Cell interactions influencing murine marrow megakaryocytes: nature of the potentiator cell in bone marrow

Auxiliary bone marrow cells are required for optimal murine megakaryocyte colony formation in addition to progenitor cells and a colony stimulating activity (CSA) present in WEHI-3 cell conditioned medium. These auxiliary cells are adherent, with a sedimentation rate of 5.8 mm hr-1 and buoyant density of 1.065-1.078 gcm-3. The activity from bone marrow cells is loss at irradiation doses above 900 rad. Bone marrow cells with these characteristics, and supernatants from lung, bone shafts, and peritoneal exudate cells were all active in enhancing megakaryocyte colony incidences in mouse bone marrow cultures above those stimulated by an obligatory activity in WEHI-3 cell conditioned medium. Certain macrophage cell lines (J774, P388D1) could elaborate the activity. This study confirms that a potentiation activity enhances CSA stimulation of megakaryocyte colony formation. The potentiator is elaborated by bone marrow cells in limiting amounts requiring either high cell concentrations or an exogenous source of the activity for optimal colony growth.     

Stromal growth factor production in irradiated lectin exposed long-term murine bone marrow cultures

Hematopoietic regulatory factors produced by adherent (stromal) cells in long-term murine bone marrow cultures have been investigated. Using an in situ double layer agar overlay system, we demonstrated that exposure of the stromal cells to 1,100-rad irradiation increased their activities in stimulating colony formation of FDC-P1, an interleukin 3 (IL 3)-responsive cell line. The colony-stimulating activities (CSAs) of the irradiated stroma also stimulated normal marrow cells to form granulocyte-macrophage, megakaryocyte, and mixed lineage colonies. Addition of the lectin pokeweed mitogen to the irradiated stroma increased the level of CSAs. The FDC-P1 CSA of the irradiated stroma was inhibited by antibodies directed against murine granulocyte- macrophage colony stimulating factor (GM-CSF) but not by those against murine IL 3. Stromal-derived CSA for marrow cells was also partially blocked by anti-GM-CSF antibodies, probably reflecting the presence of other CSAs such as CSF-1. This latter growth factor has been found to be present in conditioned media from Dexter stroma, but levels are not increased after irradiation or lectin exposure. Partially purified GM- CSF, like IL 3, stimulated FDC-P1 proliferation and granulocyte, macrophage, and megakaryocyte colony formation. These results indicate that the major terminal differentiating hormone elicited by irradiation or lectin exposure of murine marrow stromal cells is GM-CSF. This growth factor, along with CSF-1, can account for the differentiated progeny produced in this system: macrophages, granulocytes, and megakaryocytes.     

Characterization of hemopoietic activities in media conditioned by a murine marrow-derived adherent cell line, B.Ad

The hemopoietic activities present in medium conditioned by a murine bone marrow-derived adherent cell line (B.Ad) have been studied. B.Ad-conditioned medium stimulated neutrophil, neutrophil-macrophage, and macrophage colonies in agar cultures of bone marrow cells and 90% of this activity was neutralized by antimacrophage colony-stimulating factor (anti-M-CSF). The conditioned medium supported the generation and/or maintenance of spleen colony-forming units (CFU-S) in liquid cultures and synergized with multilineage colony-stimulating factor (Multi-CSF; IL-3) to stimulate colony formation by day-3 post-5-fluorouracil (FU)-treated bone marrow cells. When used as feeder layers, B.Ad cells stimulated erythroid colony-forming units (CFU-E) and markedly enhanced erythroid burst-forming units (BFU-E) stimulation more than did maximal Multi-CSF (IL-3) and Epo stimulation. No CFU-E- or BFU-E-stimulating activities were detected in medium conditioned by B.Ad cells. Similarly, B.Ad-conditioned medium was unable to stimulate Multi-CSF (IL-3) or granulocyte-macrophage (GM)-CSF-dependent cell lines. The data suggest that medium conditioned by this bone marrow-derived adherent cell line contains M-CSF and other factors not detectable as CSFs that either directly or by means of a synergistic mechanism are able to stimulate CFU-S and colony-forming cells (CFC).     

