus-host disease [21] and in some patients with acute myelogenous leukemia (AML) in remission. Interestingly, the formation of eosinophil clusters is sometimes observed in A M L in relapse [16h and personal observation]. The prognostic significance of this finding is not yet established. It has been reported that eosinophils inhibit granulopoiesis in vitro [31]. They may therefore be involved in the control of leukemic growth. The increase of eosinophil colonies in disease was not, or not adequately paralleled by eosinophilia in peripheral blood. On the other hand, in cultures from two patients with massive eosinophilia which was due to allergic disease in one, and had developed in the course of acute lymphoblastic leukemia in the other, the percentage of eosinophil colonies was not increased. From these observations one would assume that there is more than one mechanism by which the eosinophil pathway can be stimulated: One seems to act at the level of eosinophil precursor cells, and thus to be recognized in vitro by increased eosinophil colony formation. It does not necessarily produce eosinophilia in vivo. Another factor seems to promote final eosinophil maturation rather than early commitment. It is not recognized in vitro but produces eosinophilia in vivo. These observations support the hypothesis that in vitro eosinophilCSF is not necessarily identical with the in vivo "eosinophilopoietin" described above. Eosinophils are known to act as effector cells in several types of immune reactions: Killing of parasites [7] and tumor cells [25, 28] by eosinophils has been observed. Increased eosinophil commitment in vitro in malignant disease therefore probably reflects an in vivo defense mechanism.
Peripheral Blood Cultures from peripheral blood differ from bone marrow cultures by a lower concentration of colony-forming cells. In addition, the relative frequency of different colony types in peripheral blood cultures shows some characteristic features: 1. Only large, late-appearing neutrophiI colonies are seen in peripheral blood cultures, more mature neutrophil precursors with restricted proliferation capacity appear to be excluded from the circulation. 2. Macrophage colonies are rare (7 ~ compared to 30 ~ in bone marrow cultures).
Myeloid Differentiation in Cultures of Human Hemopoietic Precursor Cells
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3. The percentage of eosinophil precursors is high in peripheral blood, the relative frequency was over 50 % in six patients described by Chervenick [8]. We found a mean of 40% eosinophil colonies in 10 normals. In simultaneous bone marrow cultures the incidence was 12 %. Thus, there is a true prevalence of eosinophil precursors in the circulation. Eosinophil colonies in peripheral blood cultures are large and multicentered. Since they appear dark, they may be mistaken for erythroid bursts in cultures containing erythropoietin. The ability to form multicentered colonies is characteristic for erythroid burst-forming cells. It indicates a high proliferation capacity and thus immaturity. Likewise, "eosinophil burstforming cells" may be very primitive cells. These observations suggest that primitive myeloid precursors prevail in the circulation. The same phenomenon is known for the erythroid line. Only immature precursors (burst-forming cells) are detected in peripheral blood whereas the more mature precursors (CFU-E) are excluded [23]. Peripheral blood thus seems to be a pool of very immature hemopoietic precursors.
Conclusions and Summary Culture conditions support the early phase of myeloid differentiation. Mechanisms of regulation can therefore be recognized in vitro by differential counts of neutrophil, macrophage, and eosinophil colonies under varying conditions. From such studies information of theoretical and clinical interest has emerged: Neutrophils and macrophages mature along a common pathway, from which the eosinophil line separates at an early stage of differentiation. Specific regulators exist for each of the three pathways. Colony-stimulating factors acting at the level of neutrophil, eosinophil, and macrophage precursor cells are detectable in vitro. They initiate maturation which is probably completed by other factors in vivo. An increase of macrophage and eosinophil colonies without monocytosis and eosinophilia in vivo is frequently observed in malignant disease. This phenomenon is likely to reflect a tumor defense mechanism. The relative frequency of various precursor cells in the circulation does not reflect their distribution in the bone marrow. There seems to be a prevalence of immature cells in peripheral blood.
References 1. Baccarani, M., Ricci, P., Santucci, A. M., Tufa, S. : Proliferazione e maturazione dei precursori granulocitari umani coltivati in metil cellulosa. In: Emolinfopoiesi, fattori di regolazione e meccanismi di crescita. Brunelli, M.A., Bagnara, G.P., Castaldini, C. (eds.), pp. 299-312. IlI incontro nazionale di ematologia sperimentale, Reggio Calabria 11-12 maggio 1979, Esculapio, Bologna. 2. Bartelmez, S.H., Dodge, W.H., Bass, D.A.: Antigen-mediated release of eosinophit growth stimulating factor (Eo-GSF) from trichinella spiralis sensitized spleen cells: A comparison of various T. spiralis antigens. Exp. Hematol. [Suppl. 3] 6, 77 (1978)
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Received July 23, 1979