The production of colony stimulating activity by monocyte enriched fractions from murine continuous bone marrow culture adherent layers

The production of colony stimulating activity (GM-CSA) within murine continuous bone marrow cultures was investigated by subjecting either the nonadherent or the adherent layer cells to separation by velocity sedimentation. The presence of GM-CSA in conditioned medium was defined by the support of granulocyte/macrophage colony formation in soft agar culture. Cell free conditioned medium from weekly feedings of intact continuous marrow cultures and medium conditioned by each fraction of velocity sedimentation separated, nonadherent cells did not contain assayable GM-CSA. However, medium conditioned by fractions of adherent layer cells with a modal sedimentation velocity of 8.8 mm/h (range 6.2-10.6 mm/h) contained GM-CSA. Cytochemical studies with Wright's-Giemsa and non-specific esterase stains in addition to immunofluorescent studies with anti-collagen III, anti-collagen IV and monoclonal anti-Mac I antibodies to define fibroblasts, endothelial cells and monocytes, respectively, demonstrated that the cells within the GM-CSA producing fractions were enriched with monocytes/macrophages. Fibroblasts and a small proportion of endothelial cells were also present. GM-CSA is produced within the microenvironment (adherent layer) of murine continuous marrow cultures. Either adherent layer monocytes and/or a monocyte-endothelial cell interaction account for the GM-CSA production.     

Granulopoietic effects of human bone marrow fibroblastic cells and abnormalities in the "granulopoietic microenvironment"

The in vitro granulopoietic effects of adherent bone marrow fibroblastic cells (FC) were studied in normal humans and in patients with acute myelogenous leukemia (AML) and myeloproliferative disorders (MPD). To determine their influence on granulopoiesis, we established FC in liquid-phase cultures, overlaid the adherent FC with normal bone marrow cells in agar, and subsequently measured the growth of CFU-C. When using target marrows containing few spontaneous colonies, increased numbers of CFU-C were found above the FC obtained from normals. No growth greater than controls was observed in those areas lacking FC. If target marrows contained large numbers of spontaneous CFU-C, actual inhibition of colony formation was produced by FC co- incubation. In contrast to normals, FC obtained from untreated AML and MPD patients typically failed to enhance granulopoiesis. Regardless of source, FC were not synergistic with the effects of placenta- conditioned media (typically being inhibitory) for colony number, but were synergistic for colony size. Conditioned media obtained from FC cultures did not enhance colony formation and actually inhibited spontaneous colony formation. Thus, microenvironmental abnormalities in interactions between "stromal cells" and hematopoietic progenitors may be important in the pathogenesis and clinical expression of hematopoietic malignancies in humans.     

Effects of human marrow stromal cells on proliferation by human granulocytic (GM-CFC), erythroid (BFU-E) and mixed (Mix-CFC) colony-forming cells

S ummary . We have investigated the effects of the major components of bone marrow stroma (fibroblasts, fat cells, macrophages and endothelial cells) on colony-forming haemopoietic precursor cells. Selective cultures of the different stromal cell types, grown to confluence, were used as underlayers for agar or methylcellulose cultures containing bone marrow cells. In different experiments, colony-stimulating factor (CSF) was added to stimulated granulocyte-macrophage colony-forming cells (GM-CFC), erythropoietin was used to induce erythroid burst (BFU-E) formation or erythropoietin and medium conditioned by leucocytes in the presence of phytohae-magglutinin (PHA-LCM) was added to induce the formation of colonies of mixed myeloid lineage (Mix-CFC)
Fibroblasts grown from human marrow enhanced granulopoiesis when CSF was present in the cultures but suppressed the formation of BFU-E and mixed colonies. Treatment of the cultures with methylprednisolone induced the formation of fat cells in the fibroblast cultures and prevented both the fibroblast-mediated enhancement of granulopoiesis and the fibroblast-mediated suppression of erythropoiesis. Stromal macrophages reduced granulocyte colony formation but had little effect on the proliferation of BFU-E or mixed colony-forming cells. Endothelial cells stimulated granulopoiesis by releasing CSF into the culture supernatant: supernatant from endothelial cell cultures had no marked effects on either BFU-E or Mix-CFC.
We conclude that different components of marrow stroma have contrasting effects on erythropoiesis and granulopoiesis; thus, marrow stroma may regulate the expression of stem cell differentiation in vivo.     

Enhancement of in vitro beta-thalassemic and normal hematopoiesis by a noncytotoxic monoclonal antibody, 9.1C3: evidence for negative regulation of hematopoiesis by monocytes and natural killer cells

The enhancement of in vitro human hematopoiesis by the addition of a noncytotoxic monoclonal antibody, 9.1C3, is described. Enhancement of all aspects of in vitro hematopoiesis was observed on addition of 9.1C3 antibody to cultures of mononuclear cells from normal bone marrow, cord blood, and peripheral blood from beta-thalassemia major patients. In cultures with no exogenous colony-stimulating factor (CSF), the addition of 9.1C3 resulted in a two- to eightfold increase in nonerythroid colony formation. Similarly, for cultures maximally stimulated with CSF, the addition of 9.1C3 antibody resulted in a one- to fourfold increase in colony formation. These effects were abrogated by the removal of either adherent, Leu-M3+ or Leu-7+ cells. Colony- forming cells were shown to be present among the 9.1C3-negative cells when mononuclear cells were sorted by flow cytometry. Media conditioned in the presence of 9.1C3 and mononuclear cells were able to enhance colony formation in vitro for normal nonadherent bone marrow cells beyond that achieved with supramaximal amounts of human placental- conditioned medium and erythropoietin. The data suggest that natural killer cells interact with monocytes to exert a negative regulatory control on in vitro granulopoiesis and erythropoiesis. Consequently, the number of progenitor and multipotential cells in cultures of unfractionated cell populations may be greatly underestimated.     

Interaction between normal and leukaemic human cells in agar culture.

The influence of leukaemic cells from 12 patients with acute leukaemia on normal granulopoiesis in agar culture was investigated using leukeamic cell feeder layers. Leukaemic feeder cells from 7 of the 12 patients elicited no colony growth, while cells from the remaining 5 stimulated normal colony growth. In 3 of the 7 non-stimulatory patients release of inhibitory factors from the leukaemic cells seemed responsible for the effect on normal granulopoiesis, while inappropriate colony stimulating factor (CSF) production by the feeder cells could not be ruled out in the remaining 4 patients. When the leukaemic cells were cultured with, as well as without, conditioned medium, cells from 5 of the patients formed clusters. Growth in these cultures did not correlate to the effect found in the feeder layer experiments.     

Vascular endothelium as a regulator of granulopoiesis: production of colony-stimulating activity by cultured human endothelial cells

Colony-stimulating activity is a regulatory factor(s) that promotes differentiation of hemopoietic stem cells to mature granulocytes and macrophages; in man it has been found that blood monocytes, lymphocytes, and tissue macrophages produce it. In an effort to identify other potenitally physiologic tissue sources of colony- stimulating activity, we have studied the capacity of primary cultures of human vascular endothelial cells to produce colony-stimulating activity. Medium conditioned by incubation with endothelial cultures contained activity that promoted granulocyte-macrophage colony formation of nonadherent human and murine marrow cells. Exposure of endothelial cultures to 0.1-5.0 microgram/ml S. typhosa endotoxin for 6- 72 hr enhanced colony-stimulating activity production. Similarly, incubation of endothelial cells with lysates of human blood granulocytes, or cocultivation with intact granulocytes, resulted in increased colony-stimulating activity levels. In 7-14 day cultures, freshly isolated endothelial cells, incorporated into agar underlayers, consistently stimulated more colony formation by nonadherent human marrow cells than comparable numbers of blood monocytes. These data indicate that: (1) cultured human endothelial cells are a potent source of colony-stimulating activity; (2) they respond to endotoxin and granulocytes and their contents by producing increased amounts of CSA; and (3) they produce morea colony-stimulating activity, than human blood monocytes under standardized conditions in vitro. These observations suggest that the vascular endothelium may play a role in the physiologic regulation of granulopoiesis.     

Human tissues as source of colony-stimulating factor in human bone marrow cultures.

Human bone marrow cells were cultured by the agar method using feeder layers containing human tissue fragments of various origin. Colony stimulating factor (CSF) was shown to be released from all tissues tested (adipose tissue, skeletal muscle, peritoneum and vascular wall), and primary cultures of human fibroblasts could also stimulate colony growth. Colony growth of murine bone marrow cells was also stimulated by all feeder layers. Thus, the study demonstrates that monocytes and macrophages might not be the only source of CSF of possible importance for human granulopoiesis in vivo.     

Human Tissues as Source of Colony-Stimulating Factor in Human Bone Marrow Cultures

Human bone marrow cells were cultured by the agar method using feeder layers containing human tissue fragments of various origin. Colony stimulating factor (CSF) was shown to be released from all tissues tested (adipose tissue, skeletal muscle, peritoneum and vascular wall), and primary cultures of human fibroblasts could also stimulate colony growth. Colony growth of murine bone marrow cells was also stimulated by all feeder layers. Thus, the study demonstrates that monocytes and macrophages might not be the only source of CSF of possible importance for human granulopoiesis in vivo.     

Biologic properties in vitro of a recombinant human granulocyte- macrophage colony-stimulating factor

Recombinant human granulocyte-macrophage colony-stimulating factor (rH GM-CSF) was purified to homogeneity from medium conditioned by COS cells transfected with a cloned human GM-CSF cDNA and shown to be an effective proliferative stimulus in human marrow cultures for GM and eosinophil colony formation. The specific activity of purified rH GM- CSF in human marrow cultures was calculated to be at least 4 X 10(7) U/mg protein. Clone transfer experiments showed that this proliferation was due to direct stimulation of responding clonogenic cells. Acting alone, rH GM-CSF did not stimulate erythroid colony formation, but in combination with erythropoietin, increased erythroid and multipotential colony formation in cultures of peripheral blood cells. rH GM-CSF had no proliferative effects on adult or fetal murine hematopoietic cells, did not induce differentiation in murine myelomonocytic WEHI-3B cells, and was unable to stimulate the survival or proliferation of murine hematopoietic cell lines dependent on murine multi-CSF (IL 3). rH GM- CSF stimulated antibody-dependent cytolysis of tumor cells by both mature human neutrophils and eosinophils and increased eosinophil autofluorescence and phagocytosis by neutrophils. From a comparison of these effects with those of semipurified preparations of human CSF alpha and -beta, it was concluded that rH GM-CSF exhibited all the biologic activities previously noted for CSF alpha.     

Heterogeneity in human neutrophil, macrophage and eosinophil progenitor cells demonstrated by velocity sedimentation separation

Progenitor cells of neutrophils, monocyte-macrophages, and eosinophils in human marrow were enumerated in agar cultures stimulated by placental conditioned medium or white cell underlayers. Fractionation of marrow populations by velocity sedimentation showed that the profiles of neutrophil and macrophage colony-forming cells shifted from a peak of 8-9 mm/hr in 7-day cultures to a peak of 6-7 mm/hr in 14-day cultures. This shift was due to degeneration of some early colonies formed by rapidly sedimenting cells and the delayed formation of colonies by slowly sedimenting cells. Eosinophil colony formation was delayed until the second week of incubation. Further evidence of heterogeneity was the observation that rapidly sedimenting colony forming cells were more responsive to stimulation than more slowly sedimenting cells. In the macrophage and eosinophil populations, cluster-forming cells were partially segregatable form colony-forming cells. The observed heterogeneity was similar to the described previously in the mouse and suggests that separate subpopulations of progenitor cells may exist within each hemopoietic family that could possibly give rise to functionally different progeny.     

Release of granulocyte-specific colony-stimulating activity by human bone marrow exposed to phorbol esters

Granulocyte-macrophage colony growth depends on the presence of colony- stimulating activity (CSA). Phorbol esters induce concentration- dependent colony formation in the absence of exogenous CSA. We questioned whether phorbol esters mimicked the action of CSA by directly stimulating colony growth, or whether phorbol esters acted indirectly by inducing marrow cells to release CSA. First, after incubating human bone marrow cells with phorbol 12,13-dibutyrate (PDB) for 3 days, we separated PDB from the protein peak of the conditioned medium by Sephadex G-10 gel filtration and tested this peak for the presence of CSA. When diluted 1:10 in the agar colony assay, this material induced 133 +/- 15 colonies/10(5) bone marrow cells. Second, to determine whether bone marrow cells required the continued presence of PDB in order to release CSA, PDB was removed from bone marrow cells by washing, and these cells were reincubated in fresh medium in the absence of PDB. CSA was found in the medium of these cultures; its release was maximal after preincubation of bone marrow cells with 5 X 10(-8) M PDB for 3 days, followed by incubation for 3 days in the absence of PDB. This CSA stimulated granulopoiesis out of proportion to monocytopoiesis, with 85% +/- 17% of the colonies being granulocytic (as indicated by histochemical staining for chloroacetate esterase), and 12% +/- 3% being monocytic (as indicated by nonspecific esterase). Inhibitors of monocyte colony formation, including PGE1, were not present in the medium that contained this CSA. These studies demonstrate that normal human bone marrow cells exposed to PDB release CSA and that this CSA selectively stimulates granulopoiesis in vitro.     

Stimulation by human placental conditioned medium of hemopoietic colony formation by human marrow cells.

Medium conditioned by human placental tissue was found to stimulate granulocytic and monocytic colony formation by human marrow cells in semisolid agar cultures. The colony-stimulating activity of unfractionated conditioned medium was equivalent to the activity of standard peripheral blood underlayers. Placentas were a reliable source of active material, and one placenta provided enough material to stimulate 5,000-10,000 cultures of normal or leukemic cells. The colony-stimulating factor in human placental conditioned medium (CSFHPCM) was concentrated and purified 1800-fold using ammonium sulfate precipitation, calcium phosphate gel absorption, DEAE-cellulose batch absorption, gel filtration on Sephadex G-150, and polyarcylamide gel gel electrophoresis. The active factor behaved on gel filtration as a macromolecule with an apparent molecular weight of 30,000 daltons. The active factor in placental conditioned medium was not dependent on the presence of adherent marrow cells with endogenous colony-stimulating activity.     

Effects of culture matrix on differentiation of murine mast cells

The effects of culture matrix (agar, collagen, and methylcellulose) on differentiation of mast cells were investigated. Because berberine sulfate-positive colonies in agar were composed of macrophages but not of mast cells, the naphthol AS-D chloroacetate esterase reaction was used to identify mast-cell colonies. When bone marrow cells of WBB6F1 mice were cultured with conditioned media containing interleukin 3 (IL-3), numbers of mast-cell colonies were greater in collagen cultures than in agar and methylcellulose cultures. Electron microscopic examination revealed that mast cells developing in collagen and agar cultures were more mature than those developing in methylcellulose cultures. However, when bone marrow-derived cultured mast cells or purified peritoneal mast cells were plated, the efficiency of colony formation and size of colonies were comparable among agar, collagen, and methylcellulose cultures. Therefore, all three matrices tested had similar effects on the proliferation of mast cells. Collagen appeared to be suitable for differentiation of bone-marrow precursors and their maturation. Agar appeared to be suitable only for maturation.     

Canine cyclic haematopoiesis: bone marrow adherent cell influence of CFU-C formation

Canine cyclic haematopoiesis (CH) appears to be a multipotential stem cell defect, possibly due to an intrinsic marrow defect. The in vitro adherent marrow cells of the cyclic haematopoietic (CH) dog were cultured as underlayers beneath normal dog nonadherent marrow cells. The marrow granulocyte-committed colony forming units (CFU-C) of the normal dog nonadherent cells were cyclically influenced by the CH adherent cell underlayers. The CH adherent cells collected on the ninth or tenth day following the onset of neutropenia did not stimulate CFU-C formation while those collected on the sixth day stimulated as many as 108 colonies. The CH adherent cells collected on other cycle days supported increased CFU-C formation with the exception of cycle day 3 which inconsistently stimulated growth. These data show that the CH marrow in vitro adherent cells alternately stimulate and inhibit in vitro granulopoiesis of normal dog marrows.     

Hydrocortisone modulates colony-stimulating activity produced by human bone marrow-derived adherent cells

In an attempt to clarify the significance of hydrocortisone (HC) in human long-term bone marrow cultures, the production of colony-stimulating activity (CSA) and colony-enhancing activity (CEA) by human bone marrow-derived adherent cells (MDAC) and the modulation by HC were examined. The CSA production by MDAC was demonstrated using bilayer agar cultures. After treatment of MDAC with 10(-6) mol/l HC, the CSA production was markedly enhanced, and after treatment of macrophages with 10(-6) mol/l HC, the CSA production was inhibited. When added to a granulocyte-macrophage precursor cells (CFU-GM) assay system, HC inhibited colony formation. These results suggest that HC treatment directly stimulates CSA production by nonmacrophage cells of MDAC. The conditioned medium of the confluent layer of MDAC contained CEA, which was not influenced by the HC treatment of MDAC. Thus, HC plays an essential role in granulopoiesis in vitro, enhancing the CSA production by MDAC and inhibiting the differentiation of CFU-GM.     

Phorbol myristate acetate stimulates macrophage differentiation and replication and alters granulopoiesis and leukemogenesis in long-term bone marrow cultures

The effects of the tumor-promoter phorbol myristate acetate (PMA) on normal hemopoiesis and Friend leukemia virus (FLV) granulocytic leukemogenesis in long-term bone marrow cultures were examined. FLV- anemia-inducing strain (FLV-A) infected, Rauscher R-MuLV clone M52R infected, or uninfected control NIH Swiss mouse marrow cultures were treated weekly with PMA or 4-O-methyl-PMA at 2.0 ng/ml or 200.0 ng/ml. Addition of PMA to control uninfected or R-MuLV-infected cultures decreased production of nonadherent granulocytic cells and granulocyte- macrophage progenitor cells (GM-CFU-c), and increased the numbers of adherent macrophages. Addition of PMA to FLV-A-infected cultures did not inhibit generation of granulocytic leukemia cell lines even though the numbers of adherent adipocytes were decreased and adherent macrophages were increased. PMA treatment of freshly explanted whole bone marrow but not purified nonadherent GM-progenitor cells from long- term bone marrow cultures stimulated GM-CFU-c and cluster formation in the absence of added colony-stimulating factor (CSF). The sensitivity of purified GM-progenitor cells to L929 or WEHI-3 CSF was not altered by PMA; however, PMA treatment of bone marrow macrophages or peritoneal exudate macrophages stimulated detectable GM-CFU-c and cluster formation by purified GM-progenitor cells under conditions where equal numbers of untreated macrophages failed to be stimulatory. Thus, several PMA effects on hempoietic stem cells in vitro are mediated through indirect action on adherent stromal cells including macrophages.     

Interleukin-1 (22-K factor) induces release of granulocyte-macrophage colony-stimulating activity from human mononuclear phagocytes

An electrophoretically pure preparation of natural human interleukin-1 (IL-1) was shown to stimulate in vitro colony formation in human bone marrow cultures. Day 4 myeloid cluster-forming cells (CFC), as well as early (day 7) and late (day 10) granulocyte-macrophage colony-forming units (CFU-GM) were stimulated in a dose-dependent fashion. At optimal concentrations of IL-1, the number of day 4 CFC reached 72%, the number of day 7 CFU-GM reached 32%, and the number of day 10 CFU-GM reached 80% of the respective numbers of colonies obtained by addition of crude leukocyte-conditioned medium (LCM). The IL-1-induced stimulatory effect on CFU-GM growth could be completely neutralized by a rabbit anti-IL-1 antiserum. Colony growth was abrogated by depleting the marrow cell suspensions of phagocytic cells prior to IL-1 addition. Conversely, the effect could be reintroduced by addition of marrow-derived adherent cells to bone marrow cell suspensions that had been depleted of both phagocytic and E rosetting T cells. Furthermore, media conditioned by bone marrow-derived adherent cells or by peripheral blood mononuclear phagocytes in the presence but not in the absence of IL-1, stimulated in vitro colony growth of phagocyte-depleted bone marrow cell suspensions. These results indicate that IL-1 induces release of granulocyte-macrophage colony-stimulating activity (GM-CSA) from human mononuclear phagocytes.